Limits...
Critical Role of Autophagy in the Processing of Adenovirus Capsid-Incorporated Cancer-Specific Antigens.

Klein SR, Jiang H, Hossain MB, Fan X, Gumin J, Dong A, Alonso MM, Gomez-Manzano C, Fueyo J - PLoS ONE (2016)

Bottom Line: We found that viral antigens were processed by JNK-mediated autophagy, and that autophagy was required for their presentation.Consistent with these results, splenocytes isolated from virus-immunized mice were activated by infected cells in an MHC II-dependent manner.However, inhibition of autophagy drastically decreased the capability of the specific antibody to detect the cancer-related epitope in infected cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuro-Oncology, Brain Tumor Center, The University of Texas MD Anderson Cancer Center, Houston, Texas, United States of America.

ABSTRACT
Adenoviruses are highly immunogenic and are being examined as potential vectors for immunotherapy. Infection by oncolytic adenovirus is followed by massive autophagy in cancer cells. Here, we hypothesize that autophagy regulates the processing of adenoviral proteins for antigen presentation. To test this hypothesis, we first examined the presentation of viral antigens by infected cells using an antibody cocktail of viral capsid proteins. We found that viral antigens were processed by JNK-mediated autophagy, and that autophagy was required for their presentation. Consistent with these results, splenocytes isolated from virus-immunized mice were activated by infected cells in an MHC II-dependent manner. We then hypothesize that this mechanism can be utilized to generate an efficient cancer vaccine. To this end, we constructed an oncolytic virus encompassing an EGFRvIII cancer-specific epitope in the adenoviral fiber. Infection of cancer cells with this fiber-modified adenovirus resulted in recognition of infected cancer cells by a specific anti-EGFRvIII antibody. However, inhibition of autophagy drastically decreased the capability of the specific antibody to detect the cancer-related epitope in infected cells. Our data suggest that combination of adenoviruses with autophagy inducers may enhance the processing and presentation of cancer-specific antigens incorporated into capsid proteins.

No MeSH data available.


Related in: MedlinePlus

Infection with Δ24FvIII adenovirus induces EGFRvIII epitope expression and presentation.(a) Schematic representation of the generation of the vIII chimeric fiber. PCR-based mutagenesis was used to insert the sequence of the LEEKKGNYVVT epitope in the hypervariable region of the HI loop. A unique EcoRV restriction site was incorporated to allow the insertion of the ectopic sequence between glycine-543 and aspartic acid-544. (b) Expression of the chimeric fiber encompassing the LEEKKGNYVVT peptide was assessed in A549 cells infected with Δ24FvIII (40 MOI). Protein lysates were subjected to Western blot analysis 72 h after infection using both anti-fiber and L8A4 anti-LEEKKGNYVVT antibodies. (c) Live wild-type Atg5 and Atg5-/- MEFs were infected with Δ24FvIII at an MOI 150 for 48 h and then stained with L8A4, followed by FITC-conjugated secondary antibodies for flow cytometry analyses. Ethidium homodimer-1 staining was used to exclude dead cells. Percentages of FITC-positive live cells are indicated at the top right corner of each graph. The decrease in the number of positive cells in Δ24FvIII-infected Atg5-/- compared with Δ24FvIII-infected wild-type Atg5 cells was statistically significant. Data are shown as the mean ± SD of three experiments. *** P < 0.001 (unpaired, two-tailed Student t-test). (d) HeLa cells were infected with Δ24FvIII at an MOI of 40. IFN-γ (300 units/mL) or/and bafilomycin A1 (BA1; 100 nM) were added to the media 6 h or 24 h after infection, respectively. Live cells were stained with L8A4 48 h after infection and then incubated with FITC-conjugated secondary antibodies to visualize positive cells with a flow cytometer. Ethidium homodimer-1 staining was used to exclude dead cells. Graph represents mean values of three experiments ± SD. * P < 0.05; ** P < 0.01 (unpaired, two-tailed Student t-test).
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4836716&req=5

