PMC

Profiles of peptide release determined by the dialysis tube method comparing DPX-Survivac, w/o-Survivac, and AC-Survivac and evaluating DPX-Survivac over 1 month. Peptide antigens are retained in the DPX formulation when compared with a water-in-oil formulation and an aqueous control. Peptide antigens remain in the DPX formulation. a-g) Peptide release over 24 h, h) Peptide release over 1 month

Clearance of Antigen in DPX-R9F and w/o-R9F Detected by MRI. MR images displaying iron content of SPIO-R9F in either a), b) and c) DPX-R9F or d), e) and f) w/o-emulsion. Images were acquired one (a & d), four (b & e), or fourteen (c & f) days post-immunization. In DPX-R9F SPIO-antigen clears centrally from the vaccine site, from the middle of the site of injection (SOI) outwards, while the SPIO-R9F clears from the edges of the w/o-R9F depot. Red arrows indicate site of active clearance

Increased Local Inflammatory Response in Sap/Lip-R9F Vaccine. a) BSSFP MRI images (150um)3 isotropic voxels of representative mice (n = 2) from the sap/lip-R9F group. Red arrows demonstrate regions with edema as indicated by hyperintensity. Edema can be seen in a) and b) the SOI, c) the vaccine-draining right inguinal lymph node (RLN), and d) the right popliteal lymph node

ELISPOT data. Mice (C57BL/6, n = 10) were implanted on the left flank with C3 tumours and vaccinated 5 days later with either DPX-R9F, w/o-R9F, DPX (containing no antigen), or untreated. Vaccinations were given subcutaneously on the right flank. Mice (n = 5) were terminated 7 days (a) and 14 days (b) after immunization and IFN-γ ELISPOT performed using splenocytes. Naïve, untreated mice were also included in each experiment. Total lymph node cell counts were performed on the vaccine draining, right inguinal lymph nodes (c) and tumour draining left inguinal lymph nodes (d) at each time point

Graphs demonstrating volumetric changes in inguinal lymph nodes and tumours over the course of the study comparing DPX-R9F(n = 23) and w/o-R9F (n = 29) and Histological Evaluation of Site of Injection for DPX-R9F and w/o-R9F. a Tumour volume time course (mm³). b) Normalized % amount of SPIO-R9F remaining at the SOI. c % Right lymph node (RLN) volume increase over time (draining vaccine site). d Ratio of RLN volume over LLN volume. Data on graphs is mean ± SE. Significance was calculated using a 2-way ANOVA to compare overall differences across groups and time, and Holm-Sidak corrected t-tests to compare individual time points, * = p < 0.1, ** = p < 0.05. SOIs were extracted from 5 mice per group 14 days after immunization, sectioned and stained by H&E. Representative figures for 2 different mice are shown for DPX-R9F (e, g) and w/o-R9F (f, h)

Graphs demonstrating volumetric changes in inguinal lymph nodes and tumours over the course of the study comparing DPX-R9F (n = 5) to Squal w/o-R9F (n = 7) and Sap/Lip-R9F (n = 6) and Immune Response by ELISPOT. a Tumour volume time course (mm³). b Normalized % amount SPIO-R9F remaining at the SOI. c % Right lymph node (RLN) volume increase over time (draining vaccine site). d Ratio of RLN volume over LLN volume. Data on graphs is mean ± SE. Significance was calculated using a 2-way ANOVA to compare overall differences across groups and time, and Holm-Sidak corrected t-tests to compare group differences at individual time points, ** = p < 0.05. e Immune response by IFN-γ ELISPOT. HLA-A2 transgenic mice (n = 5) were vaccinated with a mixture of peptides derived from the survivin protein: SurA1.T, SurA2.M, SurA3.K, SurA24, SurB7 as well as a universal T helper epitope A16L. Peptides were formulated in DepoVax, w/o-emulsion with ISA 51, O/W with squalene, or aqueous liposomes. Eight days after immunization mice were euthanized and spleens removed for IFN-γ ELISPOT. Splenocytes (500,000 cells/ well) were stimulated in duplicate with media alone, an irrelevant peptide (HLA-A2 restricted peptide ALMEQQHYV), or SurA2.M peptide. Results shown as average per group +/− SEM. Statistics by one-way ANOVA comparing responses to SurA2.M stimulation, no statistical significance detected