Jeremy Juang

Comparison of the CD8+ T cell responses and antitumor effects generated by DNA vaccine administered through gene gun, biojector, and syringe

DNA vaccines have emerged as an attractive approach for antigen-specific cancer immunotherapy. We have previously linked Mycobacterium tuberculosis heat shock protein 70 (HSP70) to human papillomavirus type 16 (HPV-16) E7 in the context of a DNA vaccine. Vaccination with DNA encoding E7/HSP70 has generated a dramatic increase of E7-specific CD8+ T cell precursors and a strong antitumor effect against E7-expressing tumor (TC-1) in vaccinated mice. The success of our strategy has led to two phases I/II clinical trial proposals in patients with HPV-16 associated high-grade squamous intraepithelial lesion (HSIL) of the cervix and in patients with advanced HPV-associated head and neck squamous cell carcinoma (HNSCC). To translate our HPV DNA vaccines into the clinical domain, the efficacy of pNGVL4a-Sig/E7(detox)/HSP70 DNA vaccine and of various routes of administrations were assessed in mice. Our results indicated that pNGVL4a-Sig/E7(detox)/HSP70 DNA vaccine administered via gene gun generated the highest number of E7-specific CD8+ T cells. In addition, DNA vaccination via gene gun required the least dose to generate similar or slightly better antitumor effects compared to needle intramuscular (i.m.) and biojector administrations. Thus, our data suggest that DNA vaccination via gene gun represents the most potent regimen for DNA administration.

Enhancing DNA vaccine potency by coadministration of DNA encoding antiapoptotic proteins

Intradermal vaccination by gene gun efficiently delivers DNA vaccines into DCs of the skin, resulting in the activation and priming of antigen-specific T cells in vivo. DCs, however, have a limited life span, hindering their long-term ability to prime antigen-specific T cells. We reason that a strategy that prolongs the survival of DNA-transduced DCs will enhance priming of antigen-specific T cells and DNA vaccine potency. Here we show that codelivery of DNA encoding inhibitors of apoptosis (BCL-xL, BCL-2, XIAP, dominant negative caspase-9, or dominant negative caspase-8) with DNA encoding model antigens prolongs the survival of transduced DCs. More importantly, vaccinated mice exhibited significant enhancement in antigen-specific CD8+ T cell immune responses, resulting in a potent antitumor effect against antigen-expressing tumors. Among these antiapoptotic factors, BCL-xL demonstrated the greatest enhancement in antigen-specific immune responses and antitumor effects. Thus, coadministration of DNA vaccines with DNA encoding antiapoptotic proteins represents an innovative approach to enhance DNA vaccine potency.

Comparison of HPV DNA vaccines employing intracellular targeting strategies

Intradermal vaccination via gene gun efficiently delivers DNA vaccines into dendritic cells (DCs) of the skin, resulting in the activation and priming of antigen-specific T cells in vivo. In the context of DNA vaccines, we previously used the gene gun approach to test several intracellular targeting strategies that are able to route a model antigen, such as the human papillomavirus type-16 (HPV-16) E7, to desired subcellular compartments in order to enhance antigen processing and presentation to T cells. These strategies include the use of the sorting signal of lysosome-associated membrane protein (LAMP-1), Mycobacterium tuberculosis heat-shock protein 70 (HSP70), calreticulin (CRT) and the translocation domain (dII) of Pseudomonas aeruginosa exotoxin A (ETA). Vaccination with DNA vaccines encoding E7 antigen linked to any of these molecules all led to a significant enhancement of E7-specific CD8+ T-cell immune responses and strong antitumor effects against an E7-expressing tumor, TC-1. However, we were interested in identifying the most potent DNA vaccine for our future clinical trials. Thus, we performed a series of experiments to directly compare the potency of the various DNA vaccines. Among the DNA vaccines we tested, we found that vaccination with pcDNA3-CRT/E7 generated the highest number of E7-specific CD8+ T cells and potent long-term protection and treatment effects against E7-expressing tumors in mice. Interestingly, we observed that pcDNA3-CRT/E7 is also capable of protecting against an E7-expressing tumor with downregulated MHC class I expression, a common feature associated with most HPV-associated cervical cancers. Our data suggest that the DNA vaccine linking CRT to E7 (CRT/E7) may be a suitable candidate for human trials for the control of HPV infections and HPV-associated lesions.

Improving DNA Vaccine Potency by Linking Marek's Disease Virus Type 1 VP22 to an Antigen

ABSTRACT We have previously employed an intercellular spreading strategy using herpes simplex virus type 1 (HSV-1) VP22 protein to enhance DNA vaccine potency because DNA vaccines lack the intrinsic ability to amplify in cells. Recently, studies have demonstrated that the protein encoded by UL49 of Mareks disease virus type 1 (MDV-1) exhibits some degree of homology to the HSV-1 VP22 protein and features the property of intercellular transport. We therefore generated a DNA vaccine encoding MDV-1 VP22 linked to a model antigen, human papillomavirus type 16 E7. We demonstrated that compared with mice vaccinated with DNA encoding wild-type E7, mice vaccinated with MDV-1 VP22/E7 DNA exhibited a significant increase in number of gamma-interferon-secreting, E7-specific CD8+-T-cell precursors as well as stronger tumor prevention and treatment effects. Furthermore, our data indicated that the antitumor effect was CD8 dependent. These results suggested that the development of vaccines encoding VP22 fused to a target antigen might be a promising strategy for improving DNA vaccine potency.

