Browsing by Subject "Antibodies"
Now showing 1 - 9 of 9
- Results Per Page
- Sort Options
Item Analyzing infection-driven immune perturbations by quantitative IR-Seq(2017-09-08) Wendel, Ben Shogo; Jiang, Ning Jenny; Georgiou, George; Maynard, Jennifer; Alper, Hal; Davis, MarkImmune repertoire sequencing (IR-Seq) rapidly emerged with the advent of high-throughput sequencing as a means of characterizing the adaptive immune system. Early generations of IR-Seq were plagued by sequencing errors and low diversity coverage. We developed Molecular Identifier Clustering-based IR-Seq (MIDCIRS) to quantitatively and comprehensively measure the immune repertoire from a small amount of blood. We used naive B cells to formulate a general framework for IR-Seq experimental validation and quality control and showed that MIDCIRS can be applied to as few as 1,000 naive B cells with excellent diversity coverage. Using MIDCIRS, we studied the antibody repertoire response to acute malaria infection in young children. We found that the infant antibody repertoire is surprisingly competent at introducing somatic hypermutations (SHM) and diversifying B cell clonal lineages in response to malaria infection. Detailed analysis of memory B cell-containing lineages in malaria-experienced toddlers revealed that memory B cells further mutate upon malaria rechallenge. IgM-expressing memory B cells largely retain IgM expression upon rechallenge, but a subset class switch to IgG and IgA. Accurate antibody repertoire analysis requires not only accurate sequencing data, but also correct reference germline allele sequences. Mismatches between the reference sequence and an individual’s true germline sequence would be mistakenly counted as SHMs, inflating the SHM load and skewing the repertoire analysis. We developed a simple yet effect method for predicting novel germline allele sequences from antibody repertoire data and validating them via targeted sequencing of non-rearranged genomic DNA. HIV infection has a profound impact on the CD4⁺ T cell compartment, which can have a devastating effect on the adaptive immune system as a whole. Paradoxically, while peripheral CD4⁺ T cell counts drop with disease severity, T[subscript FH] cells show an inverse relationship. We found that these expanded T[subscript FH] cells exhibit a functionally restricted phenotype, which could contribute to ineffective antibody responses both to HIV and unrelated vaccines. Using MIDCIRS, we found that these expanded T[subscript FH] cells are enriched with HIV-specific sequences and show signs of antigen-driven convergent evolution, suggesting that HIV-specific T cells are selected and recruited into the T[subscript FH] compartment during infection.Item Antibody Repertoires in Humanized NOD-scid-IL2Rγnull Mice and Human B Cells Reveals Human-Like Diversification and Tolerance Checkpoints in the Mouse(Public Library of Science, 2012-04-27) Ippolito, Gregory C.; Hoi, Kam Hon; Reddy, Sai T.; Carroll, Sean M.; Ge, Xin; Rogosch, Tobias; Zemlin, Michael; Shultz, Leonard D.; Ellington, Andrew D.; VanDenBerg, Carla L.; Georgiou, GeorgeImmunodeficient mice reconstituted with human hematopoietic stem cells enable the in vivo study of human hematopoiesis. In particular, NOD-scid-IL2Rγnull engrafted mice have been shown to have reasonable levels of T and B cell repopulation and can mount T-cell dependent responses; however, antigen-specific B-cell responses in this model are generally poor. We explored whether developmental defects in the immunoglobulin gene repertoire might be partly responsible for the low level of antibody responses in this model. Roche 454 sequencing was used to obtain over 685,000 reads from cDNA encoding immunoglobulin heavy (IGH) and light (IGK and IGL) genes isolated from immature, naïve, or total splenic B cells in engrafted NOD-scid-IL2Rγnull mice, and compared with over 940,000 reads from peripheral B cells of two healthy volunteers. We find that while naïve B-cell repertoires in humanized mice are chiefly indistinguishable from those in human blood B cells, and display highly correlated patterns of immunoglobulin gene segment use, the complementarity-determining region H3 (CDR-H3) repertoires are nevertheless extremely diverse and are specific for each individual. Despite this diversity, preferential DH-JH pairings repeatedly occur within the CDR-H3 interval that are strikingly similar across all repertoires examined, implying a genetic constraint imposed on repertoire generation. Moreover, CDR-H3 length, charged amino-acid content, and hydropathy are indistinguishable between humans and humanized mice, with no evidence of global autoimmune signatures. Importantly, however, a statistically greater usage of the inherently autoreactive IGHV4-34 and IGKV4-1 genes was observed in the newly formed immature B cells relative to naïve B or total splenic B cells in the humanized mice, a finding consistent with the deletion of autoreactive B cells in humans. Overall, our results provide evidence that key features of the primary repertoire are shaped by genetic factors intrinsic to human B cells and are principally unaltered by differences