Search

Abstract Different innate immune pathways converge to Stimulator of interferon genes (STING) and trigger type I interferon responses after recognition of abnormal nucleic acids in the cells. This non-redundant function renders STING a major player in immunosurveillance, and an emerging target for cancer and infectious diseases therapeutics. Beyond somatic mutations that often occur in cancer, the human gene encoding STING protein, TMEM173 (STING1), holds great genetic heterogeneity; R232, HAQ (R71H-G230A-R293Q) and H232 are the most common alleles. Although some of these alleles are likely to be hypomorphic, their function is still debated, due to the available functional assessments, which have been performed in biased biological systems. Here, by using genetic background-matched models, we report on the functional evaluation of R232, HAQ and H232 variants on STING function, and on how these genotypes affect the susceptibility to clinically relevant viruses, thus supporting a potential contributing cause to differences in inter-individual responses to infections. Our findings also demonstrate a novel toll-like receptor-independent role of STING in modulating monocytic cell function and differentiation into macrophages. We further supported the interplay of STING1 variants and human biology by demonstrating how monocytes bearing the H232 allele were impaired in M1/M2 differentiation, interferon response and antigen presentation. Finally, we assessed the response to PD-1 inhibitor in a small cohort of melanoma patients stratified according to STING genotype. Given the contribution of the STING protein in sensing DNA viruses, bacterial pathogens and misplaced cancer DNA, these data may support the development of novel therapeutic options for infectious diseases and cancer.

Quality control testing of vaccines, including potency assessment, is critical to ensure equivalence of clinical lots. We developed a potency assay to support the clinical advancement of Nous-209, a cancer vaccine based on heterologous prime/boost administration of two multivalent viral vector products: GAd-209 and MVA-209. These consist of a mix of four Adeno (Great Ape Adenovirus; GAd) and four Modified Vaccinia Ankara (MVA) vectors respectively, each containing a different transgene encoding a synthetic polypeptide composed of antigenic peptide fragments joined one after the other. The potency assay employs quantitative Reverse Transcription PCR (RT-Q-PCR) to quantitatively measure the transcripts from the four transgenes encoded by each product in in vitro infected cells, enabling simultaneous detection. Results showcase the assay’s robustness and biological relevance, as it effectively detects potency loss in one component of the mixture comparably to in vivo immunogenicity testing. This report details the assay’s setup and validation, offering valuable insights for the clinical development of similar genetic vaccines, particularly those encoding synthetic polypeptides.

Background Tumor microenvironment (TME) represents a critical hurdle in cancer immunotherapy, given its ability to suppress antitumor immunity. Several efforts are made to overcome this hostile TME with the development of new therapeutic strategies modifying TME to boost antitumor immunity. Among these, cytokine-based approaches have been pursued for their known immunomodulatory effects on different cell populations within the TME. IL-12 is a potent pro-inflammatory cytokine that demonstrates striking immune activation and tumor control but causes severe adverse effects when systemically administered. Thus, local administration is considered a potential strategy to achieve high cytokine concentrations at the tumor site while sparing systemic adverse effects.Methods Modified Vaccinia Ankara (MVA) vector is a potent inducer of pro-inflammatory response. Here, we cloned IL-12 into the genome of MVA for intratumoral immunotherapy, combining the immunomodulatory properties of both the vector and the cargo. The antitumor activity of MVA-IL-12 and its effect on TME reprogramming were investigated in preclinical tumor models. RNA sequencing (RNA-Seq) analysis was performed to assess changes in the TME in treated and distal tumors and the effect on the intratumoral T-cell receptor repertoire.Results Intratumoral injection of MVA-IL-12 resulted in strong antitumor activity with the complete remission of established tumors in multiple murine models, including those resistant to checkpoint inhibitors. The therapeutic activity of MVA-IL-12 was associated with very low levels of circulating cytokine. Effective TME reprogramming was demonstrated on treatment, with the reduction of immunosuppressive M2 macrophages while increasing pro-inflammatory M1, and recruitment of dendritic cells. TME switch from immunosuppressive into immunostimulatory environment allowed for CD8 T cells priming and expansion leading to tumor attack.Conclusions Intratumoral administration of MVA-IL-12 turns immunologically ‘cold’ tumors ‘hot’ and overcomes resistance to programmed cell death protein-1 blockade.

