Synergy of Immune Checkpoint Blockade with a Novel Synthetic Consensus DNA Vaccine Targeting TERT.

Immune checkpoint blockade antibodies are setting a new standard of care for cancer patients. It is therefore important to assess any new immune-based therapies in the context of immune checkpoint blockade. Here, we evaluate the impact of combining a synthetic consensus TERT DNA vaccine that has improved capacity to break tolerance with immune checkpoint inhibitors. We observed that blockade of CTLA-4 or, to a lesser extent, PD-1 synergized with TERT vaccine, generating more robust anti-tumor activity compared to checkpoint alone or vaccine alone. Despite this anti-tumor synergy, none of these immune checkpoint therapies showed improvement in TERT antigen-specific immune responses in tumor-bearing mice. αCTLA-4 therapy enhanced the frequency of T-bet+/CD44+ effector CD8+ T cells within the tumor and decreased the frequency of regulatory T cells within the tumor, but not in peripheral blood. CTLA-4 blockade synergized more than Treg depletion with TERT DNA vaccine, suggesting that the effect of CTLA-4 blockade is more likely due to the expansion of effector T cells in the tumor rather than a reduction in the frequency of Tregs. These results suggest that immune checkpoint inhibitors function to alter the immune regulatory environment to synergize with DNA vaccines, rather than boosting antigen-specific responses at the site of vaccination.

A Novel DNA Vaccine Platform Enhances Neo-antigen-like T Cell Responses against WT1 to Break Tolerance and Induce Anti-tumor Immunity.

Tumor-associated antigens have emerged as important immunotherapeutic targets in the fight against cancer. Germline tumor antigens, such as WT1, Wilms' tumor gene 1, are overexpressed in many human malignancies but have low expression in somatic tissues. Recent vaccination approaches to target WT1 have been hampered by poor in vivo immune potency, likely due to the conserved self-antigen nature of WT1. In this study, we use a novel synthetic micro-consensus SynCon DNA vaccine approach with the goal of breaking tolerance and increasing vaccine immune potency. This approach induced new, neo-antigen-like responses that were superior to those induced by native WT1 DNA immunogens for driving T cell immunity and breaking tolerance. Non-human primates (NHPs) vaccinated with SynCon WT1 antigens elicited immune responses against native rhesus WT1 peptides. When delivered by electroporation (EP) in mice, SynCon-based WT1 constructs elicited strong CD4 and CD8 T cell responses (including IFN-γ, CD107a, and TNF-α) to both native and consensus peptides. In addition, SynCon WT1 vaccine-induced antibodies recognized native WT1 in vitro. Vaccination with the SynCon WT1 immunogens was capable of slowing tumor growth in therapeutic models in vivo. These data support the further study of synthetic consensus DNA vaccines for breaking tolerance to important germline antigens.

Intradermal DNA vaccination enhanced by low-current electroporation improves antigen expression and induces robust cellular and humoral immune responses.

DNA represents an ideal vaccine platform for HIV and many infectious diseases because of its safety, stability, and ease of manufacture. However, the immunogenicity of DNA vaccines has traditionally been low compared with viral vectors, recombinant protein, and live attenuated vaccines. The immunogenicity of DNA vaccines has been significantly enhanced by delivery with in vivo electroporation. Further improvements now allow electroporation to be performed in the dermis, which could potentially improve patient tolerability and may further enhance immunogenicity. In this study we examined how the current of intradermal vaccination impacts antigen expression, inflammation, and the induction of both humoral and cellular immunity in guinea pigs and nonhuman primates. We observed that a lower (0.1 A) current reduced inflammation and improved antigen expression compared with a 0.2 A current. The improved antigen expression resulted in a trend toward higher cellular immune responses but no impact on HIV- and influenza-specific binding titers. This study highlights the need for optimization of electroporation conditions in vivo in order to balance enhanced plasmid transfection with a loss of expression due to tissue inflammation and necrosis. These results suggest that a lower, 0.1-A current may not only improve patient tolerability but also improve immunogenicity.

An optimized SIV DNA vaccine can serve as a boost for Ad5 and provide partial protection from a high-dose SIVmac251 challenge.