pone.0153814.g004: Infection with Δ24FvIII adenovirus induces EGFRvIII epitope expression and presentation.(a) Schematic representation of the generation of the vIII chimeric fiber. PCR-based mutagenesis was used to insert the sequence of the LEEKKGNYVVT epitope in the hypervariable region of the HI loop. A unique EcoRV restriction site was incorporated to allow the insertion of the ectopic sequence between glycine-543 and aspartic acid-544. (b) Expression of the chimeric fiber encompassing the LEEKKGNYVVT peptide was assessed in A549 cells infected with Δ24FvIII (40 MOI). Protein lysates were subjected to Western blot analysis 72 h after infection using both anti-fiber and L8A4 anti-LEEKKGNYVVT antibodies. (c) Live wild-type Atg5 and Atg5-/- MEFs were infected with Δ24FvIII at an MOI 150 for 48 h and then stained with L8A4, followed by FITC-conjugated secondary antibodies for flow cytometry analyses. Ethidium homodimer-1 staining was used to exclude dead cells. Percentages of FITC-positive live cells are indicated at the top right corner of each graph. The decrease in the number of positive cells in Δ24FvIII-infected Atg5-/- compared with Δ24FvIII-infected wild-type Atg5 cells was statistically significant. Data are shown as the mean ± SD of three experiments. *** P < 0.001 (unpaired, two-tailed Student t-test). (d) HeLa cells were infected with Δ24FvIII at an MOI of 40. IFN-γ (300 units/mL) or/and bafilomycin A1 (BA1; 100 nM) were added to the media 6 h or 24 h after infection, respectively. Live cells were stained with L8A4 48 h after infection and then incubated with FITC-conjugated secondary antibodies to visualize positive cells with a flow cytometer. Ethidium homodimer-1 staining was used to exclude dead cells. Graph represents mean values of three experiments ± SD. * P < 0.05; ** P < 0.01 (unpaired, two-tailed Student t-test).

Mentions: Modification of adenoviral capsids by insertion of antigens sequences is a promising technology for the development of vaccines and anti-cancer vaccines,[31] and therefore we sought to determine whether autophagy was the main mechanism for the processing of ectopic cancer-specific epitopes encoded by adenoviral fibers. We then generated an experimental model that would allow us to determine whether a specific sequence in the adenoviral fiber could be detected in adenovirus-infected cells. To this end, we designed and constructed a chimeric adenovirus fiber encompassing the sequence of a well-characterized human epitope (Fig 4A). The resulting construct was termed Δ24FvIII, and used the Δ24 tumor-selective backbone adenovirus[32] and encoded the epitope LEEKKGNYVVT inserted into the HI-loop of the adenovirus fiber sequence. This peptide is derived from the junctional sequence of the truncated protein generated in the mutant variant III of the mutant EGFR and has been previously shown to be highly immunogenic[21] (Fig 4A).


Critical Role of Autophagy in the Processing of Adenovirus Capsid-Incorporated Cancer-Specific Antigens.

Klein SR, Jiang H, Hossain MB, Fan X, Gumin J, Dong A, Alonso MM, Gomez-Manzano C, Fueyo J - PLoS ONE (2016)

Infection with Δ24FvIII adenovirus induces EGFRvIII epitope expression and presentation.(a) Schematic representation of the generation of the vIII chimeric fiber. PCR-based mutagenesis was used to insert the sequence of the LEEKKGNYVVT epitope in the hypervariable region of the HI loop. A unique EcoRV restriction site was incorporated to allow the insertion of the ectopic sequence between glycine-543 and aspartic acid-544. (b) Expression of the chimeric fiber encompassing the LEEKKGNYVVT peptide was assessed in A549 cells infected with Δ24FvIII (40 MOI). Protein lysates were subjected to Western blot analysis 72 h after infection using both anti-fiber and L8A4 anti-LEEKKGNYVVT antibodies. (c) Live wild-type Atg5 and Atg5-/- MEFs were infected with Δ24FvIII at an MOI 150 for 48 h and then stained with L8A4, followed by FITC-conjugated secondary antibodies for flow cytometry analyses. Ethidium homodimer-1 staining was used to exclude dead cells. Percentages of FITC-positive live cells are indicated at the top right corner of each graph. The decrease in the number of positive cells in Δ24FvIII-infected Atg5-/- compared with Δ24FvIII-infected wild-type Atg5 cells was statistically significant. Data are shown as the mean ± SD of three experiments. *** P < 0.001 (unpaired, two-tailed Student t-test). (d) HeLa cells were infected with Δ24FvIII at an MOI of 40. IFN-γ (300 units/mL) or/and bafilomycin A1 (BA1; 100 nM) were added to the media 6 h or 24 h after infection, respectively. Live cells were stained with L8A4 48 h after infection and then incubated with FITC-conjugated secondary antibodies to visualize positive cells with a flow cytometer. Ethidium homodimer-1 staining was used to exclude dead cells. Graph represents mean values of three experiments ± SD. * P < 0.05; ** P < 0.01 (unpaired, two-tailed Student t-test).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4836716&req=5

