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Antigen delivery by filamentous bacteriophage fd displaying an anti-DEC-205 single-chain variable fragment confers adjuvanticity by triggering a TLR9-mediated immune response.

Sartorius R, D'Apice L, Trovato M, Cuccaro F, Costa V, De Leo MG, Marzullo VM, Biondo C, D'Auria S, De Matteis MA, Ciccodicola A, De Berardinis P - EMBO Mol Med (2015)

Bottom Line: A significant differential expression of genes involved in innate immunity, co-stimulation and cytokine production was observed.We also found that fdsc-αDEC is delivered into LAMP-1-positive compartments and co-localizes with TLR9.Thus, phage particles containing a single-strand DNA genome rich in CpG motifs delivered via DEC-205 are able to intercept and trigger the active TLR9 innate immune receptor into late endosome/lysosomes and to enhance the immunogenicity of the displayed antigenic determinants.

View Article: PubMed Central - PubMed

Affiliation: Institute of Protein Biochemistry, National Council of Research, Naples, Italy.

No MeSH data available.


Related in: MedlinePlus

Confocal microscopy analysis of BMDCs after phage uptakeA–D BMDCs were incubated with TRITC-conjugated fdWT (A, C) or fdsc-αDEC bacteriophages (B, D) for 6 h. Cells were then fixed, permeabilized, immunostained with anti-EEA-1 (A, B) or anti-LAMP-1 (C, D) (green) antibody and analyzed by confocal microscopy. Representative images are shown. Numbers at the bottom of the right panels indicate the percentage of EEA-1 (A, B) or LAMP-1 (C, D) bacteriophage co-localization (mean values ± SD, N = 2, n = 100). Scale bars, 10 mm. White arrows indicate LAMP-1-positive structures that also are positive or contain fdsc-αDEC or fdWT bacteriophages.
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fig04: Confocal microscopy analysis of BMDCs after phage uptakeA–D BMDCs were incubated with TRITC-conjugated fdWT (A, C) or fdsc-αDEC bacteriophages (B, D) for 6 h. Cells were then fixed, permeabilized, immunostained with anti-EEA-1 (A, B) or anti-LAMP-1 (C, D) (green) antibody and analyzed by confocal microscopy. Representative images are shown. Numbers at the bottom of the right panels indicate the percentage of EEA-1 (A, B) or LAMP-1 (C, D) bacteriophage co-localization (mean values ± SD, N = 2, n = 100). Scale bars, 10 mm. White arrows indicate LAMP-1-positive structures that also are positive or contain fdsc-αDEC or fdWT bacteriophages.

Mentions: In order to understand how and where fdsc-αDEC intercepts TLR9, we analyzed the intracellular fate of fdsc-αDEC and of wild-type bacteriophages by dissecting the intracellular trafficking route of fd particles (delivered or not via DEC-205) in BMDCs. BMDCs incubated with tetramethylrhodamine (TRITC)-conjugated fdWT or fdsc-αDEC for 6 h were analyzed by confocal microscopy, and their endosomal compartments were mapped using early endosomal (EEA-1) or late endosomal/lysosomal (LAMP-1) markers. We observed low co-localization of fd bacteriophages particles, either wild-type or expressing anti-DEC-205, in EEA-1-positive early endosomes (< 3% of EEA-1-positive structures contain fd wild-type (Fig4A) or fdsc-αDEC phage particles (Fig4B)). By contrast, we found that fdsc-αDEC bacteriophages, but not fd wild-type, localized in the LAMP1-positive late endosomal/lysosomal compartments. As shown in Fig4C and D, the display of anti-DEC-205 scFv on the phage surface markedly increases the localization of bacteriophages into late endolysosomal compartments. Co-localization analysis showed that 41.3% of LAMP-1-positive structures also contain fdsc-αDEC phage particles (Fig4D), while fd wild-type seems to localize with only 8.9% of the LAMP-1-positive endolysosomes (Fig4C).


