1,927 research outputs found

    Structural and Computational Biology in the Design of Immunogenic Vaccine Antigens

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    Applying bioinformatics for antibody epitope prediction using affinity-selected mimotopes – relevance for vaccine design

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    To properly characterize protective polyclonal antibody responses, it is necessary to examine epitope specificity. Most antibody epitopes are conformational in nature and, thus, cannot be identified using synthetic linear peptides. Cyclic peptides can function as mimetics of conformational epitopes (termed mimotopes), thereby providing targets, which can be selected by immunoaffinity purification. However, the management of large collections of random cyclic peptides is cumbersome. Filamentous bacteriophage provides a useful scaffold for the expression of random peptides (termed phage display) facilitating both the production and manipulation of complex peptide libraries. Immunoaffinity selection of phage displaying random cyclic peptides is an effective strategy for isolating mimotopes with specificity for a given antiserum. Further epitope prediction based on mimotope sequence is not trivial since mimotopes generally display only small homologies with the target protein. Large numbers of unique mimotopes are required to provide sufficient sequence coverage to elucidate the target epitope. We have developed a method based on pattern recognition theory to deal with the complexity of large collections of conformational mimotopes. The analysis consists of two phases: 1) The learning phase where a large collection of epitope-specific mimotopes is analyzed to identify epitope specific “signs” and 2) The identification phase where immunoaffinity-selected mimotopes are interrogated for the presence of the epitope specific “signs” and assigned to specific epitopes. We are currently using computational methods to define epitope “signs” without the need for prior knowledge of specific mimotopes. This technology provides an important tool for characterizing the breadth of antibody specificities within polyclonal antisera

    The Molecular Basis of Antibody Mediated Neutralization of Hepatitis C Virus

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    Hepatitis C virus: HCV) is positive strand, blood-borne, hepatotropic RNA virus that causes chronic infection in ~170 million people worldwide and is the leading cause of liver transplantation in the United States. HCV entry and attachment is mediated by the envelope protein E2 through interaction with several cellular receptors including CD81, scavenger receptor B1: SRB-1), claudin-1, and occludin, although the exact mechanism by which these receptors facilitate infection remains unclear, largely due to the absence of a structural model of E2. The production of neutralizing antibodies against E2 is thought to be important for controlling HCV infection, likely by blocking virus interaction with these receptors. To better understand the structural and molecular basis of antibody neutralization of HCV, which could be used to inform novel therapeutic or vaccine approaches, we generated a panel of 78 monoclonal antibodies: MAbs) against the E2 protein from HCV genotypes 1 and 2 and assessed their neutralizing activity in vitro. Using this approach and by performing mechanistic studies, we identified three neutralizing MAbs, H77.16, H77.39, and J6.36, that inhibit infection at a post-attachment step. Using a yeast display library of E2 protein variants, we mapped the critical binding residues of these MAbs to distinct regions of the E2 protein: H77.16 binds within the HVR1 and to a conserved CD81 binding region ~125 amino acid residues C-terminal to the HVR1; H77.39 binds to conserved residues upstream of the hypervariable region: HVR1); and J6.36 binds to amino acid residues within HVR1 as well a site ~150 amino acids C-terminal to HVR1. Receptor-binding inhibition studies using E2 demonstrated that H77.16 potently inhibits binding to SR-B1, H77.39 potently inhibits binding to SR-B1 and CD81, and J6.36 potently inhibits binding to SR-B1 and modestly inhibits binding to CD81. Further mechanistic studies demonstrated that MAb-mediated neutralization could be enhanced by increases in pre-incubation temperature and time and that these results were likely due to altered epitope exposure on the viral surface. Together, these data provide new insight into the mechanisms by which antibodies neutralize infection of HCV

    Automated Detection of Conformational Epitopes Using Phage Display Peptide Sequences

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    Background: Precise determination of conformational epitopes of neutralizing antibodies represents a key step in the rational design of novel vaccines. A powerful experimental method to gain insights on the physical chemical nature of conformational epitopes is the selection of linear peptides that bind with high affinities to a monoclonal antibody of interest by phage display technology. However, the structural characterization of conformational epitopes from these mimotopes is not straightforward, and in the past the interpretation of peptide sequences from phage display experiments focused on linear sequence analysis to find a consensus sequence or common sequence motifs. Results: We present a fully automated search method, EpiSearch that predicts the possible location of conformational epitopes on the surface of an antigen. The algorithm uses peptide sequences from phage display experiments as input, and ranks all surface exposed patches according to the frequency distribution of similar residues in the peptides and in the patch. We have tested the performance of the EpiSearch algorithm for six experimental data sets of phage display experiments, the human epidermal growth factor receptor-2 (HER-2/neu), the antibody mAb Bo2C11 targeting the C 2 domain of FVIII, antibodies mAb 17b and mAb b12 of the HIV envelope protein gp120, mAb 13b5 targeting HIV-1 capsid protein and 80R of the SARS coronavirus spike protein. In all these examples th

