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HPV-E7 delivered by engineered exosomes elicits a protective CD8⁺ T cell-mediated immune response.

Di Bonito P, Ridolfi B, Columba-Cabezas S, Giovannelli A, Chiozzini C, Manfredi F, Anticoli S, Arenaccio C, Federico M - Viruses (2015)

Bottom Line: These exosomes, whose limiting membrane was decorated with VSV-G, i.e., an envelope protein inducing pH-dependent endosomal fusion, proved to be as immunogenic as the cognate VLPs.It is noteworthy that the immunogenicity of the engineered exosomes remained unaltered in the absence of VSV-G.These results represent the proof-of-concept about both feasibility and efficacy of the Nefmut-based exosome platform for the induction of CD8+ T cell immunity.

View Article: PubMed Central - PubMed

Affiliation: Department of Infectious, Parasitic and Immunomediated Diseases, Istituto Superiore di Sanità, 00161 Rome, Italy. paola.dibonito@iss.it.

ABSTRACT
We developed an innovative strategy to induce a cytotoxic T cell (CTL) immune response against protein antigens of choice. It relies on the production of exosomes, i.e., nanovesicles spontaneously released by all cell types. We engineered the upload of huge amounts of protein antigens upon fusion with an anchoring protein (i.e., HIV-1 Nefmut), which is an inactive protein incorporating in exosomes at high levels also when fused with foreign proteins. We compared the immunogenicity of engineered exosomes uploading human papillomavirus (HPV)-E7 with that of lentiviral virus-like particles (VLPs) incorporating equivalent amounts of the same antigen. These exosomes, whose limiting membrane was decorated with VSV-G, i.e., an envelope protein inducing pH-dependent endosomal fusion, proved to be as immunogenic as the cognate VLPs. It is noteworthy that the immunogenicity of the engineered exosomes remained unaltered in the absence of VSV-G. Most important, we provide evidence that the inoculation in mouse of exosomes uploading HPV-E7 induces production of anti-HPV E7 CTLs, blocks the growth of syngeneic tumor cells inoculated after immunization, and controls the development of tumor cells inoculated before the exosome challenge. These results represent the proof-of-concept about both feasibility and efficacy of the Nefmut-based exosome platform for the induction of CD8+ T cell immunity.

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CD8+ T cell immune response in mice inoculated with (VSV-G)Nefmut-based VLPs or exosomes. C57 Bl/6 mice (five per group) were inoculated three times with VLPs or exosomes incorporating Nefmut/E7. As control, mice were also inoculated with equivalent amounts of both nanovesicle types incorporating Nefmut alone. Splenocytes recovered from mice were incubated with or without 5 μg/mL of either unrelated or E7-specific peptides. Afterwards, cell activation extents were evaluated by IFN-γ Elispot assay carried out in triplicate with 105 cells/well. As control, untreated cells were also incubated with 5 ng/mL of PMA and 500 ng/mL of ionomycin. Shown are the mean + SD number of IFN-γ spot-forming cells (SFU)/105 cells. The results are representative of two independent experiments. *p < 0.05.
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viruses-07-01079-f002: CD8+ T cell immune response in mice inoculated with (VSV-G)Nefmut-based VLPs or exosomes. C57 Bl/6 mice (five per group) were inoculated three times with VLPs or exosomes incorporating Nefmut/E7. As control, mice were also inoculated with equivalent amounts of both nanovesicle types incorporating Nefmut alone. Splenocytes recovered from mice were incubated with or without 5 μg/mL of either unrelated or E7-specific peptides. Afterwards, cell activation extents were evaluated by IFN-γ Elispot assay carried out in triplicate with 105 cells/well. As control, untreated cells were also incubated with 5 ng/mL of PMA and 500 ng/mL of ionomycin. Shown are the mean + SD number of IFN-γ spot-forming cells (SFU)/105 cells. The results are representative of two independent experiments. *p < 0.05.

Mentions: C57 Bl/6 mice were inoculated subcutaneously (s.c.) three times with volumes of VSV-G pseudotyped VLPs or exosomes containing 650 ng of immunogen, as determined by semi-quantitative Western blot analysis carried out using recombinant Nef as reference standard (not shown). Two weeks after the last inoculation, mouse splenocytes were isolated and cultured for 4 days in the presence or absence of either unrelated or HPV-E7-specific nonamers to selectively stimulate CD8+ T lymphocytes. Afterwards, 105 cells were seeded in IFN-γ Elispot wells, and the number of spot-forming units (SFU) was scored 16 h later. Data reported in Figure 2 show that, in terms of induction of CD8+ T cell response, E7 uploaded in exosomes was as immunogenic as that delivered by VLPs. Of note, no anti-HPV E7 antibodies were found in sera from mice inoculated with either VLPs or exosomes (not shown).