Antigen delivery by filamentous bacteriophage fd displaying an anti-DEC-205 single-chain variable fragment confers adjuvanticity by triggering a TLR9-mediated immune response.

Sartorius R, D'Apice L, Trovato M, Cuccaro F, Costa V, De Leo MG, Marzullo VM, Biondo C, D'Auria S, De Matteis MA, Ciccodicola A, De Berardinis P - EMBO Mol Med (2015)

Confocal microscopy analysis of BMDCs after phage uptakeA–D BMDCs were incubated with TRITC-conjugated fdWT (A, C) or fdsc-αDEC bacteriophages (B, D) for 6 h. Cells were then fixed, permeabilized, immunostained with anti-EEA-1 (A, B) or anti-LAMP-1 (C, D) (green) antibody and analyzed by confocal microscopy. Representative images are shown. Numbers at the bottom of the right panels indicate the percentage of EEA-1 (A, B) or LAMP-1 (C, D) bacteriophage co-localization (mean values ± SD, N = 2, n = 100). Scale bars, 10 mm. White arrows indicate LAMP-1-positive structures that also are positive or contain fdsc-αDEC or fdWT bacteriophages.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4520660&req=5

fig04: Confocal microscopy analysis of BMDCs after phage uptakeA–D BMDCs were incubated with TRITC-conjugated fdWT (A, C) or fdsc-αDEC bacteriophages (B, D) for 6 h. Cells were then fixed, permeabilized, immunostained with anti-EEA-1 (A, B) or anti-LAMP-1 (C, D) (green) antibody and analyzed by confocal microscopy. Representative images are shown. Numbers at the bottom of the right panels indicate the percentage of EEA-1 (A, B) or LAMP-1 (C, D) bacteriophage co-localization (mean values ± SD, N = 2, n = 100). Scale bars, 10 mm. White arrows indicate LAMP-1-positive structures that also are positive or contain fdsc-αDEC or fdWT bacteriophages.
Mentions: In order to understand how and where fdsc-αDEC intercepts TLR9, we analyzed the intracellular fate of fdsc-αDEC and of wild-type bacteriophages by dissecting the intracellular trafficking route of fd particles (delivered or not via DEC-205) in BMDCs. BMDCs incubated with tetramethylrhodamine (TRITC)-conjugated fdWT or fdsc-αDEC for 6 h were analyzed by confocal microscopy, and their endosomal compartments were mapped using early endosomal (EEA-1) or late endosomal/lysosomal (LAMP-1) markers. We observed low co-localization of fd bacteriophages particles, either wild-type or expressing anti-DEC-205, in EEA-1-positive early endosomes (< 3% of EEA-1-positive structures contain fd wild-type (Fig4A) or fdsc-αDEC phage particles (Fig4B)). By contrast, we found that fdsc-αDEC bacteriophages, but not fd wild-type, localized in the LAMP1-positive late endosomal/lysosomal compartments. As shown in Fig4C and D, the display of anti-DEC-205 scFv on the phage surface markedly increases the localization of bacteriophages into late endolysosomal compartments. Co-localization analysis showed that 41.3% of LAMP-1-positive structures also contain fdsc-αDEC phage particles (Fig4D), while fd wild-type seems to localize with only 8.9% of the LAMP-1-positive endolysosomes (Fig4C).

Bottom Line: A significant differential expression of genes involved in innate immunity, co-stimulation and cytokine production was observed.We also found that fdsc-αDEC is delivered into LAMP-1-positive compartments and co-localizes with TLR9.Thus, phage particles containing a single-strand DNA genome rich in CpG motifs delivered via DEC-205 are able to intercept and trigger the active TLR9 innate immune receptor into late endosome/lysosomes and to enhance the immunogenicity of the displayed antigenic determinants.

View Article: PubMed Central - PubMed

Affiliation: Institute of Protein Biochemistry, National Council of Research, Naples, Italy.

No MeSH data available.


Related in: MedlinePlus