This study presents proof of concept assays to validate gold nanoparticles loaded with the bacterial peptide 91–99 of the listeriolysin O toxin (GNP-LLO91–99 nanovaccines) as immunotherapy for bladder tumors. GNP-LLO91–99 nanovaccines showed adjuvant abilities as they induce maturation and activation of monocyte-derived dendritic cells (MoDCs) to functional antigen-presenting cells in healthy donors and patients with melanoma or bladder cancer (BC), promoting a Th1 cytokine pattern. GNP-LLO91–99 nanovaccines were also efficient dendritic cell inducers of immunogenic tumor death using different bladder and melanoma tumor cell lines. The establishment of a pre-clinical mice model of subcutaneous BC confirmed that a single dose of GNP-LLO91–99 nanovaccines reduced tumor burden 4.7-fold and stimulated systemic Th1-type immune responses. Proof of concept assays validated GNP-LLO91–99 nanovaccines as immunotherapy by comparison to anti-CTLA-4 or anti-PD-1 antibodies. In fact, GNP-LLO91–99 nanovaccines increased percentages of CD4+ and CD8+ T cells, B cells, and functional antigen-presenting DCs in tumor-infiltrated lymphocytes, while they reduced the levels of myeloid-derived suppressor cells (MDSC) and suppressor T cells (Treg). We conclude that GNP-LLO91–99 nanovaccines can work as monotherapies or combinatory immunotherapies with anti-CTLA-4 or anti-PD-1 antibodies for solid tumors with high T cell infiltration, such as bladder cancer or melanoma.
Coronavirus disease 2019 (COVID-19) is the greatest threat to global health at the present time, and considerable public and private effort is being devoted to fighting this recently emerged disease. Despite the undoubted advances in the development of vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, uncertainty remains about their future efficacy and the duration of the immunity induced. It is therefore prudent to continue designing and testing vaccines against this pathogen. In this article we computationally designed two candidate vaccines, one monopeptide and one multipeptide, using a technique involving optimizing lambda-superstrings, which was introduced and developed by our research group. We tested the monopeptide vaccine, thus establishing a proof of concept for the validity of the technique. We synthesized a peptide of 22 amino acids in length, corresponding to one of the candidate vaccines, and prepared a dendritic cell (DC) vaccine vector loaded with the 22 amino acids SARS-CoV-2 peptide (positions 50-71) contained in the NTD domain (DC-CoVPSA) of the Spike protein. Next, we tested the immunogenicity, the type of immune response elicited, and the cytokine profile induced by the vaccine, using a non-related bacterial peptide as negative control. Our results indicated that the CoVPSA peptide of the Spike protein elicits noticeable immunogenicity in vivo using a DC vaccine vector and remarkable cellular and humoral immune responses. This DC vaccine vector loaded with the NTD peptide of the Spike protein elicited a predominant Th1-Th17 cytokine profile, indicative of an effective anti-viral response. Finally, we performed a proof of concept experiment in humans that included the following groups: asymptomatic non-active COVID-19 patients, vaccinated volunteers, and control donors that tested negative for SARS-CoV-2. The positive control was the current receptor binding domain epitope of COVID-19 RNA-vaccines. We successfully developed a vaccine candidate technique involving optimizing lambda-superstrings and provided proof of concept in human subjects. We conclude that it is a valid method to decipher the best epitopes of the Spike protein of SARS-CoV-2 to prepare peptide-based vaccines for different vector platforms, including DC vaccines.
538 Background: Urothelial bladder cancer (UBC) is characterized by high immunogenicity. Multiple immunotherapies both in the perioperative and advanced setting are being developed. However, we lack good predictive biomarkers of response to immunotherapy in UBC. We aimed to characterize the immune profile of UBC patients using TURBT and serum samples. Methods: 13 UBC biopsies from TURBT samples were analyzed along with sera of these patients. Biopsy samples were divided in two equal parts. One part was formalin-fixed paraffin embedded and stained for immune histochemistry [IHC] for the following markers (CD3, CD4, CD8, CD20, CD68 and PD-L1 using SP142 antibody) and results expressed in percentages. The other part was homogenized and processed for immune cell isolation (human tumor isolation Miltenyi Kit). Tumor cells were stained with PHK and double positive CD8 and PHK were isolated using a MACSTM procedure. Different immune cell populations were analyzed by flow cytometry for the following markers (CD3, CD4, CD8, CD20 and CD68) and results expressed in percentages. Cytokines were examined in sera using the Luminex multiparametric procedure and results were expressed as pg/mL. Results: IHC and flow cytometry data were comparable and identified three groups according to the immune cells associated with the tumors (Table). Groups 1 and 2 showed high and moderate levels of tumor activated CD8+ T cells (1-4%) along with a higher expression of PD-L1 (40-20%). Group 3 lacked tumor activated CD8+ T cells and show lower expression of PD-L1 (15%). All groups showed significant levels of Th2 cytokines (IL-4, IL-6 or IL-10) while very low or lack of Th1 cytokines (IFN or IL-2) was observed except in group 2. Conclusions: TURBT biopsies and serum analysis of cytokines could represent a valid approach to further evaluate potential predictive biomarkers of response to immunotherapy in UBC. Different immune populations subgroups were identified in our series and could potentially predict for diverse responses to treatment. Further prospective validation in larger series is ongoing to confirm these results.[Table: see text]
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