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Vaccination is the most effective countermeasure to reduce the severity of influenza. Current seasonal influenza vaccines mainly elicit humoral immunity targeting hemagglutinin (HA). In particular, the amino acid residues around the receptor-binding site in the HA head domain are predominantly targeted by humoral immunity as "immunodominant" epitopes. However, mutations readily accumulate in the head domain due to high plasticity, resulting in antigenic drift and vaccine mismatch, particularly with influenza A (H3N2) viruses. A vaccine strategy that targets more conserved immunosubdominant epitopes is required to attain a universal vaccine. Here, we designed an H3 HA vaccine antigen with various amino acids at immunodominant epitopes of the HA head domain, termed scrambled HA (scrHA). In ferrets, scrHA vaccination induced lower serum neutralizing antibody levels against homologous virus compared with wild-type (WT) HA vaccination; however, similar levels of moderately neutralizing titers against antigenically distinct H3N2 viruses were observed. Ferrets vaccinated with scrHA twice and then challenged with homologous or heterologous virus showed the same level of reduced virus shedding in nasal swabs as WT HA-vaccinated animals but reduced body temperature increase, whereas WT HA-vaccinated ferrets exhibited body temperature increases similar to those of mock-vaccinated animals. scrHA elicited antibodies against HA immunodominant and -subdominant epitopes at lower and higher levels, respectively, than WT HA vaccination, whereas antistalk antibodies were induced at the same level for both groups, suggesting scrHA-induced redirection from immunodominant to immunosubdominant head epitopes. scrHA vaccination thus induced broader coverage than WT HA vaccination by diluting out the immunodominancy of HA head epitopes. IMPORTANCE Current influenza vaccines mainly elicit antibodies that target the immunodominant head domain, where strain-specific mutations rapidly accumulate, resulting in frequent antigenic drift and vaccine mismatch. Targeting conserved immunosubdominant epitopes is essential to attain a universal vaccine. Our findings with the scrHA developed in this study suggest that designing vaccine antigens that "dilute out" the immunodominancy of the head epitopes may be an effective strategy to induce conserved immunosubdominant epitope-based immune responses.
Proteasome-catalyzed peptide splicing represents an additional catalytic activity of proteasomes contributing to the pool of MHC-class I-presented epitopes. We here biochemically and functionally characterized a new melanoma gp100 derived spliced epitope. We demonstrate that the gp100(mel)47-52/40-42 antigenic peptide is generated in vitro and in cellulo by a not yet described proteasomal condensation reaction. gp100(mel)47-52/40-42 generation is enhanced in the presence of the β5i/LMP7 proteasome-subunit and elicits a peptide-specific CD8(+) T cell response. Importantly, we demonstrate that different gp100(mel)-derived spliced epitopes are generated and presented to CD8(+) T cells with efficacies comparable to non-spliced canonical tumor epitopes and that gp100(mel)-derived spliced epitopes trigger activation of CD8(+) T cells found in peripheral blood of half of the melanoma patients tested. Our data suggest that both transpeptidation and condensation reactions contribute to the frequent generation of spliced epitopes also in vivo and that their immune relevance may be comparable to non-spliced epitopes.
CD8+ T-cell responses to pathogens are directed against infected cells that present pathogen-encoded peptides on MHC class-I molecules. Although natural responses are polyclonal, the spectrum of peptides that qualify for epitopes is remarkably small even for pathogens with high coding capacity. Among those few that are successful at all, a hierarchy exists in the magnitude of the response that they elicit in terms of numbers of CD8+ T cells generated. This led to a classification into immunodominant and non-immunodominant or subordinate epitopes, IDEs and non-IDEs, respectively. IDEs are favored in the design of vaccines and are chosen for CD8+ T-cell immunotherapy. Using murine cytomegalovirus as a model, we provide evidence to conclude that epitope hierarchy reflects competition on the level of antigen recognition. Notably, high-avidity cells specific for non-IDEs were found to expand only when IDEs were deleted. This may be a host's back-up strategy to avoid viral immune escape through antigenic drift caused by IDE mutations. Importantly, our results are relevant for the design of vaccines based on cytomegaloviruses as vectors to generate high-avidity CD8+ T-cell memory specific for unrelated pathogens or tumors. We propose the deletion of vector-encoded IDEs to avoid the suppression of epitopes of the vaccine target.
