Name: expression of exogenous antigens in the mycobacterium bovis bcg vaccine via non-genetic surface decoration with the avidin-biotin system
Uploaded: 2018-01-31T13:00:00
Description: Watch this Scientific Journal Video about Expression of Exogenous Antigens in the Mycobacterium bovis BCG Vaccine via Non-genetic Surface Decoration with the Avidin-biotin System at JoVE.com

We thank Dr. R Stokes for the BCG Pasteur strain and A. Talal for technical support. We also thank GenScript for help with gene synthesis.

All animals were maintained in accordance with protocols approved by the Animal Care and Use Committees at the University of British Columbia. Experiments were approved by the Animal Care and Use Committees and performed according to the Canadian Council on Animal Care Guidelines. The animal assurance welfare number is A11-0247.
1. Generation of Monomeric Avidin Fusion Proteins Expressing Plasmids
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We reported in this study a non-genetic approach for rapid and effective display of exogenous proteins on BCG surface to add either specific antigens or specific functional properties expected to efficiently improve the bacterium&#39;s immunogenicity. We demonstrated that the BCG cell surface could be easily biotinylated for instantaneous surface decoration with avidin fusion proteins. The total procedure can be performed within 2 h, while genetic transformation and selection of positive clones requires 2 to 3 months of ...

Various strategies have been proposed to replace the current TB vaccine BCG, including protein adjuvant systems, viral vectored technologies, attenuated live M.tb strains, and genetically modified BCG strains, either to introduce genes over-expressing BCG antigens that are not sufficiently expressed during infection1 or Mtb-specific antigens not present in BCG2. Genetic engineering, however, faces many barriers including the uncertain level of safety, the time-consuming process, and the low efficiency of expression vectors4,5. With regards to improving BCG, an alternate approach is needed to improve immunogenicity without the need for uncertain genetic alternations.
In this study, we introduce a novel strategy for display of recombinant proteins of interest on the BCG cell surface that is based on the well-known high-affinity avidin interaction with biotin. This approach allows rapid and reproducible attachment of recombinant avidin fusion proteins on the surface of biotinylated BCG, which facilitates broad manipulations of BCG to achieve maximal improvement of its efficacy while maintaining its excellent safety record, observed over decades of use.
Avidin affinity for biotin is extremely high (Kd = 10&#8722;15 M) and once formed, the biotin-avidin complex is very stable and can only be disrupted under denaturing conditions6. However, for this type of interaction to serve as a gene transfer method alternative, long-term but reversible display of recombinant proteins is required. Thus, we introduced here a low affinity monomeric avidin (Kd = 10&#8722;7 M) that leads to the reversible release of protein from the surface decorated BCG once ingested inside antigen presenting cells. In order to provide a proof of concept, we tested this method using a monomeric avidin chimeric protein corresponding to a surrogate antigen derived from ovalbumin (OVA)7,8. The results showed that the BCG cell surface can be easily and rapidly decorated with monomeric avidin fusion proteins and that this binding to the BCG surface is stable and reproducible without detectable changes in bacterial growth and survival. Also, we found that BCG decorated with monomeric avidin fused with OVA (AviOVA) can induce an immune response similar to that induced by BCG genetically expressing the same antigen both in vitro and in vivo. This technology of reversible display of proteins of interest on the bacterial surface is therefore an effective replacement of traditional gene transfer approaches and can provide a platform for broad manipulations of BCG and further applications in vaccine development.

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			<td style="width:119px;">&#160;VWR 66011-675</td>
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expression of exogenous antigens in the mycobacterium bovis bcg vaccine via non-genetic surface decoration with the avidin-biotin system

Tuberculosis (TB) is a serious infectious disease and the only available vaccine M. bovis bacillus Calmette-Gu&#233;rin (BCG) is safe and effective for protection against children&#39;s severe TB meningitis and some forms of disseminated TB, but fails to protect against pulmonary TB, which is the most prevalent form of the disease. Promising strategies to improve BCG currently rely either on its transformation with genes encoding immunodominant M. tuberculosis (Mtb)-specific antigens and/or complementation with genes encoding co-factors that would stimulate antigen presenting cells. Major limitations to these approaches include low efficiency, low stability, and the uncertain level of safety of expression vectors. In this study, we present an alternative approach to vaccine improvement, which consists of BCG complementation with exogenous proteins of interest on the surface of bacteria, rather than transformation with plasmids encoding corresponding genes. First, proteins of interest are expressed in fusion with monomeric avidin in standard E. coli expression systems and then used to decorate the surface of biotinylated BCG. Animal experiments using BCG surface decorated with surrogate ovalbumin antigen demonstrate that the modified bacterium is fully immunogenic and capable of inducing specific T cell responses. Altogether, the data presented here strongly support a novel and efficient method for reshaping the current BCG vaccine that replaces the laborious conventional approach of complementation with exogenous nucleic acids.

With the general procedures described above, the feasibility of BCG surface biotinylation and decoration with surrogate antigen OVA was examined. The immunogenicity of the modified BCG was then tested in vivo. The bacterial surface was easily labeled with biotin for rapid display of avidin chimeric antigens without any detectable changes in bacterial phenotypes. The resulting modified BCG is efficiently ingested by antigen presentation cells and can induce an OVA-specific immune ...

Watch this Scientific Journal Video about Expression of Exogenous Antigens in the Mycobacterium bovis BCG Vaccine via Non-genetic Surface Decoration with the Avidin-biotin System at JoVE.com

Expression of Exogenous Antigens in the Mycobacterium bovis BCG Vaccine via Non-genetic Surface Decoration with the Avidin-biotin System

A novel technique for rapid antigen display on a bacterial surface is presented, which involves surface biotinylation followed by exposure to proteins of interest in fusion with monomeric avidin. Loading BCG with selected antigens successfully improves its immunogenicity, suggesting that surface decoration can replace traditional genetic approaches.

Expression of Exogenous Antigens in the Mycobacterium bovis BCG Vaccine via Non-genetic Surface Decoration with the Avidin-biotin System

Tuberculosis (TB) is a serious infectious disease and the only available vaccine M. bovis bacillus Calmette-Gu&#233;rin (BCG) is safe and effective for ...

Research

JoVE Journal

Immunology and Infection

A Functional Whole Blood Assay to Measure Viability of Mycobacteria, using Reporter-Gene Tagged BCG or M.Tb (BCG lux/M.Tb lux)

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Functional assays have long played a key role in measuring of immunogenicity of a given vaccine. This is conventionally expressed as serum bactericidal ...

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Development of an in vitro model system for studying the interaction of Equus caballus IgE with its high-affinity receptor Fc&#949;RI

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Dendritic cells (DCs) are important for initiating immune responses, in part through their ability to acquire and shuttle antigen to the draining lymph ...

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

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Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte ...

Purification of the Membrane Compartment for Endoplasmic Reticulum-associated Degradation of Exogenous Antigens in Cross-presentation

Dendritic cells (DCs) are highly capable of processing and presenting internalized exogenous antigens upon major histocompatibility class (MHC) I ...

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

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Reporter enzyme fluorescence (REF) utilizes substrates that are specific for enzymes present in target organisms of interest for imaging or detection by ...

Single-cell Quantitation of mRNA and Surface Protein Expression in Simian Immunodeficiency Virus-infected CD4+ T Cells Isolated from Rhesus macaques