Journal of virology
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Journal of virology · Nov 2014
Isolation of dengue virus-specific memory B cells with live virus antigen from human subjects following natural infection reveals the presence of diverse novel functional groups of antibody clones.
Natural dengue virus (DENV) infection in humans induces antibodies (Abs) that neutralize the serotype of infection in a potent and type-specific manner; however, most Abs generated in response to infection are serotype cross-reactive and poorly neutralizing. Such cross-reactive Abs may enhance disease during subsequent infection with a virus of a different DENV serotype. Previous screening assays for DENV-specific human B cells and antibodies, using viral and recombinant antigens, mainly led to the isolation of dominant nonneutralizing B cell clones. To improve upon our ability to recover and study rare but durable and potently neutralizing DENV-specific Abs, we isolated human DENV-specific B cells by using a primary screen of binding to live virus, followed by a secondary screen with a high-throughput, flow cytometry-based neutralization assay to identify DENV-specific B cell lines prior to generation of hybridomas. Using this strategy, we identified several new classes of serotype-specific and serotype-cross-neutralizing anti-DENV monoclonal Abs (MAbs), including ultrapotent inhibitory antibodies with neutralizing activity concentrations of <10 ng/ml. We isolated serotype-specific neutralizing Abs that target diverse regions of the E protein, including epitopes present only on the intact, fully assembled viral particle. We also isolated a number of serotype-cross-neutralizing MAbs, most of which recognized a region in E protein domain I/II containing the fusion loop. These data provide insights into targets of the protective Ab-mediated immune response to natural DENV infection, which will prove valuable in the design and testing of new experimental DENV vaccines. ⋯ Dengue virus infection is one of the most common mosquito-borne diseases and occurs in most countries of the world. Infection of humans with dengue virus induces a small number of antibodies that inhibit the infecting strain but also induces a large number of antibodies that can bind but do not inhibit dengue virus strains of other serotypes. We used a focused screening strategy to discover a large number of rare potently inhibiting antibodies, and we mapped the regions on the virus that were recognized by such antibodies. Our studies revealed that humans have the potential to generate very potent antibodies directed to diverse regions of the dengue virus surface protein. These studies provide important new information about protection from dengue virus infection that will be useful in the design and testing of new experimental dengue vaccines for humans.
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Journal of virology · Nov 2014
Establishment and characterization of a lethal mouse model for the Angola strain of Marburg virus.
Infections with Marburg virus (MARV) and Ebola virus (EBOV) cause severe hemorrhagic fever in humans and nonhuman primates (NHPs) with fatality rates up to 90%. A number of experimental vaccine and treatment platforms have previously been shown to be protective against EBOV infection. However, the rate of development for prophylactics and therapeutics against MARV has been lower in comparison, possibly because a small-animal model is not widely available. Here we report the development of a mouse model for studying the pathogenesis of MARV Angola (MARV/Ang), the most virulent strain of MARV. Infection with the wild-type virus does not cause disease in mice, but the adapted virus (MARV/Ang-MA) recovered from liver homogenates after 24 serial passages in severe combined immunodeficient (SCID) mice caused severe disease when administered intranasally (i.n.) or intraperitoneally (i.p.). The median lethal dose (LD50) was determined to be 0.015 50% TCID50 (tissue culture infective dose) of MARV/Ang-MA in SCID mice, and i.p. infection at a dose of 1,000× LD50 resulted in death between 6 and 8 days postinfection in SCID mice. Similar results were obtained with immunocompetent BALB/c and C57BL/6 mice challenged i.p. with 2,000× LD50 of MARV/Ang-MA. Virological and pathological analyses of MARV/Ang-MA-infected BALB/c mice revealed that the associated pathology was reminiscent of observations made in NHPs with MARV/Ang. MARV/Ang-MA-infected mice showed most of the clinical hallmarks observed with Marburg hemorrhagic fever, including lymphopenia, thrombocytopenia, marked liver damage, and uncontrolled viremia. Virus titers reached 10(8) TCID50/ml in the blood and between 10(6) and 10(10) TCID50/g tissue in the intestines, kidney, lungs, brain, spleen, and liver. This model provides an important tool to screen candidate vaccines and therapeutics against MARV infections. ⋯ The Angola strain of Marburg virus (MARV/Ang) was responsible for the largest outbreak ever documented for Marburg viruses. With a 90% fatality rate, it is similar to Ebola virus, which makes it one of the most lethal viruses known to humans. There are currently no approved interventions for Marburg virus, in part because a small-animal model that is vulnerable to MARV/Ang infection is not available to screen and test potential vaccines and therapeutics in a quick and economical manner. To address this need, we have adapted MARV/Ang so that it causes illness in mice resulting in death. The signs of disease in these mice are reminiscent of wild-type MARV/Ang infections in humans and nonhuman primates. We believe that this will be of help in accelerating the development of life-saving measures against Marburg virus infections.