Malaria is an infectious disease caused by parasites from the genus Plasmodium (P. falciparum and P. vivax are responsible for 90% of all clinical cases); it is widely distributed throughout the world's tropical and subtropical regions. The P. vivax Pv12 protein is involved in invasion, is expressed on merozoite surface and has been recognised by antibodies from individuals exposed to the disease. In this study, B- and T-cell epitopes from Pv12 were predicted and characterised to advance in the design of a peptide-based vaccine against malaria. For evaluating the humoral response of individuals exposed to natural P. vivax infection from two endemic areas in Colombia, BepiPred-1.0 software was used for selecting B-cell epitopes. B-cell epitope 39038 displayed the greatest recognition by naturally-acquired antibodies and induced an IgG2/IgG4 response. NetMHCIIpan-3.1 prediction software was used for selecting peptides having high affinity binding for HLA-DRβ1* allele lineages and this was confirmed by in-vitro binding assays. T-epitopes 39113 and 39117 triggered a memory T-cell response (Stimulation Index≥2) and significant cytokine production. Combining in-silico, in-vitro and functional assays, two Pv12 protein regions (containing peptides 39038, 39040, 39113 and 39117) have thus been characterised as promising vaccine candidates against P. vivax malaria.
Atherosclerosis and osteoporosis are epidemiologically linked and oxidation specific epitopes (OSEs), such as phosphocholine (PC) of oxidized phospholipids (PC-OxPL) and malondialdehyde (MDA), are pathogenic in both. The proatherogenic effects of OSEs are opposed by innate immune antibodies. Here we show that high-fat diet (HFD)-induced bone loss is attenuated in mice expressing a single chain variable region fragment of the IgM E06 (E06-scFv) that neutralizes PC-OxPL, by increasing osteoblast number and stimulating bone formation. Similarly, HFD-induced bone loss is attenuated in mice expressing IK17-scFv, which neutralizes MDA. Notably, E06-scFv also increases bone mass in mice fed a normal diet. Moreover, the levels of anti-PC IgM decrease in aged mice. We conclude that OSEs, whether produced chronically or increased by HFD, restrain bone formation, and that diminished defense against OSEs may contribute to age-related bone loss. Anti-OSEs, therefore, may represent a novel therapeutic approach against osteoporosis and atherosclerosis simultaneously.
Abrin, a toxin isolated from the seeds of Abrus precatorius (jequirity pea) is considered a biological threat agent by the Center for Disease Control and Prevention. To date, there is no effective postexposure treatment for abrin poisoning, and efforts are being made to develop an efficient vaccine and measures for postexposure therapy. Epitope mapping is widely applied as an efficient tool for discovering the antigenic moieties of toxins, thus providing invaluable information needed for the development of vaccines and therapies. Aiming to identify the immunodominant epitopes of abrin, several neutralizing antiabrin polyclonal antibodies were screened using a set of 15-mer peptides spanning the amino acid sequence of either the A or B subunits of abrin. Analysis of the antibody-binding pattern revealed 11 linear epitopes for the A subunit and 14 epitopes for the B subunit that are located on the surface of the toxin and thus accessible for antibody interactions. Moreover, the spatial location of several of these epitopes suggests they may block the galactose-binding pockets or the catalytic domain, thus neutralizing the toxin. These findings provide useful information and suggest a possible strategy for the development and design of an improved abrin-based vaccine and therapeutic antibodies.
