Fig 1.
iiAbs in cured mice cross-react with heterologous mouse tumor cell lines.
The indicated (A) syngeneic and (B) allogeneic tumor cells were incubated with 1% serum of AIPV-treated mice that had previous rejected B16F10 or naive mice. The cells were washed, stained with Alexa-Fluor-647-conjugated anti-mouse IgG secondary Ab, and analyzed by flow cytometry. (C) Age-matched mice (n = 5) and mice that were cured of B16F10 and rejected secondary challenge (n = 5) were inoculated with 105 MC38 cells and left untreated. (D) B16F10 (B) and MC38 (M) membrane proteins were immunoblotted with AIPV serum (CM# for each cured mouse), naive serum (N), or TA99 and detected with IRDye-800CW-conjugated anti-mouse IgG2b and IgG2c Abs. *P < 0.05.
Fig 2.
Modified 2D immunoblotting allows for the identification of tumor antigens.
(A) The schematic describes the workflow to partially transfer a 2D gel and overlay silver-stained gel on chromogenic immunoblot to identify the spots of interest. (B) B16F10 membrane proteins and biotinylated BSA, soybean trypsin inhibitor, and equine hemoglobin were immunoblotted with CM7 serum and detected with IRDye-800CW-conjugated anti-mouse IgG2b and IgG2c Abs (green) and Alexa-Fluor-647-conjugated streptavidin (red). (C) Identical sample in (B) was partially transferred and chromogenically detected with biotinylated anti-mouse IgG2b and IgG2c Abs and HRP-conjugated streptavidin. (D) The gel remaining after the partial transfer was silver-stained and overlaid on the chromogenic immunoblot. Red boxes indicate the spots of interest.
Fig 3.
eMLV gp70 is the dominant cell-surface antigen.
(A) eMLV gp70 and gag were recombinantly expressed and immunoblotted with CM6 and CM7 sera and detected as in Fig 1D. (B) B16F10 cells were incubated with 1% serum or 10 μg/ml TA99 in the presence of 50 μg/ml gp70 or gag and analyzed as in Fig 1A. (C) B16F10 or B16F10 envKO cells were incubated with 1% serum or 10 μg/ml TA99 or 1E4 and analyzed as in Fig 1A.
Fig 4.
Anti-env Ab is cross-reactive and protective against intravenous B16F10 challenge.
(A) Indicated cells were incubated with 10 μg/ml 1E4 and analyzed as in Fig 1A. (B) TA99 or 1E4 was injected i.p. into naive mice 6 h before intravenous challenge of 2.5 × 105 B16F10 cells. Shown are the images of lungs isolated 17 d after the challenge. (C) Five hundred micrograms of Alexa-Fluor-750 labeled 1E4 was intravenously injected into a mouse bearing a B16F10 tumor. Shown is a representative cryo-fluorescence tomography image 48 h after the injection.
Fig 5.
Anti-env Ab response is protective against subcutaneous B16F10 challenge.
(A) Naive mice were inoculated with 105 B16F10 cells on day 0 and treated with 200 μg TA99 (n = 5), 1E4.2.1 (n = 5), or PBS (n = 5) on days -1, 2, 5, 8, 11. Arrows indicate treatment points. Shown are tumor area curves and overall survival. (B) Age-matched mice (n = 5) and naive mice vaccinated five times with 10 μg RBD and 5 μg saponin-MPLA nanoparticles (n = 5) were challenged with 105 B16F10 cells. Shown are tumor area curves and overall survival. *P < 0.05 and **P < 0.01.