between mouse and human stromal microenvironments.Item Biochemical characterization of monoclonal antibodies to the Bordetella pertussis Filamentous hemagglutinin (FHA) and Pertussis toxin (PTx) : implications for improved acellular pertussis vaccine design(2015-08-28) Acquaye, Edith Abena; Maynard, Jennifer Anne, 1974-; Hoffman, David; Russell, Rick; Barrick, Jeffrey E.; Mukhopadhyay, SomshuvraIncidence rates of whooping cough were dramatically decreased by immunization with whole cell pertussis (wP) vaccines in the 1940s. However, concerns about the safety of the wP vaccine resulted in development of new pertussis vaccines using acellular components of B. pertussis. However, B. pertussis continues to circulate and cause whooping cough disease, even with the safer acellular pertussis (aP) vaccines. Over the last decade, in spite of the high vaccination coverage in many countries, there has been a significant rise in whooping cough infection in industrialized countries. The filamentous hemagglutinin (FHA) and pertussis toxin (PTx) are two antigens included in all currently licensed aP vaccines. Both FHA and PTx induce an immune response sufficient for protection, although anti-PTx antibodies correlate more with protection. Most studies assessing the efficacy of aP vaccines have used serum titers as the primary means to measure an antibody response to a given antigen. Single cell techniques however offer an opportunity to better understand the biochemical attributes of individual antibodies induced upon aP vaccination. In this study, we characterized the antibody responses to FHA and PTx after adult aP booster vaccination. Monoclonal antibodies derived from single B cells which responded to FHA and PTx were purified, the binding affinities to the antigens evaluated, mechanisms of neutralization of FHA and PTx toxicity evaluated, and the binding epitopes of the antibodies analyzed. This study is the first to provide insight into the anti-FHA and PTx antibody repertoire after pertussis vaccination, and also identifies useful antibodies for further elucidation of the structure and function of these antigens. In addition, the mechanisms of neutralization of two potently neutralizing anti-PTx monoclonal antibodies, 1B7 and 11E6 are presented. Antibody binding to live B. pertussis, inhibition of PTx binding to a model receptor fetuin and effects on PTx trafficking in CHO-K1 cells were evaluated. Further understanding of the molecular mechanisms of 1B7 and 11E6 neutralization provides a platform for engineering or isolating such unique anti-PTx antibodies for passive immunization therapies.Item Contributions of antibodies binding defined epitopes to protection against B. pertussis and SARS-CoV-2(2022-08-08) Silva, Rui Pedro; Maynard, Jennifer Anne, 1974-; Georgiou, George; Payne, Shelley; Gordon, Vernita; Davies, BryanWhile antibodies have the potential to reduce the severity of many infections and prevent transmission of pathogens, microbes have a corresponding capacity to undermine these immune responses. Bordetella pertussis has been the subject of several marketed vaccines, while severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), presents a new challenge. In both cases, the need for more effective vaccination and treatment strategies based on a greater understanding of molecular pathogenesis persists. B. pertussis continues to cause considerable morbidity and mortality even in countries with high vaccination rates. The widespread use of current acellular vaccines appears to have provided pertactin-deficient variants a fitness advantage. These now dominate circulation in countries using pertactin-containing acellular vaccines. To better understand the contributions of anti-pertactin antibodies to protection and bacterial fitness, we identified antibodies binding four distinct epitopes which are immunodominant and conserved. Passive immunization with these antibodies promotes bacterial clearance from mouse lungs after experimental infection in an Fc-dependent manner. We also show that an antibody neutralizing the adenylate cyclase toxin (ACT) protects leukocytes, which improves survival of mice infected with B. pertussis on its own but also synergizes with anti-pertactin antibodies to enhance protection. In addition to developing new antibody tools that can help elucidate pertactin functions, this work supports the inclusion of ACT in future pertussis vaccine formulations. COVID-19 has been responsible for millions of deaths worldwide and has subsequently disrupted every aspect of society. Extensive efforts have focused on understanding the roles of antibodies binding specific epitopes in the spike protein to develop therapeutic antibodies that prevent spike interactions with its ACE2 receptor and viral infection of a cell. We isolated and characterized an antibody binding a highly conserved epitope in the spike S2-core. While this antibody neutralizes COVID-19 and MERS pseudo-virus weakly in vitro, it mediates Fc effector functions that may contribute to protection against this virus. Understanding antibody responses against conserved epitopes on the coronavirus spike