Biochemical and proteomic characterization of retrovirus Gag based microparticles carrying melanoma antigens

Abstract

Extracellular vesicles are membraneous particles released by a variety of cells into the extracellular microenvironment. Retroviruses utilize the cellular vesiculation pathway for virus budding/assembly and the retrovirus Gag protein induces the spontaneous formation of microvesicles or virus-like particles (VLPs) when expressed in the mammalian cells. In this study, five different melanoma antigens, MAGEA4, MAGEA10, MART1, TRP1 and MCAM, were incorporated into the VLPs and their localization within the particles was determined. Our data show that the MAGEA4 and MAGEA10 proteins as well as MCAM are expressed on the surface of VLPs. The compartmentalization of exogenously expressed cancer antigens within the VLPs did not depend on the localization of the protein within the cell. Comparison of the protein content of VLPs by LC-MS/MS-based label-free quantitative proteomics showed that VLPs carrying different cancer antigens are very similar to each other, but differ to some extent from VLPs without recombinant antigen. We suggest that retrovirus Gag based virus-like particles carrying recombinant antigens have a potential to be used in cancer immunotherapy.

Figure 1. Generation and purification of VLPs.

(A) General workflow of generation of MLV Gag based VLPs. (B) Schematic representation of the purification protocol. (C) Western blot analysis of VLPs. Particles obtained with ultracentrifugation through 20% sucrose cushion were further centrifuged through stepwise sucrose density gradient (20%, 35%, 45%, 60%) at 120 000 g and 4 °C for 1.5 h in a Beckman SW55 rotor and divided into 10 fractions. The presence of VLPs in each fraction was analyzed by specific antibodies against melanoma antigens and the MLV Gag protein. (D) Physical characterization of VLPs ultracentrifuged through 20% sucrose cushion as assessed by DLS. The mean value of 4 × 10 measurements performed at 22 °C is shown. (E) Transmission electron micrograph of negatively stained MAGEA4 VLPs purified through density gradient centrifugation. Enlarged image of particle is shown on the upper-right corner of the picture.

Figure 2. Surface analysis of melanoma antigens incorporated into VLPs.

FACS analysis was performed with VLPs bound to aldehyde/sulfate latex beads as described in Materials and Methods section. The red line corresponds to VLPs carrying recombinant melanoma antigens, the blue line to VLPs without cancer antigen, and grey area shows the signal obtained with secondary Alexa488-labelled antibody. One representative experiment out of the three performed is shown. MFI = Mean fluorescence intensity.

Figure 3. Subcellular localization of melanoma antigens used in this study.

The COP5 cells transfected with plasmids encoding for the MLV Gag protein and respective cancer antigen were grown on cover slips, fixed and incubated with specific antibodies and Alexa568-conjugated secondary antibody as described in Methods section. Untransfected cells (mock control) incubated with specific antibodies are shown. DAPI was used to stain nuclei of the cells.

Figure 4. Cell surface expression of melanoma antigens transiently expressed in COP5 cells.

(A) FACS analysis was performed with immunostained living cells transfected with plasmids encoding for cancer melanoma antigens MART1, TRP1, MCAM, MAGEA4 and MAGEA10, and MLV Gag. The red line corresponds to cells expressing recombinant melanoma antigens, the blue line to cells without cancer antigen, and grey area shows the signal obtained with secondary Alexa488-labelled antibody. The percentage of gated cells is shown (blue for cells without cancer antigen and red for cells expressing recombinant melanoma antigens) on the image. The image of MLV Gag contains the data of five transfections obtained with electroporation with five different melanoma antigens. In all cases, one representative experiment out of the two performed is shown. MFI = Mean fluorescence intensity. (B) Western blot analysis of COP5 cells expressing melanoma antigens. Lane 1, mock control; lane 2, transfected cells.

Figure 5. Proteomic analysis of VLPs.

(A) The composition of MLV Gag induced VLPs. (B) Correlation of protein abundances (LFQ intensity) between VLPs carrying MAGEA4 antigen with VLPs without recombinant antigen (MLV Gag). Pearson correlation coefficient is shown on the blot. (C) Differently expressed proteins in MLV Gag VLPs and VLPs carrying melanoma antigens. (D) Comparison of LFQ intensities of VLPs carrying different cancer antigens. (E) Western blot analysis of VLPs using antibodies against MLV Gag and cellular proteins α-tubulin, Rps6, Pabp, histone H3 and Mcm6. The equal amount of total protein (100 ng for MLV Gag and 2 μg for others) was loaded on each lane.