Alphavirus-based hepatitis C virus therapeutic vaccines:can universal helper epitopes enhance HCV-specific cytotoxic T lymphocyte responses?

BACKGROUND: Antigen-specific T cell immune responses play a pivotal role in resolving acute and chronic hepatitis C virus (HCV) infections. Currently, no prophylactic or therapeutic vaccines against HCV are available. We previously demonstrated the preclinical potency of therapeutic HCV vaccines based on recombinant Semliki Forest virus (SFV) replicon particles. However, clinical trials do not always meet the high expectations of preclinical studies, thus, optimization of vaccine strategies is crucial. In efforts to further increase the frequency of HCV-specific immune responses in the candidate SFV-based vaccines, the authors assessed whether inclusion of three strong, so-called universal helper T cell epitopes, and an endoplasmic reticulum localization, and retention signal (collectively termed sigHELP-KDEL cassette) could enhance HCV-specific immune responses. METHODS: We included the sigHELP-KDEL cassette in two of the candidate SFV-based HCV vaccines, targeting NS3/4A and NS5A/B proteins. We characterized the new constructs in vitro for the expression and stability of the transgene-encoded proteins. Their immune efficacy with respect to HCV-specific immune responses in vivo was compared with the parental SFV vaccine expressing the corresponding HCV antigen. Further characterization of the functionality of the HCV-specific CD8+ T cells was assessed by surface and intracellular cytokine staining and flow cytometry analysis. RESULTS: Moderate, but significantly, enhanced frequencies of antigen-specific immune responses were achieved upon lower/suboptimal dosage immunization. In optimal dosage immunization, the inclusion of the cassette did not further increase the frequencies of HCV-specific CD8+ T cells when compared with the parental vaccines and the frequencies of effector and memory populations were identical. CONCLUSION: We hypothesize that the additional effect of the sigHELP-KDEL cassette in SFV-based vaccines depends on the immunogenicity, nature, and stability of the target antigen expressed by the vaccine

Synthesis and Study of 2‑(Pyrrolesulfonylmethyl)‑N‑arylimines: A New Class of Inhibitors for Human Glutathione Transferase A1‑1

Overexpression of human GSTA1-1 in tumor cells is part of MDR mechanisms. We report on the synthesis of 11 pyrrole derivatives as hGSTA1-1 inhibitors starting from 1-methyl-2-[(2-nitrobenzylsulfanyl]-1H-pyrrole. Molecular modeling revealed two locations in the enzyme H binding site: the catalytic primary one accommodating shorter and longer derivatives and the secondary one, where shorter derivatives can occupy. Derivative 9, displaying the highest inhibition and bearing a p-nitroarylimino moiety, and derivative 4, lacking this moiety, were studied kinetically. Derivative 9 binds (Ki(9) = 71 ± 4 μM) at the primary site competitively vs CDNB. Derivative 4 binds (Ki(4) = 135 ± 27 μM) at the primary and secondary sites, allowing the binding of a second molecule (4 or CDNB) leading to formation of unreactive and reactive complexes, respectively. The arylmethylsulfonylpyrrole core structure is a new pharmacophore for hGSTA1-1, whereas its derivative 9 may serve as a lead structure

A PBMC-Based System to Assess Human T Cell Responses to Influenza Vaccine Candidates In Vitro

Vaccine development is an expensive and time-consuming process that heavily relies on animal models. Yet, vaccine candidates that have previously succeeded in animal experiments often fail in clinical trials questioning the predictive value of animal models. Alternative assay systems that can add to the screening and evaluation of functional characteristics of vaccines in a human context before embarking on costly clinical trials are therefore urgently needed. In this study, we have established an in vitro system consisting of long-term cultures of unfractionated peripheral blood mononuclear cells (PBMCs) from healthy volunteers to assess (recall) T cell responses to vaccine candidates. We observed that different types of influenza vaccines (whole inactivated virus (WIV), split, and peptide vaccines) were all able to stimulate CD4 and CD8 T cell responses but to different extents in line with their reported in vivo properties. In-depth analyses of different T cell subsets revealed that the tested vaccines evoked mainly recall responses as indicated by the fact that the vast majority of the responding T cells had a memory phenotype. Furthermore, we observed vaccine-induced activation of T follicular helper cells, which are associated with the induction of humoral immune responses. Our results demonstrate the suitability of the established PBMC-based system for the in vitro evaluation of memory T cell responses to vaccines and the comparison of vaccine candidates in a human immune cell context. As such, it can help to bridge the gap between animal experiments and clinical trials and assist in the selection of promising vaccine candidates, at least for recall antigens

