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Role of TLR3 in the immunogenicity of replicon plasmid-based vaccines.

Diebold SS, Schulz O, Alexopoulou L, Leitner WW, Flavell RA, Reis e Sousa C - Gene Ther. (2008)

Bottom Line: Toll-like receptor 3 (TLR3) is a dsRNA innate immune receptor expressed by antigen-presenting dendritic cells (DCs).Here, we test the hypothesis that TLR3 is necessary for the immunogenicity of replicon plasmid-based DNA vaccines.We show that mouse CD8 alpha(+) DC phagocytose dying replicon plasmid-transfected cells in vitro and are activated in a TLR3-dependent manner by dsRNA present within those cells.

View Article: PubMed Central - PubMed

Affiliation: Immunobiology Laboratory, Cancer Research UK, London Research Institute, London, UK. sandra.diebold@kcl.ac.uk

ABSTRACT
Replicon plasmids encoding an alphavirus RNA replicase constitute an alternative to conventional DNA plasmids with promise for DNA vaccination in humans. Replicase activity amplifies the levels of transgene mRNA through a copying process involving double-stranded (ds) RNA intermediates, which contribute to vaccine immunogenicity by activating innate antiviral responses. Toll-like receptor 3 (TLR3) is a dsRNA innate immune receptor expressed by antigen-presenting dendritic cells (DCs). Here, we test the hypothesis that TLR3 is necessary for the immunogenicity of replicon plasmid-based DNA vaccines. We show that mouse CD8 alpha(+) DC phagocytose dying replicon plasmid-transfected cells in vitro and are activated in a TLR3-dependent manner by dsRNA present within those cells. However, we find that cytotoxic T-cell responses to a replicon plasmid intramuscular vaccine are not diminished in the absence of TLR3 in vivo. Our results underscore the potential role of TLR3 in mediating immune activation by dsRNA-bearing replicon plasmid-transfected cells and indicate that other innate sensing pathways can compensate for TLR3 absence in vivo.

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Related in: MedlinePlus

The induction of CTL responses in vivo takes place independent of TLR3-mediated activationC57BL/6 and TLR3-/- mice were vaccinated with 50μg of replicon (pSIN-ΔOVA) or conventional plasmid (pcDNA3-ΔOVA) DNA intramuscularly. Anti-CD40 antibody was injected i.p. to boost immunization. Expansion of antigen-specific T cells was determined by Pentamer staining (a) and antigen-specific killing of target cells was assessed by in vivo CTL assay (b) 21 days after vaccination. The results represent pooled data from two independent experiments.
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Figure 5: The induction of CTL responses in vivo takes place independent of TLR3-mediated activationC57BL/6 and TLR3-/- mice were vaccinated with 50μg of replicon (pSIN-ΔOVA) or conventional plasmid (pcDNA3-ΔOVA) DNA intramuscularly. Anti-CD40 antibody was injected i.p. to boost immunization. Expansion of antigen-specific T cells was determined by Pentamer staining (a) and antigen-specific killing of target cells was assessed by in vivo CTL assay (b) 21 days after vaccination. The results represent pooled data from two independent experiments.

Mentions: Because activation of CD8α+ DC by replicon-expressing cells was TLR3-dependent, we wondered whether the immunogenicity of replicon vector-based DNA vaccines also depended on TLR3. We immunized wild type and TLR3-deficient mice intramuscularly with replicon or conventional plasmid vectors expressing the model antigen ovalbumin (OVA) and used anti-CD40 antibody as adjuvant to increase the immune response. Three weeks later, we analysed the number of OVA specific CD8+ T cells in the spleen and in vivo cytoxicity against OVA peptide-pulsed targets. Despite a high variability within groups, overall numbers of OVA specific CD8+ T cells were not significantly different for replicon and conventional plasmid-vaccinated mice (Fig. 5A). In vivo cytotoxic activity against OVA was also similar between WT and TLR3-/- mice independent of the type of plasmid used for vaccination (Fig. 5B). The same result was obtained in mice vaccinated in the absence of anti-CD40 antibody (data not shown). Thus while activation of CD8α+ DC in response to replicon-expressing cells in vitro is dependent on TLR3, in vivo CTL priming upon intramuscular injection of replicon DNA is TLR3-independent.