pone.0153814.g004: Infection with Δ24FvIII adenovirus induces EGFRvIII epitope expression and presentation.(a) Schematic representation of the generation of the vIII chimeric fiber. PCR-based mutagenesis was used to insert the sequence of the LEEKKGNYVVT epitope in the hypervariable region of the HI loop. A unique EcoRV restriction site was incorporated to allow the insertion of the ectopic sequence between glycine-543 and aspartic acid-544. (b) Expression of the chimeric fiber encompassing the LEEKKGNYVVT peptide was assessed in A549 cells infected with Δ24FvIII (40 MOI). Protein lysates were subjected to Western blot analysis 72 h after infection using both anti-fiber and L8A4 anti-LEEKKGNYVVT antibodies. (c) Live wild-type Atg5 and Atg5-/- MEFs were infected with Δ24FvIII at an MOI 150 for 48 h and then stained with L8A4, followed by FITC-conjugated secondary antibodies for flow cytometry analyses. Ethidium homodimer-1 staining was used to exclude dead cells. Percentages of FITC-positive live cells are indicated at the top right corner of each graph. The decrease in the number of positive cells in Δ24FvIII-infected Atg5-/- compared with Δ24FvIII-infected wild-type Atg5 cells was statistically significant. Data are shown as the mean ± SD of three experiments. *** P < 0.001 (unpaired, two-tailed Student t-test). (d) HeLa cells were infected with Δ24FvIII at an MOI of 40. IFN-γ (300 units/mL) or/and bafilomycin A1 (BA1; 100 nM) were added to the media 6 h or 24 h after infection, respectively. Live cells were stained with L8A4 48 h after infection and then incubated with FITC-conjugated secondary antibodies to visualize positive cells with a flow cytometer. Ethidium homodimer-1 staining was used to exclude dead cells. Graph represents mean values of three experiments ± SD. * P < 0.05; ** P < 0.01 (unpaired, two-tailed Student t-test).
Mentions: Modification of adenoviral capsids by insertion of antigens sequences is a promising technology for the development of vaccines and anti-cancer vaccines,[31] and therefore we sought to determine whether autophagy was the main mechanism for the processing of ectopic cancer-specific epitopes encoded by adenoviral fibers. We then generated an experimental model that would allow us to determine whether a specific sequence in the adenoviral fiber could be detected in adenovirus-infected cells. To this end, we designed and constructed a chimeric adenovirus fiber encompassing the sequence of a well-characterized human epitope (Fig 4A). The resulting construct was termed Δ24FvIII, and used the Δ24 tumor-selective backbone adenovirus[32] and encoded the epitope LEEKKGNYVVT inserted into the HI-loop of the adenovirus fiber sequence. This peptide is derived from the junctional sequence of the truncated protein generated in the mutant variant III of the mutant EGFR and has been previously shown to be highly immunogenic[21] (Fig 4A).

Bottom Line: We found that viral antigens were processed by JNK-mediated autophagy, and that autophagy was required for their presentation.Consistent with these results, splenocytes isolated from virus-immunized mice were activated by infected cells in an MHC II-dependent manner.However, inhibition of autophagy drastically decreased the capability of the specific antibody to detect the cancer-related epitope in infected cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuro-Oncology, Brain Tumor Center, The University of Texas MD Anderson Cancer Center, Houston, Texas, United States of America.

ABSTRACT
Adenoviruses are highly immunogenic and are being examined as potential vectors for immunotherapy. Infection by oncolytic adenovirus is followed by massive autophagy in cancer cells. Here, we hypothesize that autophagy regulates the processing of adenoviral proteins for antigen presentation. To test this hypothesis, we first examined the presentation of viral antigens by infected cells using an antibody cocktail of viral capsid proteins. We found that viral antigens were processed by JNK-mediated autophagy, and that autophagy was required for their presentation. Consistent with these results, splenocytes isolated from virus-immunized mice were activated by infected cells in an MHC II-dependent manner. We then hypothesize that this mechanism can be utilized to generate an efficient cancer vaccine. To this end, we constructed an oncolytic virus encompassing an EGFRvIII cancer-specific epitope in the adenoviral fiber. Infection of cancer cells with this fiber-modified adenovirus resulted in recognition of infected cancer cells by a specific anti-EGFRvIII antibody. However, inhibition of autophagy drastically decreased the capability of the specific antibody to detect the cancer-related epitope in infected cells. Our data suggest that combination of adenoviruses with autophagy inducers may enhance the processing and presentation of cancer-specific antigens incorporated into capsid proteins.

No MeSH data available.


Related in: MedlinePlus