An alphavirus-based therapeutic cancer vaccine: from design to clinical trial

Cancer immunotherapy has greatly advanced in recent years. Most immunotherapeutic strategies are based on the use of immune checkpoint blockade to unleash antitumor immune responses or on the induction or adoptive transfer of immune effector cells. We aim to develop therapeutic vaccines based on recombinant Semliki Forest virus vectors to induce tumor-specific effector immune cells. In this review, we describe our ongoing work on SFV-based vaccines targeted against human papillomavirus- and hepatitis C virus-related infections and malignancies, focusing on design, delivery, combination strategies, preclinical efficacy and product development for a first-in-man clinical trial with an HPV-specific vaccine

Strategies to Target Tumor Immunosuppression

Several strategies, aimed at activating both innate and adaptive antitumor immunity, have been and are currently evaluated in clinical trials. In separate chapters of this book, these strategies are being reviewed and discussed. An alternative option is to (also) target intratumoral immune escape mechanisms in order to enhance intratumoral antitumor responses. Based on their overall target aim, these strategies can be divided into two main categories: Strategies that aim at enhancing intratumor homing of effector T cells and strategies to maintain the activity of these cells once they have reached the tumor site. Currently used therapeutic strategies that attempt to increase homing of effector T cells to tumors are local tumor irradiation, blockade of endothelin receptors, and taxane-based chemotherapy. Strategies that aim to enhance antitumor activity of intratumor effector T cells, either by overcoming tumor-induced tolerance or by overriding the immunosuppressive mechanisms imposed during tumor development, are depletion or functional inhibition of immunosuppressive populations, blockade of negative regulatory factors, and blockade of tumor growth factor-beta (TGF-β)-induced signaling

Strategies to Target Tumor Immunosuppression

Several strategies, aimed at activating both innate and adaptive antitumor immunity, have been and are currently evaluated in clinical trials. In separate chapters of this book, these strategies are being reviewed and discussed. An alternative option is to (also) target intratumoral immune escape mechanisms in order to enhance intratumoral antitumor responses. Based on their overall target aim, these strategies can be divided into two main categories: Strategies that aim at enhancing intratumor homing of effector T cells and strategies to maintain the activity of these cells once they have reached the tumor site. Currently used therapeutic strategies that attempt to increase homing of effector T cells to tumors are local tumor irradiation, blockade of endothelin receptors, and taxane-based chemotherapy. Strategies that aim to enhance antitumor activity of intratumor effector T cells, either by overcoming tumor-induced tolerance or by overriding the immunosuppressive mechanisms imposed during tumor development, are depletion or functional inhibition of immunosuppressive populations, blockade of negative regulatory factors, and blockade of tumor growth factor-beta (TGF-β)-induced signaling

Strategies to Target Tumor Immunosuppression

Several strategies, aimed at activating both innate and adaptive antitumor immunity, have been and are currently evaluated in clinical trials. In separate chapters of this book, these strategies are being reviewed and discussed. An alternative option is to (also) target intratumoral immune escape mechanisms in order to enhance intratumoral antitumor responses. Based on their overall target aim, these strategies can be divided into two main categories: Strategies that aim at enhancing intratumor homing of effector T cells and strategies to maintain the activity of these cells once they have reached the tumor site. Currently used therapeutic strategies that attempt to increase homing of effector T cells to tumors are local tumor irradiation, blockade of endothelin receptors, and taxane-based chemotherapy. Strategies that aim to enhance antitumor activity of intratumor effector T cells, either by overcoming tumor-induced tolerance or by overriding the immunosuppressive mechanisms imposed during tumor development, are depletion or functional inhibition of immunosuppressive populations, blockade of negative regulatory factors, and blockade of tumor growth factor-beta (TGF-β)-induced signaling

TLR9-Mediated Conditioning of Liver Environment Is Essential for Successful Intrahepatic Immunotherapy and Effective Memory Recall.