proteins can help guide design of future pan-protective spike immunogens. Overall, this work demonstrates the potential for antibodies to dissect mechanisms of pathogenesis that can support development of improved vaccines.Item Development and optimization of pH-responsive oral delivery systems for protein therapeutics(2020-12-09) Miller, Matthew Kyle; Peppas, Nicholas A., 1948-; Croyle, Maria; Maynard, Jennifer; Rosales, AdrianneA pH-responsive anionic linear polymer was synthesized via controlled radical polymerization using reversible addition-fragmentation chain transfer (RAFT) polymerization. The linear polymer was used to form pH-responsive self-assembled nanoparticles via the nanoprecipitation technique capable of loading antibodies with high efficiency at high weight loading percentages. The linear polymer system and the nanoprecipitation process were optimized using a Design of Experiments approach, resulting in a system capable of maintaining high antibody loading while exhibiting favorable release characteristics in simulated gastric, intestinal, and circulatory conditions. The optimized polymer and nanoparticle system was able to achieve a loading efficiency of 94% with a final weight loading of 32.9% protein compared to polymer. This optimized system showed minimal release in simulated gastric and intestinal conditions, while exhibiting high release at physiological pH, which is ideal for a nanocarrier designed to transit the intestinal epithelium before releasing its payload. The optimized polymer and nanoparticle system were then evaluated in a series of in vitro models to determine their cytocompatibility and transport capabilities. The linear polymer showed no cytotoxicity at concentrations up to 1 mg/mL in two model cell lines using two different assays, which is higher than any concentration likely to be achieved in vivo. Nanoparticles were conjugated with Fc ligands to evaluate if targeting the neonatal Fc receptor (FcRn) on intestinal enterocytes could enhance transport of nanoparticles across the epithelial cell barrier. The nanoparticles were then evaluated in a transepithelial transport model using Caco-2 and HT29-MTX model cell lines. Transepithelial transport was analyzed using an ELISA assay as well as confocal microscopy of fluorescently labeled antibodies. Nanoparticles conjugated with Fc ligands resulted in transepithelial protein transport up to 25x higher than unconjugated nanoparticles or unencapsulated protein based on ELISA results. Confocal microscopy proved inconclusive in differentiating transport between groups, likely due to fluorescence quenching of the FITC probe in acidic intracellular vesicles combined with high levels of background fluorescence. Finally, cell penetrating peptides (CPPs) were evaluated as an alternative means of increasing transepithelial transport of proteins. CPPs were evaluated in a pH-responsive complexation hydrogel system delivering insulin. The crosslinking density and crosslinker type were found to have a strong effect on the loading and release characteristics of CPP-insulin dual loaded microparticles. In an in situ closed-loop intestinal model in rats, the CPP L-PenetraMax showed a promising ability to increase transepithelial transport of insulin, resulting in a decrease in blood glucose levels compared to microparticles loaded with insulin alone. Future work will likely benefit from a combination of CPPs with Fc-conjugated nanoparticle delivery systems.Item Mass spectrometry combined with strategic enzymatic digestion, selective derivatization and ultraviolet photodissociation for the identification and characterization of Immunoglobulin G antibodies(2016-10-06) Cotham, Victoria Christine; Brodbelt, Jennifer S.; Crooks, Richard M; Georgiou, George; Webb, Lauren J; Zhang, YanImmunoglobulin G (IgG) antibodies represent important analytical targets both for their therapeutic properties and for their critical role in the adaptive immune response. While much of the primary structure is conserved across the IgG class, subtle changes in amino acid sequence and the presence or absence of post-translational modifications can have a profound effect on the function and therapeutic potential of a given antibody. As such, there remains a high demand for versatile analytical tools capable of both identification and complete structural characterization of IgGs. The work presented in this dissertation largely focuses on the development of mass spectrometry-based methods for the improved analysis of antibodies. This was accomplished using strategic enzymatic Brodbeltselectivity for regions of particular diagnostic value or to facilitate comprehensive structural characterization. A method based on chromophore-mediated 351 nm UVPD was developed as a means to streamline the identification of antibodies in mixtures by enhancing selectively for the third complementarity determining region of the IgG heavy chain (CDR-H3). The hypervariable sequences within this region serve as the primary determinant of antigen binding specificity and thus provide a molecular signature by which to differentiate unique antibodies. To accomplish