Insights in AAV-mediated antigen-specific immunity and a strategy for AAV vaccine dose reduction through AAV-extracellular vesicle association

Abstract

We previously showed therapeutic advantages of using a capsid-modified and encoded antigen-optimized AAV-based cancer vaccine to initiate strong antigen-specific immune responses and increase survival in a syngeneic mouse model of melanoma. In this study, we further explore AAV vaccine dose reduction and possible mechanisms of the immune response. Immunization with extracellular vesicle (EV)-associated AAV6-S663V encoded ovalbumin (OVA) or tyrosinase-related protein 1 (TRP-1) induced significantly higher levels of antigen-specific CD8⁺ T cells compared with standard AAV in mice. Importantly, a higher number of specific CD8⁺ T cells was achieved with EV-AAV several logs lower than optAAV-based doses. EV-optAAV-OVA was used in a dose 100 times lower, and EV-optTRP-1 10 times lower than optOVA and optTRP-1 correspondingly. Our data suggest that significant dose reduction for optimized AAV-based vaccines is possible without sacrificing efficiency. In addition, we studied the role of conventional type 1 dendritic cells (cDC1) in optimized AAV-based immunization using a C57BL/6-Irf8em1Kmm (Irf8 ⁺ 32^-/-) mouse model lacking cDC1. Interestingly, we found that cDC1 are not essential for conveying effector T cell responses to AAV-encoded tumor antigens.

Keywords: adeno-associated virus; antigen-specific T cell; cancer vaccine; dendritic cell; extracellular vesicles; protective immune response.

Graphical abstract

Figure 1

AAV expression cassette and EV-AAV characterization (A) Schematic outline of AAV expression cassette used in the study. (B) An example of particle size distribution measured by microfluidic resistive pulse sensing over a range of particle diameters from 50 nm to 1 μm. National Institute of Standards and Technology 150-nm-diameter standard beads were used for calibration. The model fit of the measured distribution shows scaling behavior (mode diameter ∼40 nm; scaling coefficient = 0.275) and gives a total particle concentration of 2.88 × 10¹² particles/mL. Inset: representative TEM image of an EV-AAV6 particle (diameter ∼100 nm). (C) Particle counts obtained by SP-IRIS tetraspanin phenotyping using CD63, CD81, and CD9 capture. Fluorescence images of EVs co-expressing CD63, CD81, and CD9 captured on CD63 (top), CD81 (middle), and CD9 (bottom) spots. (C and D) Western blot analysis showing bands for VP1, 2, and 3 of the AAV6 capsid and for syntenin-1 and CD9, characteristic biomarkers for the exosome subpopulation of EVs. Equal titer of EVs were loaded.

Figure 2

EV-opt-OVA generates protective immune response at lower doses than opt-OVA (A) Schematic representation of experiment timeline. (B) Representative flow analysis OVA-specific CD8⁺ T cells on mice PBMCs and (C) quantification of relative to the total number of CD8⁺ T cells on n = 4 mice. (D) IFN-γ release ELISPOT assay on spleens restimulated with OVA peptide and (E). Relative quantification and statistical analysis of IFN-γ positive spots per 10⁶ cells. (F) Schematic representation of experiment timeline. (G) Representative images of mice lungs were with B16F10 cells metastatic nodule quantification. Representative and (H) quantification based on n = 4 animals. ∗p = 0.01, ∗∗p = 0.001, ∗∗∗∗p < 0.0001.

Figure 3

EV-opt-TRP-1 stimulates a protective immune response at lower doses compared with opt-TRP-1 (A) Schematic representation of experimental timeline. (B) Representative images of IFN-γ ELISPOT assay wells conducted on TRP-1 peptide restimulated splenocytes. (C) Quantification analysis of IFN-γ positive spots on n = 4 mice. (D) Timeline of B16F10 melanoma metastatic model experiment. (E) Representative images of C57BL6 mice lungs with B16B10 cell nodules. (F) Quantification of lungs nodules on n = 4 mice. ∗p = 0.01, ∗∗p < 0.001, ∗∗∗p < 0.005, ∗∗∗∗p < 0.0001.

Figure 4

Analysis of cDC1 and cDC2 populations in wild-type C57BL/6 and Irf8⁺32^−/− (A) Schematic representation of experimental timeline. (B and C) Flow cytometry analysis of lymph nodes isolated from C57BL/6 mice injected with opt-OVA. Subpopulation of dendritic cells identified as cDC1 (CD11c⁺/MHC-II⁺/PDCA⁻/XCR-1⁺/CD11b⁻) and cDC2 (CD11c⁺/MHC-II⁺/PDCA⁻/XCR-1^–/CD11b⁺). Activation of cDC1 and cDC2 define as upregulation of we used CD40⁺ and CCR7⁺ markers. (D) Comparison analysis of cDC1 and cDC2 subpopulations in Irf8⁺32^−/− vs. C57BL/6 and Irf8⁺32^+/− mice. cDC1 (CD11c⁺/MHC-II^+high/XCR-1⁺/CD11b⁻) and cDC2 (CD11c⁺/MHC-II^+high/XCR-1^–/CD11b⁺). (E) Quantitative analysis of cDC1 subpopulation in Irf8⁺32^−/− vs. C57BL/6 and Irf8⁺32^+/− on n = 3 mice. ∗p = 0.01, ∗∗p = 0.001.

Figure 5

Irf8+32^−/− mice vaccinated with opt-TRP-1 develop specific CD8+ T cells against TRP-1 antigen with protective capabilities against metastatic spread in lungs (A) Schematic representation of experimental timeline and vector design. (B) Representative flow cytometry analysis TRP-1-specific CD8⁺ T cells extracted from opt-TRP-1-vaccinated Irf8⁺32^−/− and C57BL/6 mice and (C) quantification of n = 3 mice (Student’s t test). (D) Representative image of IFN-γ ELISPOT analysis on splenocytes extracted from opt-TRP-1-vaccinated Irf8⁺32^−/− and (E) C57BL/6 mice and quantification of n = 3 (multivariant ANOVA). (F) Schematic representation of experiment timeline. (G) Representative images of mice lungs with B16F10 cells metastatic nodules and (H) quantification of n = 3 mice (multivariant ANOVA). ∗p = 0.01, ∗∗p = 0.001.