One limitation in the development of an improved cellular response needed for an effective HIV-vaccine is the inability to induce robust effector T-cells capable of suppressing a heterologous challenge. To improve cellular immune responses, we examined the ability of an optimized DNA vaccine to boost the cellular immune responses induced by a highly immunogenic Ad5 prime. Five Chinese rhesus macaques received pVax encoding consensus (con) gag/pol/env intramuscularly (IM) with electroporation followed by the Merck Ad5 gag/pol/nef vaccine. A second group of five animals were vaccinated with Merck Ad5 gag/pol/nef followed by pVax gag/pol/env. One year following vaccination, Ad5-prime DNA-boosted monkeys and four unvaccinated controls received an intrarectal challenge with 1000 ID50 SIV(mac)251. The quality and magnitude of the T-cell response was analyzed by ELISpot and polyfunctional flow cytometry. We observed that an Ad5-prime DNA-boost resulted in significantly elevated SIV-specific T-cell responses even compared with animals receiving a DNA-prime Ad5-boost. Ad5 prime DNA boosted animals were capable of suppressing a pathogenic SIV(mac)251 challenge. Peak control correlated with the expansion of HLA-DR(+) CD8(+) T-cells two weeks post-infection. These data illustrate that high optimization of a DNA vaccine can drive of immune responses primed by a robust vector system. This previously unachievable feature of these newly optimized DNAs warrants future studies of this strategy that may circumvent issues of serology associated with viral vector prime-boost systems.

Clinical applications of DNA vaccines: current progress.

It was discovered almost 20 years ago that plasmid DNA, when injected into the skin or muscle of mice, could induce immune responses to encoded antigens. Since that time, there has since been much progress in understanding the basic biology behind this deceptively simple vaccine platform and much technological advancement to enhance immune potency. Among these advancements are improved formulations and improved physical methods of delivery, which increase the uptake of vaccine plasmids by cells; optimization of vaccine vectors and encoded antigens; and the development of novel formulations and adjuvants to augment and direct the host immune response. The ability of the current, or second-generation, DNA vaccines to induce more-potent cellular and humoral responses opens up this platform to be examined in both preventative and therapeutic arenas. This review focuses on these advances and discusses both preventive and immunotherapeutic clinical applications.

Co-delivery of PSA and PSMA DNA vaccines with electroporation induces potent immune responses.

Prostate cancer (PCa) remains a significant public health problem. Current treatment modalities for PCa can be useful, but may be accompanied by deleterious side effects and often do not confer long-term control. Accordingly, additional modalities, such as immunotherapy, may represent an important approach for PCa treatment. The identification of tissue-specific antigens engenders PCa an attractive target for immunotherapeutic approaches. Delivery of DNA vaccines with electroporation has shown promising results for prophylactic and therapeutic targets in a variety of species including humans. Application of this technology for PCa immunotherapy strategies has been limited to single antigen and epitope targets. We sought to test the hypothesis that a broader collection of antigens would improve the breadth and effectiveness of a PCa immune therapy approach. We therefore developed highly optimized DNA vaccines encoding prostate-specific antigen (PSA) and prostate-specific membrane antigen (PSMA) as a dual antigen approach to immune therapy of PCa. PSA-and PSMA-specific cellular immunogenicity was evaluated in a mouse model for co-delivery and single antigen vaccination. Mice received 2 immunizations spaced 2 weeks apart and immunogenicity was evaluated 1 week after the second vaccination. Both the PSA and PSMA vaccines induced robust antigen-specific IFNγ responses by ELISpot. Further characterization of cellular immunogenicity by flow cytometry indicated strong antigen-specific TNFα production by CD4+ T cells and IFNγ and IL-2 secretion by both CD4+ and CD8+ T cells. There was also a strong humoral response as determined by PSA-specific seroconversion. These data support further study of this novel approach to immune therapy of PCa.

A DNA vaccine against chikungunya virus is protective in mice and induces neutralizing antibodies in mice and nonhuman primates.