Immune defense against hepatotropic viruses such as hepatitis B (HBV) and hepatitis C (HCV) poses a major challenge for therapeutic approaches. Intrahepatic cytotoxic CD8 T cells that are crucial for an immune response against these viruses often become exhausted resulting in chronic infection. We elucidated the T cell response upon therapeutic vaccination in inducible transgenic mouse models in which variable percentages of antigen-expressing hepatocytes can be adjusted, providing mosaic antigen distribution and reflecting the varying viral antigen loads observed in patients. Vaccination-induced endogenous CD8 T cells could eliminate low antigen loads in liver but were functionally impaired if confronted with elevated antigen loads. Strikingly, only by conditioning the liver environment with TLR9 ligand prior and early after peripheral vaccination, successful immunization against high intrahepatic antigen density with its elimination was achieved. Moreover, TLR9 immunomodulation was also indispensable for functional memory recall after high frequency antigen challenge. Together, the results indicate that TLR9-mediated conditioning of liver environment during therapeutic vaccination or antigen reoccurrence is crucial for an efficacious intrahepatic T cell response

Synthesis and Study of 2‑(Pyrrolesulfonylmethyl)‑<i>N</i>‑arylimines: A New Class of Inhibitors for Human Glutathione Transferase A1‑1

Overexpression of human GSTA1-1 in tumor cells is part of MDR mechanisms. We report on the synthesis of 11 pyrrole derivatives as hGSTA1-1 inhibitors starting from 1-methyl-2-[(2-nitrobenzylsulfanyl]-1<i>H</i>-pyrrole. Molecular modeling revealed two locations in the enzyme H binding site: the catalytic primary one accommodating shorter and longer derivatives and the secondary one, where shorter derivatives can occupy. Derivative <b>9</b>, displaying the highest inhibition and bearing a <i>p</i>-nitroarylimino moiety, and derivative <b>4</b>, lacking this moiety, were studied kinetically. Derivative <b>9</b> binds (<i>K</i>_i(<b>9</b>) = 71 ± 4 μM) at the primary site competitively vs CDNB. Derivative <b>4</b> binds (<i>K</i>_i(<b>4</b>) = 135 ± 27 μM) at the primary and secondary sites, allowing the binding of a second molecule (<b>4</b> or CDNB) leading to formation of unreactive and reactive complexes, respectively. The arylmethylsulfonylpyrrole core structure is a new pharmacophore for hGSTA1-1, whereas its derivative <b>9</b> may serve as a lead structure

Hotspot DNA Methyltransferase 3A (<i>DNMT3A</i>) and Isocitrate Dehydrogenase 1 and 2 (<i>IDH1/2</i>) Mutations in Acute Myeloid Leukemia and Their Relevance as Targets for Immunotherapy

DNA methyltransferase 3A (DNMT3A) and isocitrate dehydrogenase 1 and 2 (IDH1/2) are genes involved in epigenetic regulation, each mutated in 7–23% of patients with acute myeloid leukemia. Here, we investigated whether hotspot mutations in these genes encode neoantigens that can be targeted by immunotherapy. Five human B-lymphoblastoid cell lines expressing common HLA class I alleles were transduced with a minigene construct containing mutations that often occur in DNMT3A or IDH1/2. From these minigene-transduced cell lines, peptides were eluted from HLA class I alleles and analyzed using tandem mass spectrometry. The resulting data are available via ProteomeXchange under the identifier PXD050560. Mass spectrometry revealed an HLA-A*01:01-binding DNMT3AR882H peptide and an HLA-B*07:02-binding IDH2R140Q peptide as potential neoantigens. For these neopeptides, peptide–HLA tetramers were produced to search for specific T-cells in healthy individuals. Various T-cell clones were isolated showing specific reactivity against cell lines transduced with full-length DNMT3AR882H or IDH2R140Q genes, while cell lines transduced with wildtype genes were not recognized. One T-cell clone for DNMT3AR882H also reacted against patient-derived acute myeloid leukemia cells with the mutation, while patient samples without the mutation were not recognized, thereby validating the surface presentation of a DNMT3AR882H neoantigen that can potentially be targeted in acute myeloid leukemia via immunotherapy