this, a highly conserved cysteine residue located in the framework preceding the CDR-H3 region was exploited for selective tagging with an Alexa Fluor 350 (AF350) thiol-selective maleimide. This site-specific tagging combined with strategic enzymatic digestion and 351 nm UVPD allowed selective dissociation of only AF350-labeled peptides for facile discrimination of CDR-H3 sequences within a high-throughput liquid chromatography-tandem mass spectrometry (LC-MS/MS) based workflow. Two variations of middle-down mass spectrometry based on either restricted Lys-C proteolysis or hinge-selective IdeS digestion combined with 193 nm UVPD were used for the characterization of monoclonal antibodies. Both strategies yielded considerably greater diagnostic sequence information when benchmarked against conventional collision- and electron-based activation methods. The Lys-C proteolysis method was found to have considerable implications for the analysis of serological antibody repertoires owing to its facile implementation into high-throughput proteomic workflows and ability to unambiguously differentiate unique CDR-H3 sequences. The development and implementation of a front-end dual spray reactor for high-throughput ion/ion-mediated bioconjugation is demonstrated for the enhanced structural characterization of unmodified and post-translationally modified peptide cations by 193 nm UVPD and CID. The ability to generate ion/ion complexes in real-time followed by efficient covalent conversion allowed integration of the dual spray reactor into a high-throughput LC-MS [superscript n] workflow for rapid derivatization of peptide mixtures.Item Nasal Delivery of an Adenovirus-Based Vaccine Bypasses Pre-Existing Immunity to the Vaccine Carrier and Improves the Immune Response in Mice(Public Library of Science, 2008-10-29) Croyle, Maria A.; Patel, Ami; Tran, Kaylie N.; Gray, Michael; Zhang, Yi; Strong, James E.; Feldmann, Heinz; Kobinger, Gary P.Pre-existing immunity to human adenovirus serotype 5 (Ad5) is common in the general population. Bypassing pre-existing immunity could maximize Ad5 vaccine efficacy. Vaccination by the intramuscular (I.M.), nasal (I.N.) or oral (P.O.) route with Ad5 expressing Ebola Zaire glycoprotein (Ad5-ZGP) fully protected naïve mice against lethal challenge with Ebola. In the presence of pre-existing immunity, only mice vaccinated I.N. survived. The frequency of IFN-γ+ CD8+ T cells was reduced by 80% and by 15% in animals vaccinated by the I.M. and P.O. routes respectively. Neutralizing antibodies could not be detected in serum from either treatment group. Pre-existing immunity did not compromise the frequency of IFN-γ+ CD8+ T cells (3.9±1% naïve vs. 3.6±1% pre-existing immunity, PEI) nor anti-Ebola neutralizing antibody (NAB, 40±10 reciprocal dilution, both groups). The number of INF-γ+ CD8+ cells detected in bronchioalveolar lavage fluid (BAL) after I.N. immunization was not compromised by pre-existing immunity to Ad5 (146±14, naïve vs. 120±16 SFC/million MNCs, PEI). However, pre-existing immunity reduced NAB levels in BAL by ~25% in this group. To improve the immune response after oral vaccination, the Ad5-based vaccine was PEGylated. Mice given the modified vaccine did not survive challenge and had reduced levels of IFN-γ+ CD8+ T cells 10 days after administration (0.3±0.3% PEG vs. 1.7±0.5% unmodified). PEGylation did increase NAB levels 2-fold. These results provide some insight about the degree of T and B cell mediated immunity necessary for protection against Ebola virus and suggest that modification of the virus capsid can influence the type of immune response elicited by an Ad5-based vaccine.Item Strategies for generating therapeutic antibodies(2012-08) Carroll, Sean Matthew; Iverson, Brent L.; Georgiou, George; Alper, Hal; Maynard, Jennifer; Tucker, PhilipMonoclonal antibodies have become essential therapeutic tools and currently dominate the therapeutic protein market. Consequently, there is continued demand for new therapeutic antibodies and their discovery techniques. In one part of this work, we report the discovery of a new therapeutic antibody candidate with a novel mechanism for inhibition of a therapeutically relevant biochemical pathway: the classical complement pathway. In order to inhibit classical complement, an antibody was developed that modulates the signaling subcomponent of the pathway initiating C1 complex, C1s. This work includes novel protocols and strategies used for discovery and characterization of antibody D, which binds and inhibits C1s protease activity. By regulating C1s activity, antibody D is shown to regulate classical complement. It is further shown that affinity maturation of antibody D results in higher levels of complement inhibition at various antibody concentrations. This work marks the first example of an antibody that specifically regulates the classical complement pathway by targeting the C1s protease on the pathway initiating C1-complex. Next, we characterize the human immune cells produced by humanized NSG mice, most notably the B and T lymphocytes, engraftment with human CD34+ HSC cells. We detected development of naïve human