    The Structural Basis of Flaviviridae Interaction with Antibodies and Receptors

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    Flaviviridae are a family of enveloped, positive-stranded RNA viruses responsible for a variety of diseases including encephalitis, hemorrhagic fever and hepatocellular carcinoma. The envelope: E) proteins that coat the outer surface of these viruses provide the molecular machinery that drives receptor interaction and membrane fusion. The assignment of biological functions to specific structural elements of these E proteins has proven crucial to the understanding of viral entry into host cells. Clearance is dependent upon the presence of neutralizing antibodies that are able to disrupt several stages of this process. Given their fundamental role in the viral life cycle, we sought to determine the structural basis for envelope protein interaction with antibodies and receptors for human pathogens of the Flaviviridae family Japanese Encephalitis Virus, Hepatitis C Virus and St. Louis Encephalitis Virus. Viruses of the Flavivirus genus within Flaviviridae are grouped into serocomplexes with similar clinical manifestations that are defined by cross-neutralization tests with polysera from heterologous infections. Japanese Encephalitis Virus: JEV) is the leading cause of viral encephalitis and prototypical member of the JEV serocomplex. We determined the 2.1Ã… resolution crystal structure of the JEV E protein ectodomain to investigate whether structural features could contribute to our understanding of serocomplex-specific pathogenesis. JEV E possesses the three domains characteristic of flavivirus envelopes and epitope mapping of neutralizing antibodies revealed residues localized to the domain I lateral ridge, fusion loop, domain III lateral ridge and domain I-II hinge. The dimer interface, however, is remarkably small and lacks several contacts present in other flavivirus E homodimers. Uniquely conserved histidines of the JEV serocomplex suggest that pH-mediated structural transitions may be assisted by lateral interactions outside the dimer interface in the icosahedral virion. Our results suggest that variation of dimer structure and stability may influence the assembly, receptor interaction and uncoating of virions. St. Louis Encephalitis Virus: SLEV) is another member of the JEV serocomplex with similar pathogenesis to JEV. We determined the 4.0 Ã… structure of the SLEV E protein in the post-fusion trimer conformation to compare it with E trimer structures from other flavivirus serocomplexes. SLEV E crystallized as a trimer in the absence of lipids or detergents, requiring only low pH. However, its domain arrangement was nearly identical to other post-fusion structures. This suggests that viruses can alter dimer assembly but the structure of the activated, fusogenic conformation may be more strictly conserved. The only member of Flaviviridae known to chronically infect humans is Hepatitits C Virus: HCV). HCV is blood borne and carried by roughly 3 percent of the world\u27s population. Clinical manifestations include hepatitis, cirrhosis and hepatocellular carcinoma. HCV envelope protein E2 mediates interaction with host receptors CD81 and scavenger receptor BI: SR-BI) and is the primary target of neutralizing antibodies. To elucidate detailed biochemical roles for these receptors\u27 interactions with E2, we determined that the E2 ectodomain: sE2) interacts with soluble CD81 large extracellular loop: CD81-LEL) with 2:2 stoichiometry, and that this interaction inhibits subsequent engagement of SR-BI. We then evaluated the affinity and kinetics of sE2:CD81-LEL binding. Interaction between these proteins was enhanced by deletion of hypervariable region 1: HVR1) of E2 and modulated by the genotype from which sE2 was generated. Furthermore, neutralization of HVR1-deleted HCV by a cross-reactive antibody was enhanced in a genotype-specific manner that correlated with sE2:CD81-LEL affinity measurements. Our results suggest that E2 cannot engage CD81 and SR-BI simultaneously, that HVR1 obscures conserved CD81 and antibody binding sites, and that genotypic variation influences HCV host receptor preference

