Design and analysis of adjuvanticity for new vaccine strategies.

The future of vaccine design relies on three major areas of research a) the characterisation of vaccine components with regards to immunostimulatory capacity and biological effects to ensure and predict safety and efficacy b) the development of novel adjuvants and vaccine delivery systems and c) the identification of new targets that can elicit protective immunity. In this thesis a series of studies were conducted addressing aspects of these issues. There is a defined requirement for the development of in vitro systems for the evaluation of the safety and efficacy of vaccines. The modified horizontal Ussing chamber was evaluated as a tissue culture tool for the rapid in vitro screening of candidate vaccines for mucosal delivery. With limitations identified, recommendations for future design improvements and areas of application are proposed. It is postulated that future vaccines will incorporate multiple adjuvants to induce appropriate, protective types of immunity. To this end, the effect of co-administration of secondary adjuvants with alum and chitosan were investigated in the context of the clinically relevant Helicobacter pylori antigen urease (rUre). It was found that chitosan enhanced the adjuvanticity of the B subunit of cholera toxin (CTB) as well as muramyl di-peptide (MDP), although MDP was ineffective when administered intramuscularly or intranasally. In addition, co-administration of alum with Quil-A or, more so, MDP enhanced immunogenicity and introduced type I response elements suggesting a synergistic mode of action between the adjuvants. In an effort to introduce a simple application approach for double stranded RNA (dsRNA) as an adjuvant in subunit vaccine research, a plasmid-based dsRNA expression system was investigated. Although such a plasmid was constructed, problems over scalable preparation due to palindrome-mediated inviability non-viability were encountered. Alternative approaches based on dual vector systems are discussed. The Staphylococcus aureus RNA III Activating Protein (RAP), a quorum sensing (QS) ligand that controls virulence and adhesion, which has been described as a protective antigen against staphylococcal disease, even antibiotic resistant strains (such as MRSA), was investigated in the context of known effective adjuvants. This data support clinical evaluation of this antigen as it was found that alum and poly-L-lactide (PLA) nanoparticles administered intramuscularly were capable of eliciting immunity similar to that obtained in protective studies; this was achieved with a fraction of the antigen quantity previously used and with non-toxic adjuvants. As elicitation of systemic immunity was not detected following nasal vaccination, the potential role of tolerance to RAP is discussed

Targeted Therapies in Melanoma

Advances in understanding the biology of melanoma have provided great insights about the mechanisms of chemoresistance and its genetic heterogeneity in parallel with advances in drug design culminating in recent major treatment breakthroughs using small molecules inhibitors in metastatic melanoma (MM). While clinical benefit of targeted therapies has been unquestionable, future advances can only be possible if we better understand the interplay between genetic aberrations and role of other crucial non-genetic changes yet to be identified by such projects as the Cancer Genome Atlas Project (TCGA) in Melanoma. Combination therapies, either among small molecule inhibitors themselves and/or with immunotherapies may be the optimal strategy to prevent development of drug resistance that is inherently linked with such targeted therapies

Use of Susceptibility-Weighted Imaging (SWI) in the Detection of Brain Hemorrhagic Metastases from Breast Cancer and Melanoma

SWI has significantly increased our sensitivity in detecting hemorrhagic brain lesions. We sought to explore the prevalence of intra-tumoral hemorrhage as detected by SWI in brain metastases from melanoma and breast cancer

Mitochondrial respiration - an important therapeutic target in melanoma

The importance of mitochondria as oxygen sensors as well as producers of ATP and reactive oxygen species (ROS) has recently become a focal point of cancer research. However, in the case of melanoma, little information is available to what extent cellular bioenergetics processes contribute to the progression of the disease and related to it, whether oxidative phosphorylation (OXPHOS) has a prominent role in advanced melanoma. In this study we demonstrate that compared to melanocytes, metastatic melanoma cells have elevated levels of OXPHOS. Furthermore, treating metastatic melanoma cells with the drug, Elesclomol, which induces cancer cell apoptosis through oxidative stress, we document by way of stable isotope labeling with amino acids in cell culture (SILAC) that proteins participating in OXPHOS are downregulated. We also provide evidence that melanoma cells with high levels of glycolysis are more resistant to Elesclomol. We further show that Elesclomol upregulates hypoxia inducible factor 1-α (HIF-1α), and that prolonged exposure of melanoma cells to this drug leads to selection of melanoma cells with high levels of glycolysis. Taken together, our findings suggest that molecular targeting of OXPHOS may have efficacy for advanced melanoma. © 2012 Barbi de Moura et al