HPV-E7 delivered by engineered exosomes elicits a protective CD8⁺ T cell-mediated immune response.

Di Bonito P, Ridolfi B, Columba-Cabezas S, Giovannelli A, Chiozzini C, Manfredi F, Anticoli S, Arenaccio C, Federico M - Viruses (2015)

CD8+ T cell immune response in mice inoculated with (VSV-G)Nefmut-based VLPs or exosomes. C57 Bl/6 mice (five per group) were inoculated three times with VLPs or exosomes incorporating Nefmut/E7. As control, mice were also inoculated with equivalent amounts of both nanovesicle types incorporating Nefmut alone. Splenocytes recovered from mice were incubated with or without 5 μg/mL of either unrelated or E7-specific peptides. Afterwards, cell activation extents were evaluated by IFN-γ Elispot assay carried out in triplicate with 105 cells/well. As control, untreated cells were also incubated with 5 ng/mL of PMA and 500 ng/mL of ionomycin. Shown are the mean + SD number of IFN-γ spot-forming cells (SFU)/105 cells. The results are representative of two independent experiments. *p < 0.05.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4379561&req=5

viruses-07-01079-f002: CD8+ T cell immune response in mice inoculated with (VSV-G)Nefmut-based VLPs or exosomes. C57 Bl/6 mice (five per group) were inoculated three times with VLPs or exosomes incorporating Nefmut/E7. As control, mice were also inoculated with equivalent amounts of both nanovesicle types incorporating Nefmut alone. Splenocytes recovered from mice were incubated with or without 5 μg/mL of either unrelated or E7-specific peptides. Afterwards, cell activation extents were evaluated by IFN-γ Elispot assay carried out in triplicate with 105 cells/well. As control, untreated cells were also incubated with 5 ng/mL of PMA and 500 ng/mL of ionomycin. Shown are the mean + SD number of IFN-γ spot-forming cells (SFU)/105 cells. The results are representative of two independent experiments. *p < 0.05.
Mentions: C57 Bl/6 mice were inoculated subcutaneously (s.c.) three times with volumes of VSV-G pseudotyped VLPs or exosomes containing 650 ng of immunogen, as determined by semi-quantitative Western blot analysis carried out using recombinant Nef as reference standard (not shown). Two weeks after the last inoculation, mouse splenocytes were isolated and cultured for 4 days in the presence or absence of either unrelated or HPV-E7-specific nonamers to selectively stimulate CD8+ T lymphocytes. Afterwards, 105 cells were seeded in IFN-γ Elispot wells, and the number of spot-forming units (SFU) was scored 16 h later. Data reported in Figure 2 show that, in terms of induction of CD8+ T cell response, E7 uploaded in exosomes was as immunogenic as that delivered by VLPs. Of note, no anti-HPV E7 antibodies were found in sera from mice inoculated with either VLPs or exosomes (not shown).

Bottom Line: These exosomes, whose limiting membrane was decorated with VSV-G, i.e., an envelope protein inducing pH-dependent endosomal fusion, proved to be as immunogenic as the cognate VLPs.It is noteworthy that the immunogenicity of the engineered exosomes remained unaltered in the absence of VSV-G.These results represent the proof-of-concept about both feasibility and efficacy of the Nefmut-based exosome platform for the induction of CD8+ T cell immunity.

View Article: PubMed Central - PubMed

Affiliation: Department of Infectious, Parasitic and Immunomediated Diseases, Istituto Superiore di Sanità, 00161 Rome, Italy. paola.dibonito@iss.it.

ABSTRACT
We developed an innovative strategy to induce a cytotoxic T cell (CTL) immune response against protein antigens of choice. It relies on the production of exosomes, i.e., nanovesicles spontaneously released by all cell types. We engineered the upload of huge amounts of protein antigens upon fusion with an anchoring protein (i.e., HIV-1 Nefmut), which is an inactive protein incorporating in exosomes at high levels also when fused with foreign proteins. We compared the immunogenicity of engineered exosomes uploading human papillomavirus (HPV)-E7 with that of lentiviral virus-like particles (VLPs) incorporating equivalent amounts of the same antigen. These exosomes, whose limiting membrane was decorated with VSV-G, i.e., an envelope protein inducing pH-dependent endosomal fusion, proved to be as immunogenic as the cognate VLPs. It is noteworthy that the immunogenicity of the engineered exosomes remained unaltered in the absence of VSV-G. Most important, we provide evidence that the inoculation in mouse of exosomes uploading HPV-E7 induces production of anti-HPV E7 CTLs, blocks the growth of syngeneic tumor cells inoculated after immunization, and controls the development of tumor cells inoculated before the exosome challenge. These results represent the proof-of-concept about both feasibility and efficacy of the Nefmut-based exosome platform for the induction of CD8+ T cell immunity.

Show MeSH
Related in: MedlinePlus