Epitope-tagging vectors for the expression and detection of recombinant proteins in mycobacteria

Spratt, Joanne M.; Ryan, Anthony A.; Britton, Warwick J.; Triccas, James A.

doi:10.1016/j.plasmid.2004.11.002

Cited by 16 publications

(14 citation statements)

References 13 publications

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“…The gene encoding M. tuberculosis Ag85B was amplified from M. tuberculosis genomic DNA and cloned into the BamHI/HindIII sites of pCDNA:mflt3L (16), yielding pFlt-85. Recombinant BCG (rBCG) that secretes soluble murine Flt3L (BCG: Flt3L) was constructed by amplifying the Flt3L gene from pCDNA:mflt3L and cloning it into the mycobacterium-Escherichia coli shuttle vector pJEX65, which allows detection of recombinant proteins by a C-terminal epitope tag (37). BCG containing pJEX65 alone (BCG:Ct) was used as the control strain for vaccination experiments.…”

Section: Methodsmentioning

confidence: 99%

Effects of DNA- and Mycobacterium bovis BCG-Based Delivery of the Flt3 Ligand on Protective Immunity to Mycobacterium tuberculosis

et al. 2007

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Tuberculosis (TB) kills 2 million individuals per year and is the greatest cause of death by a single bacterial agent (26). The worldwide incidence of TB continues to rise (26), due in part to the variable efficacy displayed by the only registered vaccine for human use, Mycobacterium bovis bacillus Calmette-Guérin (BCG) (10). This has fuelled the search for more effective anti-TB vaccines, and approaches ranging from DNA vaccines to live, attenuated strains of Mycobacterium tuberculosis have been assessed; the majority of these approaches have failed to deliver improved protective efficacy compared to BCG in animal models (5). A small number of vaccines have recently entered human trials, two of which aim to improve expansion of T cells directed against a single member of the M. tuberculosis antigen 85 (Ag85) complex (15,24). While these two approaches have shown promise in animal models and initial testing in humans, the fact that no single mycobacterial antigen is recognized by all M. tuberculosis-infected individuals (32) may limit the breadth of coverage delivered by these vaccines within the human population.One approach to improve anti-TB vaccination is to target components of the immune response required for optimal protective efficacy. We have previously demonstrated that vaccination strategies designed to augment gamma interferon (IFN-␥) production, such as codelivery of the cytokines interleukin-12 (IL-12) and IL-23 during DNA vaccination, significantly improve protection against TB (29, 39). IL-12 is critical for the induction of Th1-like CD4 ϩ cells, and humans and mice lacking the p40 chain of IL-12 or its receptors are highly susceptible to M. tuberculosis infection (2, 6, 8). M. tuberculosis infection of either murine or human dendritic cells (DCs), but not macrophages, polarizes naïve T cells to the Th1 phenotype due to production of 14). Infection of mice with BCG and subsequent analysis of DC and macrophage populations revealed that DCs exclusively produced IL-12 and presented antigen to T cells (17). After aerosol infection of mice with M. tuberculosis it was observed that DCs, and not macrophages, migrated specifically to the draining lymph nodes (DLNs) and initiated primary Th1 responses (4). These results indicate that DCs play a pivotal role in priming the adaptive immune response to counter infection with M. tuberculosis. It is possible that limited numbers of DCs at the site of antigen production is one factor hindering development of vaccines against diseases such as TB which are reliant on optimal priming of T-cell responses.The Fms-like tyrosine kinase 3 ligand (Flt3L) influences the development of multiple hematopoietic lineages, in particular DCs (23). Administration of soluble Flt3L to both mice (21) and humans (22) increases the numbers of DCs in secondary lymphoid organs and blood. We therefore reasoned that codelivery of Flt3L may be a feasible strategy to improve the efficacy of anti-TB vaccines. In this report, we demonstrate that Flt3L, delivered as plasmid DNA fused to the d...

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Section: Methodsmentioning

confidence: 99%

Effects of DNA- and Mycobacterium bovis BCG-Based Delivery of the Flt3 Ligand on Protective Immunity to Mycobacterium tuberculosis

et al. 2007

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“…To construct M. tuberculosis secreting the same OVA fragment, plasmid pJEX87 (15) was transformed into M. tuberculosis Mt103 strain (16). Expression of OVA 230 -359 was confirmed by Western blotting, using the 9E10 mAb specific for the c-myc epitope tag fused to the C-terminal end of the OVA fragment (17).…”

Section: Construction Of Recombinant Mycobacterial Strainsmentioning

confidence: 99%

Antigen Load Governs the Differential Priming of CD8 T Cells in Response to the Bacille Calmette Guérin Vaccine or Mycobacterium tuberculosis Infection

