Structure-guided Antigen Engineering Yields Pneumolysin Mutants Suitable for Vaccination against Pneumococcal Disease

Oloo, Eliud O.; Yethon, Jeremy A.; Ochs, Martina M.; Carpick, Bruce; Oomen, Ray

doi:10.1074/jbc.m110.191148

Cited by 28 publications

(31 citation statements)

References 44 publications

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“…PlyD1 is nontoxic as it exhibits substantially decreased hemolytic activity compared to full-length recombinant PLY and to the detoxified PLY variant PdB. In the investigation of Oloo et al, PdB exhibited a residual hemolytic activity of 1% while PlyD1 had less than 0.001% activity compared to that of PLY (30).…”

Section: Resultsmentioning

confidence: 99%

“…PlyD1, a highly detoxified PLY variant, was generated by site-directed mutagenesis and differs from the wild-type form by three amino acid substitutions of T65C, G293C, and C428A. Recombinant PlyD1 protein was expressed in E. coli as soluble protein and column purified (30).…”

Section: Methodsmentioning

confidence: 99%

“…For this reason, we developed a highly detoxified pneumolysin mutant designated PlyD1 (30). A significant advantage of PlyD1 is that it was designed to possess a dual mechanism of detoxification.…”

mentioning

confidence: 99%

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Neutralizing Antibodies Elicited by a Novel Detoxified Pneumolysin Derivative, PlyD1, Provide Protection against Both Pneumococcal Infection and Lung Injury

et al. 2012

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ABSTRACTStreptococcus pneumoniaepneumolysin (PLY) is a virulence factor that causes toxic effects contributing to pneumococcal pneumonia. To date, deriving a PLY candidate vaccine with the appropriate detoxification and immune profile has been challenging. A pneumolysin protein that is appropriately detoxified and that retains its immunogenicity is a desirable vaccine candidate. In this study, we assessed the protective efficacy of our novel PlyD1 detoxified PLY variant and investigated its underlying mechanism of protection. Results have shown that PlyD1 immunization protected mice against lethal intranasal (i.n.) challenge with pneumococci and lung injury mediated by PLY challenge. Protection was associated with PlyD1-specific IgG titers andin vitroneutralization titers. Pretreatment of PLY with PlyD1-specific rat polyclonal antiserum prior to i.n. delivery of toxin reduced PLY-mediated lung lesions, interleukin-6 (IL-6) production, and neutrophil infiltration into lungs, indicating that protection from lung lesions induced by PLY is antibody mediated. Preincubation of PLY with a neutralizing monoclonal PLY antibody also specifically reduced the cytotoxic effects of PLY after i.n. inoculation in comparison to nonneutralizing monoclonal antibodies. These results indicate that the induction of neutralizing antibodies against PLY can contribute to protection against bacterial pneumonia by preventing the development of PLY-induced lung lesions and inflammation. Our detoxified PlyD1 antigen elicits such PLY neutralizing antibodies, thus serving as a candidate vaccine antigen for the prevention of pneumococcal pneumonia.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Neutralizing Antibodies Elicited by a Novel Detoxified Pneumolysin Derivative, PlyD1, Provide Protection against Both Pneumococcal Infection and Lung Injury

et al. 2012

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“…Recombinant pneumococcal histidine triad protein (PhtD), choline-binding protein (PcpA), and a genetic derivative of pneumolysin (PlyD1) were provided by Sanofi Pasteur. The mutations in PlyD1 are T65C, G293C, and C428A (34). PlyD1 lacks hemolytic activity and induces neutralizing antibodies against the wild-type toxin (35).…”

Section: Methodsmentioning

confidence: 99%

Human Antibodies to PhtD, PcpA, and Ply Reduce Adherence to Human Lung Epithelial Cells and Murine Nasopharyngeal Colonization by Streptococcus pneumoniae

et al. 2014

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bStreptococcus pneumoniae adherence to human epithelial cells (HECs) is the first step in pathogenesis leading to infections. We sought to determine the role of human antibodies against S. pneumoniae protein vaccine candidates PhtD, PcpA, and Ply in preventing adherence to lung HECs in vitro and mouse nasopharyngeal (NP) colonization in vivo. Human anti-PhtD, -PcpA, and -Ply antibodies were purified and Fab fragments generated. Fabs were used to test inhibition of adherence of TIGR4 and nonencapsulated strain RX1 to A549 lung HECs. The roles of individual proteins in adherence were tested using isogenic mutants of strain TIGR4. Anti-PhtD, -PcpA, and -Ply human antibodies were assessed for their ability to inhibit NP colonization in vivo by passive transfer of human antibody in a murine model. Human antibodies generated against PhtD and PcpA caused a decrease in adherence to A549 cells (P < 0.05). Anti-PhtD but not anti-PcpA antibodies showed a protective role against mouse NP colonization. To our surprise, anti-Ply antibodies also caused a significant (P < 0.05) reduction in S. pneumoniae colonization. Our results support the potential of PhtD, PcpA, and Ply protein vaccine candidates as alternatives to conjugate vaccines to prevent non-serotype-specific S. pneumoniae colonization and invasive infection.

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“…14 The proposed absence of pro-inflammatory activity is based on the observations that following its addition to neutrophils, Δ6 PLY, at concentrations as high as 1000 ng/ml caused only a modest Ca 2+ influx and failed to activate either the release of MMP-9 or the generation of LTB 4. Both of these are potent effectors of neutrophil-mediated inflammation and tissue damage.…”

Section: Discussionmentioning

confidence: 99%

Characterization of the interactions of the pneumolysoid, Δ6 PLY, with human neutrophils in vitro

Cockeran

Steel

Theron

et al. 2011

Vaccine

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The pneumolysin toxoid, Δ6 PLY, is a prototype pneumococcal protein vaccine candidate. However, its potentially detrimental residual proinflammatory interactions with human neutrophils are unknown. In the current study the effects of the toxoid (8-1000 ng/ml) have been compared with those of wild-type pneumolysin (WT/PLY, 8 ng/ml) on neutrophil cytosolic Ca 2+ fluxes, generation of leukotriene B 4 (LTB 4 ), and release of matrix metalloproteinase-9 (MMP-9), using spectrofluorimetric, and ELISA procedures (LTB 4 and MMP-9) respectively. Exposure of neutrophils to WT/PLY resulted in influx of Ca 2+ and significant (P<0.05) release of MMP-9 and generation of LTB 4 . However, treatment of the cells with Δ6 PLY at concentrations of up to 1000 ng/ml had only trivial effects on Ca 2+ influx and no effects on either release of MMP-9 or LTB 4 production. The observed absence of pro-inflammatory interactions of Δ6 PLY with neutrophils is clearly an important property of this pneumococcal protein vaccine candidate.

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Structure-guided Antigen Engineering Yields Pneumolysin Mutants Suitable for Vaccination against Pneumococcal Disease

Cited by 28 publications

References 44 publications

Neutralizing Antibodies Elicited by a Novel Detoxified Pneumolysin Derivative, PlyD1, Provide Protection against Both Pneumococcal Infection and Lung Injury

Neutralizing Antibodies Elicited by a Novel Detoxified Pneumolysin Derivative, PlyD1, Provide Protection against Both Pneumococcal Infection and Lung Injury

Human Antibodies to PhtD, PcpA, and Ply Reduce Adherence to Human Lung Epithelial Cells and Murine Nasopharyngeal Colonization by Streptococcus pneumoniae

Characterization of the interactions of the pneumolysoid, Δ6 PLY, with human neutrophils in vitro

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