    Novel in vitro approaches to delineate prion strain conformational variation

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    2019 Fall.Includes bibliographical references.Prions cause in invariably lethal, transmissible neurodegenerative diseases. There are no effective treatments or cures for prion diseases. Unlike other known pathogens, prions replicate in the absence of nucleic acids. Prion diseases stem from the conformational corruption of the cellular prion protein (PrPC) by the pathogenic form (PrPSc) (Prusiner, 1982). The prion phenomenon, protein-templated misfolding, is no longer limited to the prion protein (PrP). Other neurodegenerative disorders, including but not limited to Alzheimer's, Parkinson's, Huntington's are now being recognized as prion-like disorders (Soto, 2012). By exploring the intricacies of prion protein- misfolding, therapeutic approaches might emerge that will be useful in treating other neurodegenerative protein-misfolding disorders. Although the structure of PrPC has been solved (Riek et al 1997, Zahn et al 2000, Garcia et al 2000, Donne et al 2007, Antonyuk et al 2009), the three-dimensional structure of PrPSc has yet to be resolved. A confounding issue to identifying PrPSc structure is the existence of prion strains (Bett et al 2012). In the absence of nucleic acids, prion strain properties are propagated though variations in the conformational structure of PrPSc (Telling et al 1996). As such, prion strains can be defined as an infectious prion protein particle with a specific tertiary conformation that produces a specific neurodegenerative phenotype (Colby et al., 2009). Specifically, a prion strain can be considered to have a strain-specific (Peretz et al 2001) disease phenotype (Collinge et al 1996) based on the prion's ability to be stably propagated, fidelity to neuropathology, disease length, glycosylation profile, molecular weight of PK-resistant PrPSc, resistance to denaturation, amyloid seeding potential and other molecular characteristics. Ultimately, revealing PrPSc structure will provide better understanding of the basis of strains, species adaption and ultimately the species barrier. The traditional methodologies to examine prion strains are costly, time consuming, and do not provide adequate resolution of the PrPSc structure. The overarching aim of my research is to better understand how prions encrypt strain information. In Chapter 1, I outline essential background regarding prions and prion strains. In Chapter 2 and 3, I address the creation of the expanded Cell-Based Conformational Stability Assay, Epitope Stability Assay, and use of a new 7-5 ELISA Conformational Stability Assay. These represent novel tools that use chaotropic agents to probe epitope-mapped regions to identify subtle differences in prion strain structure. The prion strains evaluated were cervid (deer and elk) chronic wasting disease, murine- adapted scrapie (RML, 22L, 139A), murine-adapted chronic wasting disease (mD10) and cervid-adapted (deer and elk) RML. These techniques revealed subtle but significant prion strain structural variations within and between these strains. In Chapter 4, the techniques were used to better understand drug-induced prion evolution and strain evolution in cell culture. Drug-induced prion evolution of PrPSc structure was subtle but detectable within 24 hours of treatment. Additionally, the structural changes were not stable, but in flux. Prion strains evolve in cell culture through serial passaging, they do not recapitulate molecular characteristics of a biological prion infection. Moreover, the prion structure is not stably passaged into naïve cells, or transgenic mice. This makes reliance on chronically infected cells as a basis for anti- prion therapeutic testing inadvisable. In conclusion, the subtle variations encoded in prion strain structure can be detected with the three new techniques in this dissertation: C-CSA, ESA, and 7-5 ELISA-CSA

    HIV-1 Glycoprotein Immunogenicity

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    Crystal structure of a dimerized cockroach allergen Bla g 2 complexed with a monoclonal antibody

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    The crystal structure of a 1:1 complex between the German cockroach allergen Bla g 2 and the Fab' fragment of a monoclonal antibody 7C11 was solved at 2.8-Å resolution. Bla g 2 binds to the antibody through four loops that include residues 60-70, 83-86, 98-100, and 129-132. Cation-p interactions exist between Lys-65, Arg-83, and Lys-132 in Bla g 2 and several tyrosines in 7C11. In the complex with Fab', Bla g 2 forms a dimer, which is stabilized by a quasi-four-helix bundle comprised of an a-helix and a helical turn from each allergen monomer, exhibiting a novel dimerization mode for an aspartic protease. A disulfide bridge between C51a and C113, unique to the aspartic protease family, connects the two helical elements within each Bla g 2 monomer, thus facilitating formation of the bundle. Mutation of these cysteines, as well as the residues Asn-52, Gln-110, and Ile-114, involved in hydrophobic interactions within the bundle, resulted in a protein that did not dimerize. The mutant proteins induced less ß-hexosaminidase release from mast cells than the wild-type Bla g 2, suggesting a functional role of dimerization in allergenicity. Because 7C11 shares a binding epitope with IgE, the information gained by analysis of the crystal structure of its complex provided guidance for site-directed mutagenesis of the allergen epitope. We have now identified key residues involved in IgE antibody binding; this information will be useful for the design of vaccines for immunotherapy
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