Role of TLR3 in the immunogenicity of replicon plasmid-based vaccines.

Diebold SS, Schulz O, Alexopoulou L, Leitner WW, Flavell RA, Reis e Sousa C - Gene Ther. (2008)

The induction of CTL responses in vivo takes place independent of TLR3-mediated activationC57BL/6 and TLR3-/- mice were vaccinated with 50μg of replicon (pSIN-ΔOVA) or conventional plasmid (pcDNA3-ΔOVA) DNA intramuscularly. Anti-CD40 antibody was injected i.p. to boost immunization. Expansion of antigen-specific T cells was determined by Pentamer staining (a) and antigen-specific killing of target cells was assessed by in vivo CTL assay (b) 21 days after vaccination. The results represent pooled data from two independent experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2655288&req=5

Figure 5: The induction of CTL responses in vivo takes place independent of TLR3-mediated activationC57BL/6 and TLR3-/- mice were vaccinated with 50μg of replicon (pSIN-ΔOVA) or conventional plasmid (pcDNA3-ΔOVA) DNA intramuscularly. Anti-CD40 antibody was injected i.p. to boost immunization. Expansion of antigen-specific T cells was determined by Pentamer staining (a) and antigen-specific killing of target cells was assessed by in vivo CTL assay (b) 21 days after vaccination. The results represent pooled data from two independent experiments.
Mentions: Because activation of CD8α+ DC by replicon-expressing cells was TLR3-dependent, we wondered whether the immunogenicity of replicon vector-based DNA vaccines also depended on TLR3. We immunized wild type and TLR3-deficient mice intramuscularly with replicon or conventional plasmid vectors expressing the model antigen ovalbumin (OVA) and used anti-CD40 antibody as adjuvant to increase the immune response. Three weeks later, we analysed the number of OVA specific CD8+ T cells in the spleen and in vivo cytoxicity against OVA peptide-pulsed targets. Despite a high variability within groups, overall numbers of OVA specific CD8+ T cells were not significantly different for replicon and conventional plasmid-vaccinated mice (Fig. 5A). In vivo cytotoxic activity against OVA was also similar between WT and TLR3-/- mice independent of the type of plasmid used for vaccination (Fig. 5B). The same result was obtained in mice vaccinated in the absence of anti-CD40 antibody (data not shown). Thus while activation of CD8α+ DC in response to replicon-expressing cells in vitro is dependent on TLR3, in vivo CTL priming upon intramuscular injection of replicon DNA is TLR3-independent.

Bottom Line: Toll-like receptor 3 (TLR3) is a dsRNA innate immune receptor expressed by antigen-presenting dendritic cells (DCs).Here, we test the hypothesis that TLR3 is necessary for the immunogenicity of replicon plasmid-based DNA vaccines.We show that mouse CD8 alpha(+) DC phagocytose dying replicon plasmid-transfected cells in vitro and are activated in a TLR3-dependent manner by dsRNA present within those cells.

View Article: PubMed Central - PubMed

Affiliation: Immunobiology Laboratory, Cancer Research UK, London Research Institute, London, UK. sandra.diebold@kcl.ac.uk

ABSTRACT
Replicon plasmids encoding an alphavirus RNA replicase constitute an alternative to conventional DNA plasmids with promise for DNA vaccination in humans. Replicase activity amplifies the levels of transgene mRNA through a copying process involving double-stranded (ds) RNA intermediates, which contribute to vaccine immunogenicity by activating innate antiviral responses. Toll-like receptor 3 (TLR3) is a dsRNA innate immune receptor expressed by antigen-presenting dendritic cells (DCs). Here, we test the hypothesis that TLR3 is necessary for the immunogenicity of replicon plasmid-based DNA vaccines. We show that mouse CD8 alpha(+) DC phagocytose dying replicon plasmid-transfected cells in vitro and are activated in a TLR3-dependent manner by dsRNA present within those cells. However, we find that cytotoxic T-cell responses to a replicon plasmid intramuscular vaccine are not diminished in the absence of TLR3 in vivo. Our results underscore the potential role of TLR3 in mediating immune activation by dsRNA-bearing replicon plasmid-transfected cells and indicate that other innate sensing pathways can compensate for TLR3 absence in vivo.

Show MeSH
Related in: MedlinePlus