Evaluation of Selected Borrelia burgdorferi lp54 Plasmid-Encoded Gene Products Expressed during Mammalian Infection as Antigens To Improve Serodiagnostic Testing for Early Lyme Disease

Weiner, Zachary P.; Crew, Rebecca; Brandt, Kevin S.; Ullmann, Amy J.; Schriefer, Martin E.; Molins, Claudia R.; Gilmore, Robert D.

doi:10.1128/cvi.00399-15

Cited by 6 publications

(14 citation statements)

References 40 publications

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“…The BBA66 coding sequence minus the signal sequence and lipidation motif was amplified by PCR from B . burgdorferi B31 using primers designed for expression cloning into the pETite N-His vector according to the T7 Expresso system instructions (Lucigen, Middleton, WI) [ 32 ]. The constructed expression plasmids were transformed into E .…”

Section: Methodsmentioning

confidence: 99%

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Human neuroglial cells internalize Borrelia burgdorferi by coiling phagocytosis mediated by Daam1

2018

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Borrelia burgdorferi, the agent of Lyme borreliosis, can elude hosts’ innate and adaptive immunity as part of the course of infection. The ability of B. burgdorferi to invade or be internalized by host cells in vitro has been proposed as a mechanism for the pathogen to evade immune responses or antimicrobials. We have previously shown that B. burgdorferi can be internalized by human neuroglial cells. In this study we demonstrate that these cells take up B. burgdorferi via coiling phagocytosis mediated by the formin, Daam1, a process similarly described for human macrophages. Following coincubation with glial cells, B. burgdorferi was enwrapped by Daam1-enriched coiling pseudopods. Coiling of B. burgdorferi was significantly reduced when neuroglial cells were pretreated with anti-Daam1 antibody indicating the requirement for Daam1 for borrelial phagocytosis. Confocal microscopy showed Daam1 colocalizing to the B. burgdorferi surface suggesting interaction with borrelial membrane protein(s). Using the yeast 2-hybrid system for identifying protein-protein binding, we found that the B. burgdorferi surface lipoprotein, BBA66, bound the FH2 subunit domain of Daam1. Recombinant proteins were used to validate binding by ELISA, pull-down, and co-immunoprecipitation. Evidence for native Daam1 and BBA66 interaction was suggested by colocalization of the proteins in the course of borrelial capture by the Daam1-enriched pseudopodia. Additionally, we found a striking reduction in coiling for a BBA66-deficient mutant strain compared to BBA66-expressing strains. These results show that coiling phagocytosis is a mechanism for borrelial internalization by neuroglial cells mediated by Daam1.

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Section: Methodsmentioning

confidence: 99%

“…Proteins were dialyzed into phosphate buffered saline (PBS, pH 8.0) and quantified by bicinchoninic acid (BCA) assay (Thermo-Fisher Scientific, Rockford, IL). Recombinant OspC was generated in same manner as described previously [ 32 ].…”

Section: Methodsmentioning

confidence: 99%

Human neuroglial cells internalize Borrelia burgdorferi by coiling phagocytosis mediated by Daam1

2018

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“…By inclusion of a recombinant OspC protein from another genotype, such as type K, we may be able to improve the sensitivity (34). Inclusion of other diagnostic antigens that have shown strong potential may also improve the assay (35).…”