Understanding where antibodies recognize antigens can help define mechanisms of action and provide insights into progression of immune responses. We investigate the extent to which information about binding specificity implicitly encoded in amino acid sequence can be leveraged to identify antibody epitopes. In computationally-driven epitope localization, possible antibody-antigen binding modes are modeled, and targeted panels of antigen variants are designed to experimentally test these hypotheses. Prospective application of this approach to two antibodies enabled epitope localization using five or fewer variants per antibody, or alternatively, a six-variant panel for both simultaneously. Retrospective analysis of a variety of antibodies and antigens demonstrated an almost 90% success rate with an average of three antigen variants, further supporting the observation that the combination of computational modeling and protein design can reveal key determinants of antibody-antigen binding and enable efficient studies of collections of antibodies identified from polyclonal samples or engineered libraries.
Study of the function of epitopes of Mycobacterium tuberculosis antigens contributed significantly toward better understanding of the immunopathogenesis and to efforts for improving infection and disease control. Characterization of genetically permissively presented immunodominant epitopes has implications for the evolution of the host-parasite relationship, development of immunodiagnostic tests, and subunit prophylactic vaccines. Knowledge of the determinants of cross-sensitization, relevant to other pathogenic or environmental mycobacteria and to host constituents has advanced. Epitope-defined IFNγ assay kits became established for the specific detection of infection with tubercle bacilli both in humans and cattle. The CD4 T-cell epitope repertoire was found to be more narrow in patients with active disease than in latently infected subjects. However, differential diagnosis of active TB could not be made reliably merely on the basis of epitope recognition. The mechanisms by which HLA polymorphism can influence the development of multibacillary tuberculosis (TB) need further analysis of epitopes, recognized by Th2 helper cells for B-cell responses. Future vaccine development would benefit from better definition of protective epitopes and from improved construction and formulation of subunits with enhanced immunogenicity. Epitope-defined serology, due to its operational advantages is suitable for active case finding in selected high disease incidence populations, aiming for an early detection of infectious cases and hence for reducing the transmission of infection. The existing knowledge of HLA class I binding epitopes could be the basis for the construction of T-cell receptor-like ligands for immunotherapeutic application. Continued analysis of the functions of mycobacterial epitopes, recognized by T cells and antibodies, remains a fertile avenue in TB research.
Abnormal mitochondrial function is a widely reported contributor to neurodegenerative disease including Alzheimer's disease (AD), however, a mechanistic link between mitochondrial dysfunction and the initiation of neuropathology remains elusive. In AD, one of the earliest hallmark pathologies is neuropil threads comprising accumulated hyperphosphorylated microtubule-associated protein (MAP) tau in neurites. Rod-like aggregates of actin and its associated protein cofilin (AC rods) also occur in AD. Using a series of antibodies--AT270, AT8, AT100, S214, AT180, 12E8, S396, S404 and S422--raised against different phosphoepitopes on tau, we characterize the pattern of expression and re-distribution in neurites of these phosphoepitope labels during mitochondrial inhibition. Employing chick primary neuron cultures, we demonstrate that epitopes recognized by the monoclonal antibody 12E8, are the only species rapidly recruited into AC rods. These results were recapitulated with the actin depolymerizing drug Latrunculin B, which induces AC rods and a concomitant increase in the 12E8 signal measured on Western blot. This suggests that AC rods may be one way in which MAP redistribution and phosphorylation is influenced in neurons during mitochondrial stress and potentially in the early pathogenesis of AD.
To analyze the immunochemical structure of Escherichia coli ribosomal protein S13 and its organization in situ, we have generated and characterized 22 S13-specific monoclonal antibodies. We used a competitive enzyme-linked immunosorbent assay to divide them into groups based on their ability to inhibit binding of one another. The discovery of five groups with distinct binding properties suggested that a minimum of five distinct determinants on S13 are recognized by our monoclonal antibodies. The locations of the epitopes detected by these monoclonal antibodies have been mapped on S13 peptides. Three monoclonal antibodies bind a S13 C-terminal 34-residue segment. All the other 19 monoclonal antibodies bind a S13 N-terminal segment of about 80 residues. The binding sites of these 19 monoclonal antibodies have been further mapped to subfragments of peptides. Two monoclonal antibodies recognized S131-22; three monoclonal antibodies bound to S131-40; the binding sites of three other antibodies have been located in S1323-80, with epitopes possibly associated with residues 40-80. The remaining 11 monoclonal antibodies did not bind to these subfragments. These data provide molecular basis to the structure of S13 epitopes, whose in situ accessibility may reveal the S13 organization on the ribosome.
