Preclinical development of highly effective and safe DNA vaccines directed against HPV 16 E6 and E7

Abstract

To allow vaccination irrespective of HLA type, DNA vaccines encoding full-length antigens are required. However, here, we demonstrate that the immunogenicity of DNA vaccines encoding the full-length human papillomavirus (HPV) type 16 E7 and E6 proteins is highly reduced compared to vaccines encoding only the immunodominant epitope. Furthermore, the low remaining immunogenicity is essentially lost for both E7 and E6 when a nononcogenic ''gene-shuffled" variant is utilized. To address these issues, we tested whether alterations in transgene design can restore the immunogenicity of full-length and geneshuffled DNA vaccines. Remarkably, genetic fusion of E7 with tetanus toxin fragment C (TTFC) resulted in a dramatic increase in immunogenicity both for the full-length and the gene-shuffled version of E7. Moreover, the TTFC fusion vaccines were more immunogenic than a vaccine encoding a fusion of E7 and mycobacterial heat shock protein-70, which has recently been tested in a clinical trial. Interestingly, vaccination with these TTFC fusion vaccines also resulted in extremely persistent T-cell responses. The E7-specific CD8 1 T cells induced by TTFC fusion vaccines were functional in terms of IFN-c production,formation of immunological memory, in vivo cytolytic activity and tumor eradication. Finally, we show that genetic fusion with TTFC also improves the immunogenicity of a gene-shuffled E6 DNA vaccine. These data demonstrate that genetic fusion with tetanus toxin fragment C can dramatically improve the immunogenicity of full-length and gene-shuffled DNA vaccines. The DNA fusion vaccines developed here will be evaluated for the treatment of HPV-positive carcinomas in future studies.Persistent infection with ''high-risk'' human papillomavirus (HPV) genotypes is strongly associated with the development of anogenital cancers. 1,2 Of the ''high-risk'' genotypes, HPV 16 alone is known to be responsible for about half of the cervical cancer cases worldwide.3 Because persistent expression of the oncogenic HPV proteins E6 and E7 is required for carcinogenesis, these viral antigens are ideal targets for immunotherapeutic interventions. As E6 and E7 are solely expressed intracellularly, such therapeutic interventions should induce cellular immune responses to control existing HPV-induced lesions. 3,4 DNA vaccination forms an attractive approach for the induction of cellular immune responses as the DNA-encoded antigens are by definition produced intracellularly. Furthermore, DNA vaccines are safe, easy to produce, stable and do not suffer from the drawback of preexisting immunity or induction of antivector immunity. 5,6 In murine models, numerous DNA vaccines directed against either HPV 16 E6 or E7 have been tested with promising results.7-13 However, to date, the clinical translation of these approaches has met little success.14,15 Recently, we developed a novel DNA vaccination strategy named DNA tattoo vaccination that can potentially overcome this translational block. This strategy was shown to lead to the rapid induction of...

Tumor endothelial marker 1–specific DNA vaccination targets tumor vasculature

“…In previous mouse studies, DNA vaccination with the tumorassociated antigens AH1 and E7 induced CD8 + cell-dependent regression of established tumors that expressed those antigens (33,74). In the studies presented here, fusion of the TEM1 antigen to the TT adjuvant increased the immunogenicity of the target antigen and the efficacy of tumor control.…”

An Enhanced Heterologous Virus-Like Particle for Human Papillomavirus Type 16 Tumour Immunotherapy

“…Cells were resuspended in FACS buffer (PBS, pH 7.2 containing 1% BSA and 0.1% sodium azide) and stained with Alexa 488-conjugated anti-CD8α mAb (BD Pharmingen) and allophycocyanin-conjugated H-2K b E6 48–57 (EVYDFAFRDL) tetramer [32](NIH Tetramer Facility, Atlanta, GA, USA) for 30 min at 4°C. Cells were washed and resuspended in FACS buffer and samples were acquired using a FACSaria (Becton Dickinson, CA, USA).…”

Potent therapeutic efficacy of an alphavirus replicon DNA vaccine expressing human papilloma virus E6 and E7 antigens

“…23 The low immunogenicity of pVAX-E7SH is rescued in these studies with the addition of helper epitopes and a retention signal with insertion of SigHelp-KDEL. 33 In a previous study using VREP particles, the insertion of SigHelp-KDEL resulted in a higher percentage of E7-specific effector T cells at both early and late time points after immunization. 24 Significant differences in the effector memory T cell phenotype was only significantly higher early after immunization.…”

“…However, as seen by others pVAX was also not immunogenic at a dose of 10 µg or less, regardless of the administration method. 23,33 In those studies there were also no detectable responses observed with repeated administrations of 20 µg of pVAX, with anti-tumor control only demonstrated within the context of an optimized DNA vector. 23 …”