IntroductionVirus vectored genetic vaccines (Vvgv) represent a promising approach for eliciting immune protection against infectious diseases and cancer. However, at variance with classical vaccines to date, no adjuvant has been combined with clinically approved genetic vaccines, possibly due to the detrimental effect of the adjuvant-induced innate response on the expression driven by the genetic vaccine vector. We reasoned that a potential novel approach to develop adjuvants for genetic vaccines would be to “synchronize” in time and space the activity of the adjuvant with that of the vaccine.MethodsTo this aim, we generated an Adenovirus vector encoding a murine anti-CTLA-4 monoclonal antibody (Ad-9D9) as a genetic adjuvant for Adenovirus based vaccines.ResultsThe co-delivery of Ad-9D9 with an Adeno-based COVID-19 vaccine encoding the Spike protein resulted in stronger cellular and humoral immune responses. In contrast, only a modest adjuvant effect was achieved when combining the vaccine with the same anti-CTLA-4 in its proteinaceous form. Importantly, the administration of the adjuvant vector at different sites of the vaccine vector abrogates the immunostimulatory effect. We showed that the adjuvant activity of Ad-α-CTLA-4 is independent from the vaccine antigen as it improved the immune response and efficacy of an Adenovirus based polyepitope vaccine encoding tumor neoantigens.DiscussionOur study demonstrated that the combination of Adenovirus Encoded Adjuvant (AdEnA) with an Adeno-encoded antigen vaccine enhances immune responses to viral and tumor antigens, representing a potent approach to develop more effective genetic vaccines.

The aim of personalized cancer vaccines is to elicit potent and tumor-specific immune responses against neoantigens specific to each patient and to establish durable immunity, while minimizing the adverse events. Over recent years, there has been a renewed interest in personalized cancer vaccines, primarily due to the advancement of innovative technologies for the identification of neoantigens and novel vaccine delivery platforms. Here, we review the emerging field of personalized cancer vaccination, with a focus on the use of viral vectors as a vaccine platform. The recent advancements in viral vector technology have led to the development of efficient production processes, positioning personalized viral vaccines as one of the preferred technologies. Many clinical trials have shown the feasibility, safety, immunogenicity and, more recently, preliminary evidence of the anti-tumor activity of personalized vaccination, fostering active research in the field, including further clinical trials for different tumor types and in different clinical settings.

Vaccination against neo-antigens has resulted in an effective antitumor response in several models. Here, the authors show that delivery of larger sets of neo-antigens using an adenovirus-based vaccination platform, results in much better tumor protection when combined with checkpoint blockade in a mouse model of advanced disease.

Neoantigens are tumor-specific antigens able to induce T-cell responses, generated by mutations in protein-coding regions of expressed genes. Previous studies demonstrated that only a limited subset of mutations generates neoantigens in microsatellite stable tumors. We developed a method, called VENUS (Vaccine-Encoded Neoantigens Unrestricted Selection), to prioritize mutated peptides with high potential to be neoantigens. Our method assigns to each mutation a weighted score that combines the mutation allelic frequency, the abundance of the transcript coding for the mutation, and the likelihood to bind the patient’s class-I major histocompatibility complex alleles. By ranking mutated peptides encoded by mutations detected in nine cancer patients, VENUS was able to select in the top 60 ranked peptides, the 95% of neoantigens experimentally validated including both CD8 and CD4 T cell specificities. VENUS was evaluated in a murine model in the context of vaccination with an adeno vector encoding the top ranked mutations prioritized in the MC38 cell line. Efficacy studies demonstrated anti tumoral activity of the vaccine when used in combination with checkpoint inhibitors. The results obtained highlight the importance of a combined scoring system taking into account multiple features of each tumor mutation to improve the accuracy of neoantigen prediction.

Abstract The Cyclophilin A (CypA)/Apoptosis Inducing Factor (AIF) complex is implicated in the DNA degradation in response to various cellular stress conditions, such as oxidative stress, cerebral hypoxia-ischemia and traumatic brain injury. The pro-apoptotic form of AIF (AIF(Δ1-121)) mainly interacts with CypA through the amino acid region 370–394. The AIF(370-394) synthetic peptide inhibits complex formation in vitro by binding to CypA and exerts neuroprotection in a model of glutamate-mediated oxidative stress. Here, the binding site of AIF(Δ1-121) and AIF(370-394) on CypA has been mapped by NMR spectroscopy and biochemical studies, and a molecular model of the complex has been proposed. We show that AIF(370-394) interacts with CypA on the same surface recognized by AIF(Δ1-121) protein and that the region is very close to the CypA catalytic pocket. Such region partially overlaps with the binding site of cyclosporin A (CsA), the strongest catalytic inhibitor of CypA. Our data point toward distinct CypA structural determinants governing the inhibitor selectivity and the differential biological effects of AIF and CsA, and provide new structural insights for designing CypA/AIF selective inhibitors with therapeutic relevance in neurodegenerative diseases.

