1. Volume 25, Issue 4, Pages 976-988 (April 2017)
A Novel DNA Vaccine Platform Enhances Neo-antigen-like T Cell Responses against WT1 to Break Tolerance and Induce Anti-tumor Immunity 
Jewell N. Walters, Bernadette Ferraro, Elizabeth K. Duperret, Kimberly A. Kraynyak, Jaemi Chu, Ashley Saint-Fleur, Jian Yan, Hy Levitsky, Amir S. Khan, Niranjan Y. Sardesai, David B. Weiner 
Molecular Therapy 
Volume 25, Issue 4, Pages (April 2017)
DOI: /j.ymthe
Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

2. Figure 1
In Vitro Expression of Mouse WT1 and Synthetic WT1 DNA Vaccines
(A) Schematic outline of pMuWT1-opt, pMuWT1-nonopt, and pSynConWT1-S. (B) Detection of pMuWT1-opt, pMuWT1-nonopt, and pSynConWT1-S expression by immunoblotting. (C) Immunofluorescence assay of pMuWT1-opt, pMuWT1-nonopt, and pSynConWT1-S. Transfected RD cells expressing WT1 protein showed typical phalloidin fluorescence using a commercial WT1 polyclonal antibody.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

3. Figure 2
Characterization of Consensus WT1-Specific IFN-γ Responses and Immunodominant Epitopes for pSynConWT1-S Vaccine
(A) DNA vaccine immunization schedule showing the dosage of vaccine. C57BL/6 (B6) mice (n = 5 per group) were immunized at weeks 0, 2, 4, and 6 with pSynConWT1-S, pMuWT1-opt, and pMuWT1-nonopt via i.m. injection or EP. (B) Frequency of WT1-specific IFN-γ spot-forming units (SFUs) per million splenocytes isolated from vaccinated mice, determined by IFN-γ ELISpot assay using consensus peptides. (C) Matrix mapping to determine the WT1 consensus-specific immunodominant epitopes, comparing naive and pSynConWT1-S vaccinated mice. (D) List of immunodominant epitopes identified in the matrix mapping in (C), and comparison to the corresponding native mouse sequence. Error bars represent the average ± SEM.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

4. Figure 3
Characterization of Mouse WT1-Specific IFN-γ Responses and Immunodominant Epitopes for pSynConWT1-S Vaccine
(A) DNA vaccine immunization schedule showing the dosage of vaccine. C57BL/6 (B6) mice (n = 5 per group) were immunized at weeks 0, 2, 4, and 6 with pSynConWT1-S, pMuWT1-opt, and pMuWT1-nonopt via i.m. injection or EP. (B) Frequency of WT1-specific IFN-γ spot-forming units (SFUs) per million splenocytes isolated from vaccinated mice, determined by IFN-γ ELISpot assay using mouse peptides. (C) Matrix mapping to determine the WT1 mouse-specific immunodominant epitopes, comparing naive and pSynConWT1-S vaccinated mice. (D) List of immunodominant epitopes identified in the matrix mapping in (C), and comparison to the corresponding SynCon sequence. Error bars represent the average ± SEM.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

5. Figure 4
CD4 and CD8 Cytokine Responses to Consensus and Mouse WT1 Peptides for pSynConWT1-S Vaccine
(A and B) Expression of CD107a, IFN-γ, and TNF-α was assessed by intracellular staining of splenocytes stimulated with consensus WT1 peptides (A) or native mouse peptides (B) for 5 hr. Background-subtracted percentages of WT1-specific CD4+ or CD8+ T cells producing CD107a, IFN-γ, and TNF-α were calculated. Error bars represent the average ± SEM.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

6. Figure 5 pSynConWT1-S Induces Robust Antibody Responses
(A) WT1-specific IgG endpoint binding titers, performed using ELISA. (B) Immunoblot assay of HEK293T cells with sera from vaccinated mice. (C) Immunohistochemical analysis of WT1 expression in human renal, lung, skin, ovarian, and cervical cancer using naive sera, a commercial WT1 antibody, or pSynConWT1-S mouse sera.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

7. Figure 6 Anti-tumor Immunity Elicited by pSynConWT1-S
(A) Mice were challenged with 106 mWT1-C1498 tumor cells injected subcutaneously and were vaccinated weekly starting 3 days post-tumor implant. Tumor measurements are reported in terms of tumor volume only for surviving mice until day 28. (B) Survival data from the tumor therapeutic challenge in (A). Vaccination with pSynConWT1-S extended survival in tumor-bearing mice. (C) Representative image of tumor size in naive or pSynConWT1-S vaccinated groups at day 14 post-mWT1-C1498 implantation. Error bars represent the average ± SEM.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

8. Figure 7
Vaccination with pSynConWT1-L-Induced WT1-Specific Immune Responses in Rhesus Macaques
(A) DNA vaccine immunization schedule showing the number of vaccinations and bleeds. Rhesus macaques (n = 5 per group) were immunized at weeks 0, 4, 8, and 12 with 2 mg of pSynConWT1-L intramuscularly, followed by EP. (B) PBMCs were isolated and stimulated with native rhesus WT1 peptides for 24 hr. Shown is the frequency of WT1-specific IFN-γ secreting cells per million PBMCs, determined by IFN-γ ELISpot assay at week 14.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions