A Supramolecular Vaccine Platform Based on α‑Helical Peptide Nanofibers

A supramolecular peptide vaccine system was designed in which epitope-bearing peptides self-assemble into elongated nanofibers composed almost entirely of α-helical structure. The nanofibers were readily internalized by antigen presenting cells and produced robust antibody, CD4+ T-cell, and CD8+ T-cell responses without supplemental adjuvants in mice. Epitopes studied included a cancer B-cell epitope from the epidermal growth factor receptor class III variant (EGFRvIII), the universal CD4+ T-cell epitope PADRE, and the model CD8+ T-cell epitope SIINFEKL, each of which could be incorporated into supramolecular multiepitope nanofibers in a modular fashion

Effect of Different Enhancement Thresholds.

<p>A) Axial T1-weighted post-contrast image after volumetric analysis has been performed which shows in green the detected enhancing tumor volume using a 25% threshold level. B) Detected enhancing tumor volume using a 40% threshold level. C) While increasing the threshold decreases the calculated tumor volume, the volumes across different threshold levels are highly correlated.</p

Detection of Enhancing Tumor Volume Despite Resection Cavity Collapse.

<p>A) T1-weighted post-contrast axial image showing a resection cavity with rim enhancement. RECIST measurement would be A and Macdonald measurement would be “A * B”. B) T1-weighted post-contrast axial image showing the same patient 3 months postoperatively who had collapse of his resection cavity. RECIST measurement would be “a” and Macdonald measurement would be “a * b”, both of which would be smaller than the measurements from the initial scan above, but this change would be describing only the resection cavity configuration and not the underlying tumor burden.</p

Detection of Enhancement that is Obscured by Blood Products.

<p>A) Uncontrasted T1-weighted axial image showing resection cavity blood products (bright on T1). B) T1-weighted post-contrast axial image showing the difficulty in determining residual enhancing tumor. C) Our volumetric analysis is able to detect the obscured enhancing tumor tissue (shown in green). D) T1-weighted post-contrast axial image at 2.5 months later after the blood has resolved verifying the underlying enhancing tumor volume.</p

Traditional Non-Volumetric Measurements do not Adequately Describe Residual Enhancement in Surgical Resection Cavities.

<p>A) This schematic resection cavity has residual rim enhancement in gray. RECIST criteria measurement ‘A’ or ‘a’ or ‘b’ or Macdonald criteria measurement ‘A*B’ or ‘a*b’ would not adequately describe residual tumor volume and additional tumor growth around the rim or collapse of the resection cavity may be over- or under-interpreted. B) Differences in axial slice acquisition also impact measurements made by traditional criteria more than volumentric measurements. One scan could obtain axial slice ‘c’ with enhancing tumor measurement ‘x’ but a subsequent scan in the same patient could obtain axial slice ‘d’, causing an incorrect assessment of tumor response.</p

Automated Assessment of Enhancing Tumor Volume.

<p>A) T1-weighted post-contrast axial images are automatically fused with the pre-contrast sequences. B) The tumor region of interest (blue area) and nearby normal brain (purple area) are outlined roughly by hand. C) The enhancing nasal mucosa region is automatically detected with a built-in anatomic atlas (red area) and serves as a threshold for enhancement. D) Tissue that is present on the post-contrast images but not the pre-contrast that is above the enhancement threshold appears in yellow. This includes enhancing tissue such as vasculature, tumor, and superficial structures. Enhancing tumor volume is defined as the green area within the manually-defined blue tumor region of interest.</p

Effect of Inter-observer Differences in Definition of Tumor Volume.

<p>A) Axial T1-weighted post-contrast image showing a limited user-defined tumor region of interest. B) The same axial image now showing a large user-defined tumor region of interest that encompasses the meningeal enhancement. C) While including the meninges increases the enhancing volume, similar trends in changes of volume over time are seen.</p

Myeloablative TMZ enhances T-cell function yet IL-2 remains limiting.

<p>To compare the levels of IL-2 secretion by OT-I T-cells relative to IFNγ and TNFα levels in the context of NMA (A) and MA (B) TMZ regimens we performed CBA analysis. Splenocytes from mice receiving TMZ followed by OT-I transfer and vaccine, were harvested on day 7 after vaccination and cultured in the presence of 1 µM SIINFEKL chicken Ovalbumin epitope (cognate) or negative peptide control (non-cognate) for 72 hours in T-cell media alone. Supernatant was harvested and samples were subjected to to CBA analysis as specified by the manufacturer for murine IFNγ, TNFα and IL-2. Representative experiments are shown; experiments were performed in at least duplicate. Statistical analysis was performed using unpaired, two-tailed student’s t-tests. Statistical significance was determined at a *p value ≤0.05.</p

Myeloablative TMZ results in protracted depletion of CD4⁺Foxp3⁺

<p><b>T cells.</b> To evaluate, the impact of different TMZ regimens on CD4⁺Foxp3⁺ T_Regs C57BL/6 mice (n = 5) were treated with vehicle, NMA or MA TMZ regimens as stated before in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059082#pone-0059082-g001" target="_blank">Figure 1</a>. Mice were bled retro-orbitally on days 2, 7, 14 and 28 after termination of TMZ administration and submitted to flow cytometry analysis. Immediate effects of TMZ on CD4⁺Foxp3⁺ T_Regs (A), on day 2 and the recovery kinetics over time are shown (B). Absolute numbers per microliter of blood were calculated using Flowcount^® beads from Beckman Coulter^©. Representative experiments are shown; all experiments were performed in at least duplicate. Statistical analysis was performed using Mann-Whitney test. Statistical significance was determined at a *p value ≤0.05.</p

Myeloablative TMZ is required for efficacious immunotherapy against intracerebral tumor.

<p>Antitumor efficacy of a vaccination in the context of lymphopenia induced by the different TMZ regimens and the impact of these TMZ regimens as a single agent were investigated in wild-type C57BL/6 mice (n = 7) inoculated with an intracerebral injection of 5×10³ B16F10-OVA melanoma cells. These mice received vehicle, NMA or MA TMZ treatment followed by vaccine (A) or no vaccine (B). On day 3 after tumor implantation, dose-intensifying TMZ regimens were initiated and administered for 5 consecutive days intraperitoneally. Vaccinated mice received an intravenous adoptive transfer of 1×10⁷ splenocytes consisting of 1∶1 ratio of OT-I and C57BL/6 splenocytes on the day after termination of TMZ treatment. Mice underwent subsequent intradermal vaccination with 100 µg of SIINFEKL chicken ovalbumin epitope emulsified in CFA. (B) In order to assess the effects of TMZ alone, mice were also treated with TMZ in the absence of vaccine. Mice were monitored for morbidity end-points approved by the Duke University IACUC and sacrificed when end points were met. The experiment was performed three times. Survival analysis was performed using the Gehan-Breslow-Wilcoxon Test. Statistical significance was determined at a *p value ≤0.05.</p