We previously demonstrated that apoptosis induced by tocotrienols (γ and δT3) in HeLa cells is preceded by Ca2+ release from the endoplasmic reticulum. This event is eventually followed by the induction of specific calcium-dependent signals, leading to the expression and activation of the gene encoding for the IRE1α protein and, in turn, to the alternative splicing of the pro-apoptotic protein sXbp1 and other molecules involved in the unfolded protein response, the core pathway coping with EndoR stress. Here, we showed that treatment with T3s induces the expression of a specific set of miRNAs in HeLa cells. Data interrogation based on the intersection of this set of miRNAs with a set of genes previously differentially expressed after γT3 treatment provided a few miRNA candidates to be the effectors of EndoR-stress-induced apoptosis. To identify the best candidate to act as the effector of the Xbp1-mediated apoptotic response to γT3, we performed in silico analysis based on the evaluation of the highest ∆ in Gibbs energy of different mRNA–miRNA–Argonaute (AGO) protein complexes. The involvement of the best candidate identified in silico, miR-190b, in Xbp1 splicing was confirmed in vitro using T3-treated cells pre-incubated with the specific miRNA inhibitor, providing a preliminary indication of its role as an effector of EndoR-stress-induced apoptosis.

Zinc deficiency predisposes to a wide spectrum of chronic diseases. The human Zn proteome was predicted to represent about 10% of the total human proteome, reflecting the broad array of metabolic functions in which this micronutrient is known to participate. In the thyroid, Zn was reported to regulate cellular homeostasis, with a yet elusive mechanism. The Fischer Rat Thyroid Cell Line FRTL-5 cell model, derived from a Fischer rat thyroid and displaying a follicular cell phenotype, was used to investigate a possible causal relationship between intracellular Zn levels and thyroid function. A proteomic approach was applied to compare proteins expressed in Zn deficiency, obtained by treating cells with the Zn-specific chelator N,N,N′,N′-tetrakis (2-pyridylmethyl) ethylene-diamine (TPEN), with Zn repleted cells. Quantitative proteomic analysis of whole cell protein extracts was performed using stable isotope dimethyl labelling coupled to nano-ultra performance liquid chromatography-mass spectrometry (UPLC-MS). TPEN treatment led to almost undetectable intracellular Zn, while decreasing thyroglobulin secretion. Subsequent addition of ZnSO4 fully reversed these phenotypes. Comparative proteomic analysis of Zn depleted/repleted cells identified 108 proteins modulated by either treatment. Biological process enrichment analysis identified functions involved in calcium release and the regulation of translation as the most strongly regulated processes in Zn depleted cells.

Epidemiological studies suggest that moderate and prolonged consumption of coffee is associated with a reduced risk of developing type 2 diabetes but the molecular mechanisms underlying this effect are not known. In this study, we report the effects of physiological concentrations of caffeic acid, easily achievable by normal dietary habits, in endothelial cells cultured in 25 mM of glucose (high glucose, HG). In HG, the presence of 10 nM caffeic acid was associated with a decrease of glucose uptake but not to changes of GLUT-1 membrane localization or mRNA levels. Moreover, caffeic acid countered HG-induced loss of barrier integrity, reducing actin rearrangement and FITC-dextran passage. The decreased flux of glucose associated to caffeic acid affected HG induced apoptosis by down-regulating the expression of initiator (caspase 8 and 9) and effector caspases (caspase 7 and 3) and by increasing the levels of phosphorylated Bcl-2. We also observed that caffeic acid in HG condition was associated to a reduction of p65 subunit nuclear levels with respect to HG alone. NF-κB activation has been shown to lead to apoptosis in HG treated cells and the analysis of the expression of a panel of about 90 genes related to NF-κB signaling pathway revealed that caffeic acid significantly influenced gene expression changes induced by HG. In conclusion, our results suggest that caffeic acid, decreasing the metabolic stress induced by HG, allows the activation of survival mechanisms mediated by a different modulation of NF-κB-related signaling pathways and to the activation of anti-apoptotic proteins.

Rett syndrome (RTT) is mainly caused by mutations in the X-linked methyl-CpG binding protein (MeCP2) gene. By binding to methylated promoters on CpG islands, MeCP2 protein is able to modulate several genes and important cellular pathways. Therefore, mutations in MeCP2 can seriously affect the cellular phenotype. Today, the pathways that MeCP2 mutations are able to affect in RTT are not clear yet. The aim of our study was to investigate the gene expression profiles in peripheral blood lymphomonocytes (PBMC) isolated from RTT patients to try to evidence new genes and new pathways that are involved in RTT pathophysiology. LIMMA (Linear Models for MicroArray) and SAM (Significance Analysis of Microarrays) analyses on microarray data from 12 RTT patients and 7 control subjects identified 482 genes modulated in RTT, of which 430 were upregulated and 52 were downregulated. Functional clustering of a total of 146 genes in RTT identified key biological pathways related to mitochondrial function and organization, cellular ubiquitination and proteosome degradation, RNA processing, and chromatin folding. Our microarray data reveal an overexpression of genes involved in ATP synthesis suggesting altered energy requirement that parallels with increased activities of protein degradation. In conclusion, these findings suggest that mitochondrial-ATP-proteasome functions are likely to be involved in RTT clinical features.