Ryan

Nambiar

Wozniak

et al. 2009

The Journal of Immunology

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One reason proposed for the failure of Mycobacterium bovis bacille Calmette Guérin (BCG) vaccination to adequately control the spread of tuberculosis is a limited ability of the vaccine to induce effective CD8 T cell responses. However, the relative capacity of the BCG vaccine and virulent Mycobacterium tuberculosis to induce activation of CD8 T cells, and the factors that govern the initial priming of these cells after mycobacterial infection, are poorly characterized. Using a TCR transgenic CD8 T cell transfer model, we demonstrate significant activation of Ag-specific CD8 T cells by BCG, but responses were delayed and of reduced magnitude compared with those following infection with M. tuberculosis. The degree of CD8 T cell activation was critically dependent on the level of antigenic stimulation, as modifying the infectious dose to achieve comparable numbers of BCG or M. tuberculosis in draining lymph nodes led to the same pattern of CD8 T cell responses to both strains. Factors specific to M. tuberculosis infection did not influence the priming of CD8 T cells, as codelivery of M. tuberculosis with BCG did not alter the magnitude of BCG-induced T cell activation. Following transfer to RAG-1−/− recipients, BCG and M. tuberculosis-induced CD8 T cells conferred equivalent levels of protection against M. tuberculosis infection. These findings demonstrate that BCG is able to prime functional CD8 T cells, and suggest that effective delivery of Ag to sites of T cell activation by vaccines may be a key requirement for optimal CD8 T cell responses to control mycobacterial infection.

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“…Expression is driven by a mycobacterial hsp60 promoter and was verified by sequencing, emitting green fluorescence by flow cytommetry and activating OT1 and OT2 TCR transgenic T cells in vitro. 85B-OVA rBCG was generated by Dr. J. Triccas, Centenary Institute of Cancer Medicine and Cell Biology, Australia [7]. 85B-OVA rBCG was transfected with the integrative plasmid pJEX87 and expressed OVA 250-345 attached to the signal sequence for 85B and driven by the same mycobacterial hsp60 promoter in GFP-OVA rBCG.…”

Section: Methodsmentioning

confidence: 99%

The same well-characterized T cell epitope SIINFEKL expressed in the context of a cytoplasmic or secreted protein in BCG induces different CD8+ T cell responses

et al. 2010

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SummaryMycobacterium bovis BCG is still the most widely used vaccine against tuberculosis and CD8 + T cells play important roles in fighting infection. We investigated how well antigen is processed and presented to CD8 + T cells using the same well-characterized CD8 + T cell epitope SIINFEKL expressed in either a cytoplasmic (GFP-OVA) or secreted (85B-OVA) context from BCG. We report that secreted SIINFEKL from 85B-OVA BCG is presented better than cytoplasmic SIINFEKL expressed by GFP-OVA BCG. IntroductionBCG is the most widely used human vaccine and has been administered to more than 3 billion people to protect against tuberculosis (TB) [1]. There are currently over 2 billion people infected with latent TB, and over 9 million incident cases of TB were reported in 2007 [2]. There is a great need to improve the efficacy of BCG since its protection is limited and variable against active pulmonary disease. While BCG is an excellent inducer of Th1 CD4 + T cell responses, it is less effective at inducing CD8 + T cell responses, in contrast to Mtb, which induces strong responses from both. Because CD8 + T cells provide additional mechanisms to combat mycobacteria, efforts were aimed at increasing CD8 + responses induced by BCG antigens, such as creating recombinant BCG (rBCG) that secretes listeriolysin, which perforates the phagosome allowing antigens greater access to the cytosol for classical MHC I processing and presentation [3,4]. The difference between CD8 + T cell activation by cytoplasmic or secreted BCG antigens is poorly understood. rBCG expressing the well-

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Epitope-tagging vectors for the expression and detection of recombinant proteins in mycobacteria

Cited by 16 publications

References 13 publications

Effects of DNA- and Mycobacterium bovis BCG-Based Delivery of the Flt3 Ligand on Protective Immunity to Mycobacterium tuberculosis

Effects of DNA- and Mycobacterium bovis BCG-Based Delivery of the Flt3 Ligand on Protective Immunity to Mycobacterium tuberculosis

Antigen Load Governs the Differential Priming of CD8 T Cells in Response to the Bacille Calmette Guérin Vaccine or Mycobacterium tuberculosis Infection

The same well-characterized T cell epitope SIINFEKL expressed in the context of a cytoplasmic or secreted protein in BCG induces different CD8+ T cell responses

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