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1.
Biomolecules ; 10(11)2020 11 14.
Article in English | MEDLINE | ID: mdl-33202679

ABSTRACT

The Gram-negative bacterium Yersinia pestis causes plague, a fatal flea-borne anthropozoonosis, which can progress to aerosol-transmitted pneumonia. Y. pestis overcomes the innate immunity of its host thanks to many pathogenicity factors, including plasminogen activator, Pla. This factor is a broad-spectrum outer membrane protease also acting as adhesin and invasin. Y. pestis uses Pla adhesion and proteolytic capacity to manipulate the fibrinolytic cascade and immune system to produce bacteremia necessary for pathogen transmission via fleabite or aerosols. Because of microevolution, Y. pestis invasiveness has increased significantly after a single amino-acid substitution (I259T) in Pla of one of the oldest Y. pestis phylogenetic groups. This mutation caused a better ability to activate plasminogen. In paradox with its fibrinolytic activity, Pla cleaves and inactivates the tissue factor pathway inhibitor (TFPI), a key inhibitor of the coagulation cascade. This function in the plague remains enigmatic. Pla (or pla) had been used as a specific marker of Y. pestis, but its solitary detection is no longer valid as this gene is present in other species of Enterobacteriaceae. Though recovering hosts generate anti-Pla antibodies, Pla is not a good subunit vaccine. However, its deletion increases the safety of attenuated Y. pestis strains, providing a means to generate a safe live plague vaccine.


Subject(s)
Plasminogen Activators/metabolism , Protein Interaction Maps/physiology , Yersinia pestis/metabolism , Animals , Antigens, Bacterial/chemistry , Antigens, Bacterial/genetics , Antigens, Bacterial/metabolism , Humans , Plague/genetics , Plague/metabolism , Plague/prevention & control , Plague Vaccine/administration & dosage , Plague Vaccine/genetics , Plague Vaccine/metabolism , Plasminogen Activators/chemistry , Plasminogen Activators/genetics , Point Mutation/physiology , Protein Structure, Secondary , Yersinia pestis/classification , Yersinia pestis/genetics
2.
Clin Exp Immunol ; 196(3): 287-304, 2019 06.
Article in English | MEDLINE | ID: mdl-30985006

ABSTRACT

Plague caused by the Gram-negative bacterium, Yersinia pestis, is still endemic in parts of the world today. Protection against pneumonic plague is essential to prevent the development and spread of epidemics. Despite this, there are currently no licensed plague vaccines in the western world. Here we describe the means of delivering biologically active plague vaccine antigens directly to mucosal sites of plague infection using highly stable microvesicles (outer membrane vesicles; OMVs) that are naturally produced by the abundant and harmless human commensal gut bacterium Bacteroides thetaiotaomicron (Bt). Bt was engineered to express major plague protective antigens in its OMVs, specifically Fraction 1 (F1) in the outer membrane and LcrV (V antigen) in the lumen, for targeted delivery to the gastrointestinal (GI) and respiratory tracts in a non-human primate (NHP) host. Our key findings were that Bt OMVs stably expresses F1 and V plague antigens, particularly the V antigen, in the correct, immunogenic form. When delivered intranasally V-OMVs elicited substantive and specific immune and antibody responses, both in the serum [immunoglobulin (Ig)G] and in the upper and lower respiratory tract (IgA); this included the generation of serum antibodies able to kill plague bacteria. Our results also showed that Bt OMV-based vaccines had many desirable characteristics, including: biosafety and an absence of any adverse effects, pathology or gross alteration of resident microbial communities (microbiotas); high stability and thermo-tolerance; needle-free delivery; intrinsic adjuvanticity; the ability to stimulate both humoral and cell-mediated immune responses; and targeting of primary sites of plague infection.