Adipose tissue is a central regulator of metabolism and an important pharmacological target to treat the metabolic consequences of obesity, such as insulin resistance and dyslipidemia. Among the various cellular compartments, the adipocyte cell surface is especially appealing as a drug target as it contains various proteins that when activated or inhibited promote adipocyte health, change its endocrine function and eventually maintain or restore whole-body insulin sensitivity. In addition, cell surface proteins are readily accessible by various drug classes. However, targeting individual cell surface proteins in adipocytes has been difficult due to important functions of these proteins outside adipose tissue, raising various safety concerns. Thus, one of the biggest challenges is the lack of adipose selective surface proteins and/or targeting reagents. Here, we discuss several receptor families with an important function in adipogenesis and mature adipocytes to highlight the complexity at the cell surface and illustrate the problems with identifying adipose selective proteins. We then discuss that, while no unique adipocyte surface protein might exist, how splicing, posttranslational modifications as well as protein/protein interactions can create enormous diversity at the cell surface that vastly expands the space of potentially unique epitopes and how these selective epitopes can be identified and targeted.
The gut microbiome is the largest source of intrinsic non-self-antigens that are continuously sensed by the immune system but typically do not elicit lymphocyte responses. CD4+ T cells are critical to sustain uninterrupted tolerance to microbial antigens and to prevent intestinal inflammation. However, clinical interventions targeting commensal bacteria-specific CD4+ T cells are rare, because only a very limited number of commensal-derived epitopes have been identified. Here, we used a new approach to study epitopes and identify T cell receptors expressed by CD4+Foxp3+ (Treg) cells specific for commensal-derived antigens. Using this approach, we found that antigens from Akkermansia muciniphila reprogram naïve CD4+ T cells to the Treg lineage, expand preexisting microbe specific Tregs, and limit wasting disease in the CD4+ T cell transfer model of colitis. These data suggest that the administration of specific commensal epitopes may help to widen the repertoire of specific Tregs that control intestinal inflammation.
The use of serological tests is valuable to diagnose Zika virus (ZIKV) infection and carry out epidemiological surveillance. However, ZIKV serological tests may result in false positives due to cross-reactivity between antibodies against other Flavivirus, especially dengue virus that worldwide disseminated. We used three online tools to predict amino acid sequences of B-cell epitopes. We selected and synthetized two epitopes that showed appropriate features in the molecular dynamic simulation and demonstrated to be suitable for serological assays.
Immunoglobulin molecules specifically recognize particular areas on the surface of proteins. These areas are commonly dubbed B-cell epitopes. The identification of epitopes in proteins is important both for the design of experiments and vaccines. Additionally, the interactions between epitopes and antibodies have often served as a model for protein-protein interactions. One of the main obstacles in creating a database of antigen-antibody interactions is the difficulty in distinguishing between antigenic and non-antigenic interactions. Antigenic interactions involve specific recognition sites on the antibody's surface, while non-antigenic interactions are between a protein and any other site on the antibody. To solve this problem, we performed a comparative analysis of all protein-antibody complexes for which structures have been experimentally determined. Additionally, we developed a semi-automated tool that identified the antigenic interactions within the known antigen-antibody complex structures. We compiled those interactions into Epitome, a database of structure-inferred antigenic residues in proteins. Epitome consists of all known antigen/antibody complex structures, a detailed description of the residues that are involved in the interactions, and their sequence/structure environments. Interactions can be visualized using an interface to Jmol. The database is available at http://www.rostlab.org/services/epitome/.