Chikungunya virus (CHIKV) is an emerging mosquito-borne alphavirus indigenous to tropical Africa and Asia. Acute illness is characterized by fever, arthralgias, conjunctivitis, rash, and sometimes arthritis. Relatively little is known about the antigenic targets for immunity, and no licensed vaccines or therapeutics are currently available for the pathogen. While the Aedes aegypti mosquito is its primary vector, recent evidence suggests that other carriers can transmit CHIKV thus raising concerns about its spread outside of natural endemic areas to new countries including the U.S. and Europe. Considering the potential for pandemic spread, understanding the development of immunity is paramount to the development of effective counter measures against CHIKV. In this study, we isolated a new CHIKV virus from an acutely infected human patient and developed a defined viral challenge stock in mice that allowed us to study viral pathogenesis and develop a viral neutralization assay. We then constructed a synthetic DNA vaccine delivered by in vivo electroporation (EP) that expresses a component of the CHIKV envelope glycoprotein and used this model to evaluate its efficacy. Vaccination induced robust antigen-specific cellular and humoral immune responses, which individually were capable of providing protection against CHIKV challenge in mice. Furthermore, vaccine studies in rhesus macaques demonstrated induction of nAb responses, which mimicked those induced in convalescent human patient sera. These data suggest a protective role for nAb against CHIKV disease and support further study of envelope-based CHIKV DNA vaccines.

A highly optimized DNA vaccine confers complete protective immunity against high-dose lethal lymphocytic choriomeningitis virus challenge.

Protection against infection is the hallmark of immunity and the basis of effective vaccination. For a variety of reasons there is a great demand to develop new, safer and more effective vaccine platforms. In this regard, while 'first-generation' DNA vaccines were poorly immunogenic, new genetic 'optimization' strategies and the application of in vivo electroporation (EP) have dramatically boosted their potency. We developed a highly optimized plasmid DNA vaccine that expresses the lymphocytic choriomeningitis virus (LCMV) nucleocapsid protein (NP) and evaluated it using the LCMV challenge model, a gold standard for studying infection and immunity. When administered intramuscularly with EP, robust NP-specific cellular and humoral immune responses were elicited, the magnitudes of which approached those following acute LCMV infection. Furthermore, these responses were capable of providing 100% protection against a high-dose, normally lethal virus challenge. This is the first non-infectious vaccine conferring complete protective immunity up to 8 weeks after vaccination and demonstrates the potential of 'next-generation' DNA vaccines.

Unique Th1/Th2 phenotypes induced during priming and memory phases by use of interleukin-12 (IL-12) or IL-28B vaccine adjuvants in rhesus macaques.

Adjuvant compounds are usually included in vaccinations in order to bolster total vaccine-specific responses or to tailor an immune response toward a desired endpoint, such as the production of gamma interferon or an increase in antibody titers. While most adjuvants are studied in regard to their impact on vaccine-specific responses during and just after the vaccination period, a detailed analysis of how adjuvants skew the Th1/Th2 axis at more distant time points is not often undertaken. In the current study, we present data that suggests that adjuvants differ in their relative abilities to bolster and skew immune responses in the short term compared with more distant time points. To that end, we have employed interleukin-12 (IL-12) and IL-28B as adjuvants for DNA vaccination of rhesus macaques. While both adjuvants were able to bolster Th1-biased responses, our analysis shows that this skewing was achieved through different mechanisms. Moreover, analysis 3 months after the final immunization revealed the activity of the IL-12 adjuvant to be short lived, while the IL-28B adjuvant continued to exert its influence on the immune system. Taken together, these data suggest that the scientific and medical communities would benefit from a more detailed analysis of adjuvant function, including the determination of long-term influences of administered adjuvants.

Ki-67 staining for determination of rhesus macaque T cell proliferative responses ex vivo.

The capacity for robust proliferation upon re-infection is a hallmark of adaptive immunity and the basis of vaccination. A widely used animal model for the study of human disease is the rhesus macaque (RM), where capacity for proliferation can be assessed ex vivo using carboxyfluorescein succinimidyl ester (CFSE)-based dilution assays. However, we show over the course of the standard ex vivo proliferation assay that CFSE-labeling at commonly used dye concentrations induces significant cell death, but that this phenomenon is dose-dependent. Here, we describe an alternative semiquantitative method for estimating T cell proliferative responses that avoids the putative biases associated with chemical modification. RM peripheral blood mononuclear cells were stimulated ex vivo with cognate peptides for 5 days, immunostained for intracellular Ki-67, and then analyzed by flow cytometry. We describe a gating strategy using Ki-67 and side light scatter, also a marker of blastogenesis, which correlates strongly with data from CFSE dilution. We show that this method is a valid tool for measuring RM antigen-specific cellular proliferation ex vivo and can be used as an alternative to CFSE dilution assays.