B and T cells and their various subtypes, as well as other human immune cells from engrafted mice. However, attempts to generate a robust antibody response to antigens were unsuccessful. Therefore, we conclude that NSG humanized mice developed in this study are suitable for studying the antibody repertoire of naïve B cells, however they are not suitable for the analysis of activated B-cells. Last, we introduce a novel strategy for the generation of polarized antibody repertoires for use in therapeutic monoclonal antibody discovery. This technique combines targeted antigen delivery to a specific lymph node and a frequency based antibody selection approach in order to directly select antigen specific antibodies in silico. By directly selecting antigen-specific antibodies, this approach circumvents laborious and time consuming screening techniques. We expect that this work will be the foundation of an overall improved protocol for monoclonal antibody discovery that accelerates the speed and enhances the simplicity of discovery techniques.Item Structural basis for receptor binding and antibody-mediated neutralization by Bordetella adenylate cyclase toxin(2023-08-15) Goldsmith, Jory A.; McLellan, Jason S.; Maynard, Jennifer; Davies, Bryan; Dickinson, Daniel; Payne, ShelleyBordetella pertussis, the gram-negative pathogen that causes whooping cough, caused 89,000 deaths in 2018 globally. In the United States, introduction of killed whole-cell B. pertussis (wP) vaccines in the 1940s reduced the number of yearly cases from over 100,000 to less than 10,000 in 1965. However, public fears about the reactogenicity of wP vaccine led many countries to switch to less effective acellular B. pertussis (aP) vaccines, containing specific purified antigens. An improved version of aP vaccine is desired, which could be achieved by including better or more optimized antigens. Notably, the adenylate cyclase toxin (ACT) of B. pertussis has been hypothesized to be an effective antigen to include in the aP vaccine as it is essential for lung colonization in mouse intranasal models and has elicited protective immunity in mice. However, ACT was never included in human vaccines as it was too difficult to produce. ACT is a virulence factor secreted by B. pertussis that inserts into host leukocytes and translocates an adenylate cyclase enzyme into the target cell cytosol. The rapid formation of cAMP in host leukocytes inhibits their bactericidal activities, thus promoting survival of B. pertussis in the respiratory epithelium. As a ‘pore-forming RTX toxin’, ACT has a C-terminal calcium-binding ‘RTX’ domain that mediates secretion. However, biochemical data had previously shown that the RTX domain of ACT contains the binding site for the ACT receptor, integrin αMβ₂. The RTX domain was also shown to harbor the epitopes for neutralizing antibodies that prevent αMβ₂ binding, suggesting that this interaction could be targeted with an RTX domain immunogen. However, the structural determinants of the ACT RTX domain’s interaction with αMβ₂ were not well understood prior to this work. Specifically, it was not known how the ACT RTX domain had been adapted to engage in protein-protein interactions, which regions of the RTX domain contacted the αMβ₂ receptor as well as neutralizing antibodies, and how binding of ACT to αMβ₂ favors membrane insertion of ACT. In addition, ACT preferentially binds to the inactive conformation of αMβ₂, and the structural basis for this preference is not understood. Therefore, this work set out to structurally define the interaction of ACT with αMβ₂ and with neutralizing antibodies, to understand key epitopes for mediating protection against B. pertussis. Chapter 1 provides an outline of the epidemiology of B. pertussis and the known details of the ACT intoxication and receptor-binding mechanism. In Chapter 2, a crystal structure of an engineered RTX domain fragment harboring the αMβ₂-binding site bound to neutralizing antibodies is described. This structure showed neutralizing antibodies bound to the variable “linker” regions of the RTX domain, which had a previously unknown fold and connect the segments of the RTX domain containing consensus 9-residue repeats known to form a helical calcium-binding structure. This suggested that the RTX linkers were involved in binding αMβ₂ as these antibodies prevent αMβ₂ binding to the ACT RTX domain. In Chapter 3, a cryo-electron microscopy structure of the ACT RTX domain in complex with the αMβ₂ ectodomain is reported. This structure showed that the RTX linkers, shown in Chapter 2 to harbor the neutralizing epitopes bound by antibodies M2B10 and M1H5, each form a separate binding site on αMβ₂, and can only contact both of these sites when αMβ₂ is in the bent, inactive conformation. This structure also showed that αMβ₂ binding would position lysine residues that require post-translational acylation for ACT activity directly at the host cell membrane, coupling receptor binding to membrane insertion. This work defined RTX linkers as key protein-protein interaction modules of the ACT RTX domain and provides a framework for the design of RTX domain vaccines.