Development of reagents and assays for the detection of pathogenic Burkholderia species

Qazi, Omar; Rani, Mridula; Gnanam, Annie J.; Cullen, Thomas W.; Stead, Christopher M.; Kensing, Haley; McCaul, Kate C; Ngugi, Sarah A.; Prior, Joann L.; Lipka, Alexandria; Nagy, Judit; Whitlock, Gregory C.; Judy, Barbara M.; Harding, Sarah V.; Titball, Richard W.; Sidhu, Sachdev S.; Trent, M. Stephen; Kitto, G. Barrie; Torres, Alfredo G.; Estes, D. Mark; Iverson, Brent L.; Georgiou, George; Brown, Katherine A.

doi:10.1039/c005422b

Cited by 4 publications

(2 citation statements)

References 38 publications

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“…OMV-, LPS-, and CPS-specific IgG, IgG1, and IgG2a serum antibody titers were measured by ELISAs using microtiter plates coated with 500 ng of OMV, LPS, or CPS, as we previously described (14). Purified LPS was provided by Kate Brown (University of Texas at Austin) and was prepared from acapsular Burkholderia thailandensis strain E264 using a modified hot phenol extraction method, as previously described (18). Purified CPS was provided by Don Woods (University of Calgary) and was prepared from an O-antigen-deficient B. pseudomallei mutant strain by using methods described by Perry et al (19).…”

Section: Methodsmentioning

confidence: 99%

A Burkholderia pseudomallei Outer Membrane Vesicle Vaccine Provides Protection against Lethal Sepsis

Nieves

Petersen

Judy

et al. 2014

Clin. Vaccine Immunol.

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The environmental Gram-negative encapsulated bacillus Burkholderia pseudomallei is the causative agent of melioidosis, a disease associated with high morbidity and mortality rates in areas of Southeast Asia and northern Australia in which the disease is endemic. B. pseudomallei is also classified as a tier I select agent due to the high level of lethality of the bacterium and its innate resistance to antibiotics, as well as the lack of an effective vaccine. Gram-negative bacteria, including B. pseudomallei, secrete outer membrane vesicles (OMVs) which are enriched with multiple protein, lipid, and polysaccharide antigens. Previously, we demonstrated that immunization with multivalent B. pseudomallei-derived OMVs protects highly susceptible BALB/c mice against an otherwise lethal aerosol challenge. In this work, we evaluated the protective efficacy of OMV immunization against intraperitoneal challenge with a heterologous strain because systemic infection with phenotypically diverse environmental B. pseudomallei strains poses another hazard and a challenge to vaccine development. We demonstrated that B. pseudomallei OMVs derived from strain 1026b afforded significant protection against septicemic infection with B. pseudomallei strain K96243. OMV immunization induced robust OMV-, lipopolysaccharide-, and capsular polysaccharide-specific serum IgG (IgG1, IgG2a, and IgG3) and IgM antibody responses. OMV-immune serum promoted bacterial killing in vitro, and passive transfer of B. pseudomallei OMV immune sera protected naive mice against a subsequent challenge. These results indicate that OMV immunization provides antibody-mediated protection against acute, rapidly lethal sepsis in mice. B. pseudomallei-derived OMVs may represent an efficacious multivalent vaccine strategy against melioidosis.

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Section: Methodsmentioning

confidence: 99%

A Burkholderia pseudomallei Outer Membrane Vesicle Vaccine Provides Protection against Lethal Sepsis

Nieves

Petersen

Judy

et al. 2014

Clin. Vaccine Immunol.

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“…10 Scale-up of candidate selection is, of course, not limited to nucleic acids, and the availability of phage display libraries as a central resource could allow for rapid antibody selection for more traditional diagnostics; detection of pathogens by this route was demonstrated at this Faraday Discussion. 11 A counterpart to any selective binding is the surface tailoring of the transduction surface. Amperometry has been a mainstay of sensing, but impedimetric analysis offers a further tool that is less often used, and which may be better suited to an era of smart and reactive materials.…”

Section: Sensing Strategiesmentioning

confidence: 99%

Concluding remarks

Vadgama

2011

Faraday Discuss.

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Two Synthetic Antibodies that Recognize and Neutralize Distinct Proteolytic Forms of the Ebola Virus Envelope Glycoprotein

et al. 2012

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Ebola Virus (EBOV) is a highly pathogenic member of the family Filoviridae of viruses that causes severe hemorrhagic fever. Infection proceeds through fusion of the host cell and viral membranes, a process that is mediated by the viral envelope glycoprotein (GP). Following endosomal uptake, a key step in viral entry is the proteolytic cleavage of GP by host endosomal cysteine proteases. Cleavage exposes a binding site for the host cell receptor Niemann-Pick C1 (NPC1) and may induce conformational changes in GP leading to membrane fusion. However, the precise details of the structural changes in GP associated with proteolysis and the role of these changes in viral entry have not been established. Here, we have employed synthetic antibody technology to identify antibodies targeting EBOV GP prior to and following proteolysis (i.e. in the “uncleaved” [GPUNCL] and “cleaved” [GPCL] forms). We identified antibodies with distinct recognition profiles: FabCL bound preferentially to GPCL (EC50 = 1.7 nM), whereas FabUNCL bound specifically to GPUNCL (EC50 = 75 nM). Neutralization assays with GP-containing pseudotyped viruses indicated that these antibodies inhibited GPCL or GPUNCL mediated viral entry with specificity matching their recognition profiles (IC50: 87 nM for IgGCL; 1 μM for FabUNCL). Competition ELISAs indicate that FabCL binds an epitope distinct from that of KZ52, a well-characterized EBOV GP antibody, and from that of the luminal domain of NPC1. The binding epitope of FabUNCL was also distinct from that of KZ52, suggesting that FabUNCL binds a novel neutralization epitope on GPUNCL. Furthermore, the neutralizing ability of FabCL suggests that there are targets on GPCL available for neutralization. This work showcases the applicability of synthetic antibody technology to the study of viral membrane fusion, and provides new tools for dissecting intermediates of EBOV entry.

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Development of reagents and assays for the detection of pathogenic Burkholderia species

Cited by 4 publications

References 38 publications

A Burkholderia pseudomallei Outer Membrane Vesicle Vaccine Provides Protection against Lethal Sepsis

A Burkholderia pseudomallei Outer Membrane Vesicle Vaccine Provides Protection against Lethal Sepsis

Concluding remarks

Two Synthetic Antibodies that Recognize and Neutralize Distinct Proteolytic Forms of the Ebola Virus Envelope Glycoprotein

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