Enhancing DNA vaccine potency by combining a strategy to prolong dendritic cell life with intracellular targeting strategies

We have recently shown that intradermal coadministration of DNA encoding Ag with DNA encoding inhibitors of apoptosis, including Bcl-xL, prolongs dendritic cell (DC) life and thereby enhances the potency of DNA vaccines in vivo. We have also demonstrated that DNA vaccines targeting Ag to subcellular compartments, using proteins such as Mycobacterium tuberculosis heat shock protein 70, calreticulin, or the sorting signal of the lysosome-associated membrane protein type 1 (LAMP-1), enhanced DNA vaccine potency. In this study, we reasoned that the combination of a strategy to prolong DC life with intracellular targeting strategies might produce a more effective DNA vaccine against human papillomavirus E7. We showed that coadministration of DNA encoding Bcl-xL with DNA encoding E7/heat shock protein 70, calreticulin/E7, or Sig/E7/LAMP-1 resulted in further enhancement of the E7-specific CD8+ T cell response for all three constructs. Of these strategies, mice vaccinated with Sig/E7/LAMP-1 DNA mixed with Bcl-xL DNA showed the greatest increase in E7-specific CD8+ T cells (∼13-fold increase). This combination of strategies resulted in increased CD8+ T cell functional avidity, an increased E7-specific CD4+ Th1 cell response, enhanced tumor treatment ability, and stronger long-term tumor protection when compared with mice vaccinated without Bcl-xL DNA. Therefore, DNA vaccines that combine strategies to enhance intracellular Ag processing and prolong DC life have potential clinical implications for control of viral infection and neoplasia.

Vaccination with a DNA Vaccine Encoding Herpes Simplex Virus Type 1 VP22 Linked to Antigen Generates Long-Term Antigen-Specific CD8-Positive Memory T Cells and Protective Immunity

Intradermal vaccination with DNA encoding herpes simplex virus type 1 (HSV-1) VP22 linked to antigen leads to spread of antigen within the epithelium and results in enhanced antigen-specific CD8+ T cell immune responses in vaccinated mice. In this study, we characterized the number of antigen-expressing dendritic cells (DCs) in the draining lymph nodes of vaccinated mice and determined whether the linkage of VP22 to antigen would influence the ability of antigen-expressing DCs to activate antigen-specific CD8+ T cells in vivo. Vaccination with DNA encoding HSV-1 VP22 linked to human papillomavirus type 16 E7 antigen generated more antigen-expressing DCs in the draining lymph nodes of vaccinated mice than E7 alone. In addition, the linkage of VP22 to E7 improved the MHC class I presentation of E7 in transfected DCs and led to enhanced activation of E7-specific CD8+ T cells. We also observed that vaccination with DNA encoding VP22 linked to E7 generated more E7-specific CD8+ memory T cells, and enhanced long-term protective antitumor immunity against an E7-expressing tumor in vaccinated mice compared with vaccination with DNA encoding E7 alone. Thus, administration of DNA encoding VP22 linked to antigen represents a plausible approach for the development of protective DNA vaccines.

CD8 + T cells, NK cells and IFN-γ are important for control of tumor with downregulated MHC class I expression by DNA vaccination

One of the major hurdles facing cancer immunotherapy is that cancers may downregulate expression of MHC class I molecules. The development of a suitable tumor model with downregulated MHC class I expression is critical for designing vaccines and immunotherapeutic strategies to control such tumors. We developed an E7-expressing murine tumor model with downregulated MHC class I expression, TC-1 P3 (A15). Using this model, we tested DNA and vaccinia vaccines for their ability to control tumors with downregulated MHC class I expression. We found that vaccination with DNA encoding E7 linked to Mycobacterial heat shock protein 70 (HSP70) generated a significant antitumor effect against TC-1 P3 (A15), while vaccination with E7/HSP70 vaccinia did not generate an appreciable antitumor effect. Lymphocyte depletion experiments revealed that both CD8+ T cells and NK cells were essential for the antitumor effect generated by E7/HSP70 DNA against TC-1 P3 (A15). Furthermore, tumor protection experiments using IFN-γ knockout mice revealed that IFN-γ was essential for the antitumor effect generated by E7/HSP70 DNA against TC-1 P3 (A15). Our results demonstrate that vaccination with E7/HSP70 DNA results in a significant antitumor effect against a neoplasm with downregulated MHC class I expression and the importance of CD8+ T cells, NK cells, and IFN-γ in generating this antitumor effect.