Subject(s)
Antigens, Bacterial/metabolism , Bacterial Outer Membrane/metabolism , Bacteroides thetaiotaomicron/metabolism , Plague Vaccine/immunology , Plague/immunology , Pore Forming Cytotoxic Proteins/metabolism , Transport Vesicles/immunology , Yersinia pestis/physiology , Administration, Intranasal , Animals , Antibodies, Bacterial/blood , Antigens, Bacterial/genetics , Bacteroides thetaiotaomicron/genetics , Bioengineering , Cell Death , Cells, Cultured , Gastrointestinal Microbiome/genetics , Humans , Immunity, Cellular , Immunity, Humoral , Immunoglobulin A/metabolism , Immunoglobulin G/blood , Macaca , Plague/prevention & control , Plague Vaccine/metabolism , Pore Forming Cytotoxic Proteins/genetics , Transport Vesicles/metabolism
3.
J Plant Physiol ; 168(2): 174-80, 2011 Jan 15.
Article in English | MEDLINE | ID: mdl-20655621

ABSTRACT

Expression of the protective F1 and V antigens of Yersinia pestis, as a fusion protein, in carrot was pursued in an effort to develop an alternative vaccine production system against the serious plague disease. Transgenic carrot plants carrying the F1-V encoding gene were developed via Agrobacterium-mediated transformation. Presence, integration, and expression of the F1-V encoding gene were confirmed by polymerase chain reaction (PCR), DNA gel blot analysis, and reverse-transcriptase (RT)-PCR analyses, respectively. An ELISA assay confirmed the antigenicity of the plant-derived F1-V fusion protein. Immunogenicity was evaluated subcutaneously in mice using a soluble protein extract of freeze-dried transgenic carrot. Significant antibody levels were detected following immunization. These results demonstrated that the F1-V protein could be expressed in carrot tap roots, and that the carrot F1-V recombinant protein retained its antigenicity and immunogenicity.


Subject(s)
Antigens, Bacterial/metabolism , Bacterial Proteins/metabolism , Daucus carota/metabolism , Plant Roots/metabolism , Plants, Genetically Modified/metabolism , Pore Forming Cytotoxic Proteins/metabolism , Recombinant Fusion Proteins/metabolism , Yersinia pestis/metabolism , Animals , Antigens, Bacterial/genetics , Bacterial Proteins/genetics , Daucus carota/genetics , Enzyme-Linked Immunosorbent Assay , Mice , Mice, Inbred BALB C , Plague/immunology , Plague Vaccine/genetics , Plague Vaccine/immunology , Plague Vaccine/metabolism , Plant Roots/genetics , Plants, Genetically Modified/genetics , Pore Forming Cytotoxic Proteins/genetics , Recombinant Fusion Proteins/genetics , Reverse Transcriptase Polymerase Chain Reaction , Yersinia pestis/genetics
4.
Planta ; 232(2): 409-16, 2010 Jul.
Article in English | MEDLINE | ID: mdl-20461403

ABSTRACT

Yersinia pestis is a pathogenic agent that causes the bubonic and pneumonic plague. The development of an efficient and low-cost oral vaccine against these diseases is highly desirable. In this study, the immunogenic fusion protein F1-V from Y. pestis was introduced into lettuce via Agrobacterium-mediated transformation, and putative transgenic lines were developed. The presence of the transgene in these putative transgenic lines was determined using polymerase chain reaction (PCR), and transgene integration and transgene copy number were confirmed following Southern blot analysis. The presence of specific F1-V transcripts was confirmed by reverse-transcriptase (RT)-PCR. Using monoclonal antibodies, ELISA and western blot analysis revealed that the expected antigenic F1-V protein was successfully expressed in transgenic lines. Mice immunized subcutaneously with lettuce expressing the F1-V antigen developed systemic humoral responses as 'proof of concept' of using lettuce as a production platform for the F1-V immunogen that could be used as a candidate plant-based vaccine against plague.


Subject(s)
Antigens, Bacterial/immunology , Antigens, Bacterial/metabolism , Lactuca/metabolism , Plague Vaccine/immunology , Plague Vaccine/metabolism , Plague/immunology , Pore Forming Cytotoxic Proteins/immunology , Pore Forming Cytotoxic Proteins/metabolism , Yersinia pestis/immunology , Animals , Antigens, Bacterial/genetics , Blotting, Western , Enzyme-Linked Immunosorbent Assay , Lactuca/genetics , Mice , Mice, Inbred BALB C , Plague/microbiology , Plague Vaccine/genetics , Plants, Genetically Modified/genetics , Plants, Genetically Modified/metabolism , Polymerase Chain Reaction , Pore Forming Cytotoxic Proteins/genetics , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/immunology , Recombinant Fusion Proteins/metabolism , Rhizobium/genetics , Yersinia pestis/pathogenicity
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