Cell Biology & Molecular Genetics

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    Extraction of Membrane Components from Neisseria gonorrhoeae Using Catanionic Surfactant Vesicles: A New Approach for the Study of Bacterial Surface Molecules
    (MDPI, 2020-08-20) Stein, Daniel C.; Stocker, Lenea H.; Powell, Abigail E.; Kebede, Salsawi; Watts, David; Williams, Emma; Soto, Nicholas; Dhabaria, Avantika; Fenselau, Catherine; Ganapati, Shweta; DeShong, Philip
    Identification of antigens is important for vaccine production. We tested extraction protocols using cetyltrimethylammonium tosylate (CTAT) and sodium dodecylbenzenesulfonate (SDBS) to formulate surfactant vesicles (SVs) containing components from Neisseria gonorrhoeae. Carbohydrate and protein assays demonstrated that protein and carbohydrates were incorporated into the vesicle leaflet. Depending on the extraction protocol utilized, 100–400 µg of protein/mL of SVs solution was obtained. Gel electrophoresis followed by silver staining demonstrated that SV extracts contained lipooligosaccharide and a subset of bacterial proteins and lipoproteins. Western blotting and mass spectral analysis indicated that the majority of the proteins were derived from the outer membrane. Mass spectrometric and bioinformatics analysis of SVs identified 29 membrane proteins, including porin and opacity-associated protein. Proteins embedded in the SVs leaflet could be degraded by the addition of trypsin or proteinase K. Our data showed that the incorporation of CTAT and SDBS into vesicles eliminated their toxicity as measured by a THP-1 killing assay. Incorporation of gonococcal cell surface components into SVs reduced toxicity as compared to the whole cell extracts, as measured by cytokine induction, while retaining the immunogenicity. This process constitutes a general method for extracting bacterial surface components and identification of antigens that might be included in vaccines.
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    Deconstructing The Polyclonal Antibody Response To Dengue Virus
    (2015) VanBlargan, Laura Anne; Pierson, Theodore C; Simon, Anne E; Molecular and Cell Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Dengue viruses are mosquito-borne flaviviruses that circulate in nature as four related serotypes (DENV1-4). These emerging pathogens are responsible for an estimated 390 million human infections each year. The outcome of human DENV infection ranges from clinically inapparent disease (~75% of infections), to a self-limiting febrile illness, to severe disease characterized by hemorrhage and shock. Severe clinical manifestations of disease are predominantly associated with secondary infections by a heterotypic DENV serotype. The increased risk of severe disease in DENV-sensitized populations significantly complicates vaccine development, as a vaccine should confer protection against all four DENV serotypes. As the development of a neutralizing antibody response is a correlate of protection for successful vaccines for several other flaviviruses, eliciting a protective tetravalent neutralizing antibody response is a major goal of ongoing DENV vaccine development efforts. Understanding the neutralizing antibody response to infection and vaccination is an important step toward the development and evaluation of safe DENV vaccines. While considerable insights have been gained from studies of monoclonal antibodies, the individual contributions and dynamics of the repertoire of circulating antibody specificities elicited by infection and vaccination are poorly understood on a functional and molecular level. We studied polyclonal antibody responses elicited by monovalent DENV1 vaccination and sought to identify epitopes recognized by serotype-specific neutralizing antibodies. DENV1 structural gene variants were produced and screened for reduced sensitivity to neutralization by DENV1 sera but unaltered sensitivity to control antibodies. We identified amino acid residues that contribute significantly to type-specific recognition by polyclonal DENV1 immune sera. These findings provide an important step towards deconvoluting the functional complexity of DENV serology following vaccination.
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    Reverse Genetics of Influenza B and the Development of a Novel LAIV Vaccine
    (2014) Finch, Courtney LaPaglia; Perez, Daniel R; Molecular and Cell Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Due to the disease burden of influenza virus types A and B, vaccines, which are manufactured as formalin-inactivated killed virus (KV) and live-attenuated virus (LAIV), are produced to provide coverage against currently circulating influenza A (IAV) and B (IBV) viruses. Although the licensed LAIV vaccine provides enhanced coverage over the KV vaccine, it is only licensed for immunocompetent individuals ages 2-49 years without pre-existing conditions, so individuals who are most at risk cannot receive it. Previously, our lab showed that incorporation of an 8 amino acid HA tag in frame at the C-terminus of the PB1 open-reading frame (ORF) in addition to the mutations found in the PB2 and PB1 segments of the licensed LAIV vaccine yielded a stable, efficacious alternative LAIV vaccine for IAV; however, to develop a complete vaccine, a corresponding IBV candidate is required. Towards this goal, a contemporary IBV strain, B/Brisbane/60/2008, was cloned and recovered by reverse genetics (RG-B/Bris). Subsequently, it was demonstrated that the parental and RG-B/Bris show similar growth kinetics in vitro. An initial vaccine attempt, which combined PB2 cap-binding mutants with the HA tag in PB1, was made but led to the realization of the PB2 cap-binding mutations, PB2 W359F and F406Y, as virulence factors. In a subsequent vaccine attempt, mutations analogous to those found only in segment 2 of the A/Ann Arbor/6/60 cold-adapted LAIV backbone were introduced into the homologous segment of RG-B/Bris. The following mutations were introduced into the PB1 gene segment of RG-B/Bris, either in the presence or absence of a C-terminal HA tag: K391E, E580G, and S660A. Two viruses were rescued, referred to as RG-B/Bris ts and RG-B/Bris att, both containing the set of three amino acid mutations but differing in the absence or presence of the HA tag, respectively. Both viruses showed ideal attenuation, safety, and immunogenicity in DBA/2 mice and conferred protection against lethal IBV challenge. More importantly, RG-B/Bris att, but not RG-B/Bris ts, showed ideal stability with no reverting mutations over 8 passages in eggs. Taken together, a stable, immunogenic, and live attenuated virus alternative to the current live influenza B virus vaccine was produced.
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    The development and characterization of transgenic Leishmania major expressing murine CD40L
    (2007-05-18) Field, Ann Elizabeth; Mosser, David M; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Leishmanization is the inoculation of live Leishmania into the host to vaccinate against subsequent infections. This approach has been largely discontinued due to safety concerns. We have previously shown that combining CD40L with Leishmania antigen preferentially induces a type 1 immune response and provides some protection to vaccinated mice. In the present study, we developed transgenic L. major which express and secrete the extracellular portion of CD40L (L. major CD40LE). We hypothesized that these organisms would be less virulent but more immunogenic than wild-type organisms, and therefore be more effective at leishmanization. Transgenic parasites expressing CD40L mRNA and protein were developed. These parasites had similar growth characteristics to wild-type organisms. Susceptible BALB/c mice infected with these parasites developed significantly smaller lesions containing fewer parasites than animals infected with wild-type organisms. Infection of C57BL/6 CD40L-/- mice with transgenic L. major resulted in significantly smaller lesions than infection with wild-type L. major, indicating in vivo biological activity of the transgenic protein. Infection of resistant C57BL/6 mice with low doses of transgenic parasites induced a significant amount of protection against subsequent high dose infection with wild-type organisms. These results demonstrate that transgenic organisms expressing CD40L are less virulent than wild-type organisms while retaining full immunogenicity. The implications of this study are that parasites expressing immune-modulatory molecules may be improved alternatives to traditional leishmanization.