Importance of glycolysis and oxidative phosphorylation in advanced melanoma

Serum lactate dehydrogenase (LDH) is a prognostic factor for patients with stage IV melanoma. To gain insights into the biology underlying this prognostic factor, we analyzed total serum LDH, serum LDH isoenzymes, and serum lactate in up to 49 patients with metastatic melanoma. Our data demonstrate that high serum LDH is associated with a significant increase in LDH isoenzymes 3 and 4, and a decrease in LDH isoenzymes 1 and 2. Since LDH isoenzymes play a role in both glycolysis and oxidative phosphorylation (OXPHOS), we subsequently determined using tissue microarray (TMA) analysis that the levels of proteins associated with mitochondrial function, lactate metabolism, and regulators of glycolysis were all elevated in advanced melanomas compared with nevic melanocytes. To investigate whether in advanced melanoma, the glycolysis and OXPHOS pathways might be linked, we determined expression of the monocarboxylate transporters (MCT) 1 and 4. Analysis of a nevus-to-melanoma progression TMA revealed that MCT4, and to a lesser extend MCT1, were elevated with progression to advanced melanoma. Further analysis of human melanoma specimens using the Seahorse XF24 extracellular flux analyzer indicated that metastatic melanoma tumors derived a large fraction of energy from OXPHOS. Taken together, these findings suggest that in stage IV melanomas with normal serum LDH, glycolysis and OXPHOS may provide metabolic symbiosis within the same tumor, whereas in stage IV melanomas with high serum LDH glycolysis is the principle source of energy

Melanoma central nervous system metastases: current approaches, challenges, and opportunities

Melanoma central nervous system metastases are increasing, and the challenges presented by this patient population remain complex. In December 2015, the Melanoma Research Foundation and the Wistar Institute hosted the First Summit on Melanoma Central Nervous System (CNS) Metastases in Philadelphia, Pennsylvania. Here, we provide a review of the current status of the field of melanoma brain metastasis research; identify key challenges and opportunities for improving the outcomes in patients with melanoma brain metastases; and set a framework to optimize future research in this critical area

DNA methylation profiles in primary cutaneous melanomas are associated with clinically significant pathologic features

DNA methylation studies have elucidated a methylation signature distinguishing primary melanomas from benign nevi and provided new insights about genes that may be important in melanoma development. However, it is unclear whether methylation differences among primary melanomas are related to tumor pathologic features with known clinical significance. We utilized the Illumina Golden Gate Cancer Panel array to investigate the methylation profiles of 47 primary cutaneous melanomas. Array-wide methylation patterns revealed a positive association of methylation with Breslow thickness and mutated BRAF, a negative association with mitotic rate, and a weak association with ulceration. Hierarchical clustering on CpG sites exhibiting the most variable methylation (n=235) divided the melanoma samples into three clusters, including a highly-methylated cluster that was positively associated with Breslow thickness and an intermediately-methylated cluster associated with Breslow thickness and mitotic rate. Our findings provide support for the existence of methylation-defined subsets in melanomas, with increased methylation associated with Breslow thickness

Pathologic and gene expression features of metastatic melanomas to the brain: Biology of Metastatic Melanomas to the Brain

The prognosis of MBM is variable with prolonged survival in a subset. It is unclear whether MBMs differ from extracranial metastases (EcM) and primary melanomas (PrM)

PD-1 and Tim-3 Regulate the Expansion of Tumor Antigen-Specific CD8+ T Cells Induced by Melanoma Vaccines

Although melanoma vaccines stimulate tumor antigen (TA)-specific CD8+ T cells, objective clinical responses are rarely observed. To investigate this discrepancy, we evaluated the character of vaccine-induced CD8+ T cells with regard to the inhibitory T cell co-receptors PD-1 and Tim-3 in metastatic melanoma patients who were administered tumor vaccines. The vaccines included incomplete Freund's adjuvant (IFA), CpG oligodeoxynucleotide (CpG) and the HLA-A2-restricted analog peptide NY-ESO-1 157-165V, either by itself or in combination with the pan-DR epitope NY-ESO-1 119-143. Both vaccines stimulated rapid TA-specific CD8+ T-cell responses detected ex vivo, however, TA-specific CD8+ T cells produced more IFN-γ and exhibited higher lytic function upon immunization with MHC class I and class II epitopes. Notably, the vast majority of vaccine-induced CD8+ T cells upregulated PD-1 and a minority also upregulated Tim-3. Levels of PD-1 and Tim-3 expression by vaccine-induced CD8+ T cells at the time of vaccine administration correlated inversely with their expansion in vivo. Dual blockade of PD-1 and Tim-3 enhanced the expansion and cytokine production of vaccine-induced CD8+ T cells in vitro. Collectively, our findings support the use of PD-1 and Tim-3 blockades with cancer vaccines to stimulate potent antitumor T cell responses and increase the likelihood of clinical responses in advanced melanoma patients