Intradermal delivery of plasmid VEGF(165) by electroporation promotes wound healing.

Skin flaps are extensively used in reconstructive surgeries to repair large defects and deep wounds, but severe ischemia and necrosis often results in loss of the transplanted tissue. Thus, skin flap models are often used to study the biology of healing and necrosis of acute ischemic wounds. Delivery of exogenous vascular endothelial growth factor (VEGF) to areas of ischemia has shown promise for promoting therapeutic angiogenesis, but its expression must be tightly regulated to avoid adverse effects. In this study, plasmid DNA encoding VEGF(165) (pVEGF) was delivered to the ischemic skin of a rat skin flap model by intradermal injection followed by electroporation (EP) (pVEGFE+). Treatment with pVEGFE+ significantly increased VEGF expression for 5 days after delivery compared to injection of pVEGF without EP (pVEGFE-). The short-term increase in VEGF was sufficient to mediate an upregulation of endothelial nitric oxide synthase, an angiogenic factor that increases vascular permeability. pVEGFE+ significantly increased skin flap perfusion at both days 10 and 14 postoperatively. The observed increase in perfusion with pVEGFE+ correlated with an increase in skin flap healing and survival. Our results demonstrate that pVEGFE+ is a potential nonviral noninvasive therapy to increase perfusion and healing of skin flaps and ischemic wounds.

EGR1 predicts PTEN and survival in patients with non-small-cell lung cancer.

PURPOSE: The zinc finger transcription factor early growth response gene 1 (EGR1) is underexpressed in non-small-cell lung cancer (NSCLC) compared with normal lung. EGR1 expression has been linked to tumor suppression as a result of cell cycle arrest and apoptosis through regulation of tumor suppressor pathways including PTEN. For these reasons, we hypothesized that reduced levels of EGR1 would correlate with inferior outcome in patients with NSCLC. PATIENTS AND METHODS: Patients who underwent surgical resection for NSCLC had RNA extracted from tumor tissue and EGR1 gene expression was quantified by real-time quantitative polymerase chain reaction. The levels of EGR1 expression were examined in relationship to patient characteristics, histology, tumor stage, PTEN expression, and overall and disease-free survival. RESULTS: EGR1 expression strongly correlated with PTEN expression (P < .0001). No correlation of EGR1 with histology or stage was detected. Patients with high levels of EGR1 had better overall and disease-free survival compared with patients with low levels of EGR1 (P = .040 and P = .096, respectively). In a stratified log-rank test, low EGR1 expression was predictive of poor survival independent of tumor stage. CONCLUSION: EGR1 gene expression predicts PTEN levels and survival after surgical resection of NSCLC. Consistent with its known tumor suppressor properties, lower levels of EGR1 are associated with poor outcome. Identification of patients with low EGR1 therefore may identify patients at high risk for disease recurrence and may also identify patients who have tumors resistant to therapy secondary to loss of pathways such as PTEN.

Stat3 regulates genes common to both wound healing and cancer.

Wound healing and cancer are both characterized by cell proliferation, remodeling of extracellular matrix, cell invasion and migration, new blood vessel formation, and modulation of blood coagulation. The mechanisms that link wound healing and cancer are poorly understood. We report here that Stat3, a common signaling mechanism involved in oncogenesis and tissue injury, regulates a common set of genes involved in wound healing and cancer. Using oligonucleotide gene arrays and quantitative real-time PCR, we evaluated changes in global gene expression resulting from expression of Stat3 in lung epithelial cells. We report here previously uncharacterized genes induced by Stat3 implicated in signaling pathways common to both wound healing and cancer including cell invasion and migration, angiogenesis, modulation of coagulation, and repression of interferon-inducible genes. Consistent with these results, we found increased Stat3 activity associated with wound healing in chronically inflamed mouse lungs and increased Stat3 activity was identified at the leading edge of lung tumors invading adjacent nontumor stroma. These findings provide a molecular basis for understanding cancer as a deregulation of normal wound healing processes.