Antibodies are important for immunity and exist in several classes (IgM, IgD, IgA, IgG, IgE). They are composed of symmetric dimeric molecules with two antigen binding regions (Fab) and a constant part (Fc), usually depicted as Y-shaped molecules. Rheumatoid factors found in patients with rheumatoid arthritis are autoantibodies binding to IgG and paradoxically appear to circulate in blood alongside with their antigen (IgG) without reacting with it. Here, it is shown that rheumatoid factors do not react with native IgG in solution, and that their epitopes only become accessible upon certain physico-chemical treatments (e.g. heat treatment at 57 °C), by physical adsorption on a hydrophobic surface or by antigen binding. Moreover, chemical cross-linking in combination with mass spectrometry showed that the native state of IgG is a compact (closed) form and that the Fab parts of IgG shield the Fc region and thereby control access of rheumatoid factors and presumably also some effector functions. It can be inferred that antibody binding to pathogen surfaces induces a conformational change, which exposes the Fc part with its effector sites and rheumatoid factor epitopes. This has strong implications for understanding antibody structure and physiology and necessitates a conceptual reformulation of IgG models.
Bioinformatic tools and databases for glycobiology and glycomics research are playing increasingly important roles in functional studies. However, to verify hypotheses generated by computational glycomics with empirical functional assays is only an emerging field. In this study, we predicted glycan epitopes expressed by a cancer-derived mucin, MUC1, by computational glycomics. MUC1 is expressed by tumor cells with a deficiency in glycosylation. Although numerous diagnostic reagents and cancer vaccines have been designed based on abnormally glycosylated MUC1 sequences, the glycan and peptide sequences responsible for immune responses in vivo are poorly understood. The immunogenicity of synthetic MUC1 glycopeptides bearing Tn or sialyl-Tn antigens have been studied in mouse models, while authentic glyco-epitopes expressed by tumor cells remain unclear. To examine the immunogenicity of authentic cancer derived MUC1 glyco-epitopes, we expressed membrane bound forms of MUC1 tandem repeats in Jurkat, a mutant cancer cell line deficient of mucin-type core-1 β1-3 galactosyltransferase activity, and immunized mice with cancer cells expressing authentic MUC1 glyco-epitopes. Antibody responses to individual glyco-epitopes were determined by chemically synthesized candidate MUC1 glycopeptides predicted through computational glycomics. Monoclonal antibodies can be generated toward chemically synthesized glycopeptide sequences. With RPAPGS(Tn)TAPPAHG as an example, a monoclonal antibody 16A, showed 25-fold higher binding to glycosylated peptide (EC50=9.278±1.059 ng/ml) compared to its non-glycosylated form (EC(50)=247.3±16.29 ng/ml) as measured by ELISA experiments with plate-bound peptides. A library of monoclonal antibodies toward authentic MUC1 glycopeptide epitopes may be a valuable tool for studying glycan and peptide sequences in cancer, as well as reagents for diagnosis and therapy.
Epitopes are a hallmark of the antigen specific immune response. The identification and characterization of epitopes is essential for modern immunologic studies, from investigating cellular responses against tumors to understanding host/pathogen interactions especially in the case of bacteria with intracellular residence. Here, we have utilized a novel approach to identify T cell epitopes exploiting the exquisite ability of particulate antigens, in the form of beads, to deliver exogenous antigen to both MHC class I and class II pathways for presentation to T cell hybridomas. In the current study, we coupled this functional assay with two distinct protein expression libraries to develop a methodology for the characterization of T cell epitopes. One set of expression libraries containing single amino acid substitutions in a defined epitope sequence was interrogated to identify epitopes with enhanced T cell stimulation for a MHC class I epitope. The second expression library is comprised of the majority of open reading frames from the intracellular pathogen and potential biowarfare agent, Francisella tularensis. By automating aspects of this technology, we have been able to functionally screen and identify novel T cell epitopes within F. tularensis. We have also expanded upon these studies to generate a novel expression vector that enables immunization of recombinant protein into mice, which has been utilized to facilitate T cell epitope discovery for proteins that are critically linked to Francisella pathogenicity. This methodology should be applicable to a variety of systems and other pathogens.
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