Section: Discussionmentioning

confidence: 99%

Five-Antigen Fluorescent Bead-Based Assay for Diagnosis of Lyme Disease

Embers

Hasenkampf

Barnes

et al. 2016

Clin Vaccine Immunol

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The systematically difficult task of diagnosing Lyme disease can be simplified by sensitive and specific laboratory tests. The currently recommended two-tier test for serology is highly specific but falls short in sensitivity, especially in the early acute phase. We previously examined serially collected serum samples from Borrelia burgdorferi-infected rhesus macaques and defined a combination of antigens that could be utilized for detection of infection at all phases of disease in humans. The five B. burgdorferi antigens, consisting of OspC, OspA, DbpA, OppA2, and the C6 peptide, were combined into a fluorescent cytometric beadbased assay for the detection of B. burgdorferi antigen-specific IgG antibodies. Samples from Lyme disease patients and controls were used to determine the diagnostic value of this assay. Using this sample set, we found that our five-antigen multiplex IgG assay exhibited higher sensitivity (79.5%) than the enzyme immunoassay (EIA) (76.1%), the two-tier test (61.4%), and the C6 peptide enzyme-linked immunosorbent assay (ELISA) (77.2%) while maintaining specificity over 90%. When detection of IgM was added to the bead-based assay, the sensitivity improved to 91%, but at a cost of reduced specificity (78%). These results indicate that the rational combination of antigens in our multiplex assay may offer an improved serodiagnostic test for Lyme disease. With an estimated 300,000 new cases each year, Lyme disease is the most common vector-borne disease in North America (http://www.cdc.gov/lyme/stats/humanCases.html) (1). The burden of this disease in many parts of Europe is also staggering (1, 2). Treatment with antibiotics is generally effective, even more so when employed soon after infection (3). Therefore, early and reliable laboratory diagnosis is critical for effective cure of Lyme disease patients.Patient serum antibodies specific for Borrelia burgdorferi antigens are detected with currently recommended laboratory tests for Lyme disease. While the detection of antigen may be preferred for early diagnosis, this is encumbered by the absence of detectable spirochetes or spirochetal antigen in the bloodstream once the organism has disseminated. Thus, the use of antigen detection from a blood or skin biopsy specimen has not demonstrated favorable sensitivity (4). The two most commonly used tests for diagnosis of Lyme disease in North America are (i) the two-tier test that includes an enzyme-linked immunosorbent assay (ELISA) and confirmatory Western blotting and (ii) the C6 test, where antibodies to a specific peptide within a conserved region of VlsE, the B. burgdorferi antigen, are detected (5-8). Both the twotier and C6 tests exhibit high specificity and are most sensitive for patients in the disseminated phases of disease (5, 8-10). In addition, the C6 test has been evaluated as an indicator of treatment outcome in the United States (11, 12), with a Ն4-fold decline in antibody titer (up to 6 months after treatment) in a majority of patients. The Western blot procedure is technically dema...

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“…The bba64, −65, −66, −69, −70, −73, and ospC coding sequences minus the signal peptide were cloned for recombinant protein expression using the Expresso T7 Cloning and Expression System (Lucigen Corporation, Middleton, WI). The genes were amplified from B. burgdorferi B31 genomic DNA with primers designed to ligate into the linearized plasmid pETite N-His Kan vector with soluble expressed proteins purified from E. coli as described [19].…”

Section: Preparation Of Recombinant Proteinsmentioning

confidence: 99%

“…CD-1 mice were immunized subcutaneously with approximately 15-35 μg (for single antigen) or approximately 2-20 ug each (for multi-antigen) recombinant protein solubilized in Imject (1:1) (Thermo Scientific) followed by two booster injections 3 weeks apart. Mice were bled 14 days following the final boost, and ELISA was performed on serum samples against recombinant protein to assess antibody titer as described [19].…”

Section: Immunization Of Mice With Recombinant Proteins and Assessmenmentioning

confidence: 99%

Immunization of mice with Borrelia burgdorferi lp54 gene encoded recombinant proteins does not provide protection against tick transmitted infectious challenge

Brandt

Gilmore

2017

Vaccine

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The Borrelia burgdorferi outer surface membrane proteins BBA65, BBA66, BBA69, BBA70, and BBA73 were tested for their ability to confer protection against B. burgdorferi infection challenge. Mice were immunized with recombinant forms of the proteins singly or in combinations. Following initial protein inoculation and booster injections, seroconversion was confirmed prior to B. burgdorferi challenge by tick bite. Despite mice having high antibody titers for each antigen, no significant protections against the challenge infections were observed. These results demonstrate that these recombinant proteins were not protective and reflects the challenges confronted to identify effective novel vaccine candidates for Lyme disease.

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Evaluation of Selected Borrelia burgdorferi lp54 Plasmid-Encoded Gene Products Expressed during Mammalian Infection as Antigens To Improve Serodiagnostic Testing for Early Lyme Disease

Cited by 6 publications

References 40 publications

Human neuroglial cells internalize Borrelia burgdorferi by coiling phagocytosis mediated by Daam1

Human neuroglial cells internalize Borrelia burgdorferi by coiling phagocytosis mediated by Daam1

Five-Antigen Fluorescent Bead-Based Assay for Diagnosis of Lyme Disease

Immunization of mice with Borrelia burgdorferi lp54 gene encoded recombinant proteins does not provide protection against tick transmitted infectious challenge

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