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SignificanceΔNp63 is a master regulator of skin homeostasis since it finely controls keratinocyte differentiation and proliferation. Here, we provide cellular and molecular evidence demonstrating the functional role of a ΔNp63 interactor, the R-loop-resolving enzyme Senataxin (SETX), in fine-tuning keratinocyte differentiation. We found that SETX physically binds the p63 DNA-binding motif present in two early epidermal differentiation genes, Keratin 1 (KRT1) and ZNF750, facilitating R-loop removal over their 3' ends and thus allowing efficient transcriptional termination and gene expression. These molecular events translate into the inability of SETX-depleted keratinocytes to undergo the correct epidermal differentiation program. Remarkably, SETX is dysregulated in cutaneous squamous cell carcinoma, suggesting its potential involvement in the pathogenesis of skin disorders.
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Diferenciação Celular , DNA Helicases/metabolismo , Epiderme/metabolismo , Queratinócitos/metabolismo , Enzimas Multifuncionais/metabolismo , RNA Helicases/metabolismo , Fatores de Transcrição/metabolismo , Terminação da Transcrição Genética , Proteínas Supressoras de Tumor/metabolismo , DNA Helicases/genética , Humanos , Queratina-1/biossíntese , Queratina-1/genética , Células MCF-7 , Enzimas Multifuncionais/genética , RNA Helicases/genética , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética , Proteínas Supressoras de Tumor/biossíntese , Proteínas Supressoras de Tumor/genéticaRESUMO
The COVID-19 pandemic and the need for additional safe, effective, and affordable vaccines gave new impetus into development of vaccine genetic platforms. Here we report the findings from the phase 1, first-in-human, dose-escalation study of COVID-eVax, a DNA vaccine encoding the receptor binding domain (RBD) of the SARS-CoV-2 spike protein. Sixty-eight healthy adults received two doses of 0.5, 1, or 2 mg 28 days apart, or a single 2-mg dose, via intramuscular injection followed by electroporation, and they were monitored for 6 months. All participants completed the primary safety and immunogenicity assessments after 8 weeks. COVID-eVax was well tolerated, with mainly mild to moderate solicited adverse events (tenderness, pain, bruising, headache, and malaise/fatigue), less frequent after the second dose, and it induced an immune response (binding antibodies and/or T cells) at all prime-boost doses tested in up to 90% of the volunteers at the highest dose. However, the vaccine did not induce neutralizing antibodies, while particularly relevant was the T cell-mediated immunity, with a robust Th1 response. This T cell-skewed immunological response adds significant information to the DNA vaccine platform and should be assessed in further studies for its protective capacity and potential usefulness also in other therapeutic areas, such as oncology.
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COVID-19 , Vacinas de DNA , Adulto , Humanos , Anticorpos Neutralizantes , Anticorpos Antivirais , COVID-19/prevenção & controle , Vacinas contra COVID-19/efeitos adversos , Método Duplo-Cego , Pandemias/prevenção & controle , SARS-CoV-2 , Vacinas de DNA/efeitos adversosRESUMO
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), has caused more than 760 million cases and over 6.8 million deaths as of March 2023. Vaccination has been the main strategy used to contain the spread of the virus and to prevent hospitalizations and deaths. Currently, two mRNA-based vaccines and one adenovirus-vectored vaccine have been approved and are available for use in the U.S. population. The versatility, low cost, and rapid production of DNA vaccines provide important advantages over other platforms. Additionally, DNA vaccines efficiently induce both B- and T-cell responses by expressing the antigen within transfected host cells, and the antigen, after being processed into peptides, can associate with MHC class I or II of antigen-presenting cells (APCs) to stimulate different T cell responses. However, the efficiency of DNA vaccination needs to be improved for use in humans. Importantly, in vivo DNA delivery combined with electroporation (EP) has been used successfully in the field of veterinary oncology, resulting in high rates of response after electrochemotherapy. Here, we evaluate the safety, immunogenicity, and protective efficacy of a novel linear SARS-CoV-2 DNA vaccine candidate delivered by intramuscular injection followed by electroporation (Vet-ePorator™) in ferrets. The linear SARS-CoV-2 DNA vaccine candidate did not cause unexpected side effects. Additionally, the vaccine elicited neutralizing antibodies and T cell responses on day 42 post-immunization using a low dose of the linear DNA construct in a prime-boost regimen. Most importantly, vaccination significantly reduced shedding of infectious SARS-CoV-2 through oral and nasal secretions in a ferret model.
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COVID-19 , Vacinas de DNA , Vacinas Virais , Humanos , Animais , Vacinas contra COVID-19 , SARS-CoV-2 , COVID-19/prevenção & controle , Vacinas de DNA/genética , Furões , Eliminação de Partículas Virais , Anticorpos Antivirais , Anticorpos Neutralizantes , DNA , Glicoproteína da Espícula de Coronavírus/genética , Imunogenicidade da VacinaRESUMO
The COVID-19 pandemic caused by SARS-CoV-2 has made the development of safe and effective vaccines a critical priority. To date, four vaccines have been approved by European and American authorities for preventing COVID-19, but the development of additional vaccine platforms with improved supply and logistics profiles remains a pressing need. Here we report the preclinical evaluation of a novel COVID-19 vaccine candidate based on the electroporation of engineered, synthetic cDNA encoding a viral antigen in the skeletal muscle. We constructed a set of prototype DNA vaccines expressing various forms of the SARS-CoV-2 spike (S) protein and assessed their immunogenicity in animal models. Among them, COVID-eVax-a DNA plasmid encoding a secreted monomeric form of SARS-CoV-2 S protein receptor-binding domain (RBD)-induced the most potent anti-SARS-CoV-2 neutralizing antibody responses (including against the current most common variants of concern) and a robust T cell response. Upon challenge with SARS-CoV-2, immunized K18-hACE2 transgenic mice showed reduced weight loss, improved pulmonary function, and lower viral replication in the lungs and brain. COVID-eVax conferred significant protection to ferrets upon SARS-CoV-2 challenge. In summary, this study identifies COVID-eVax as an ideal COVID-19 vaccine candidate suitable for clinical development. Accordingly, a combined phase I-II trial has recently started.
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Vacinas contra COVID-19/administração & dosagem , COVID-19/prevenção & controle , Imunização/métodos , Modelos Animais , SARS-CoV-2/isolamento & purificação , Glicoproteína da Espícula de Coronavírus/imunologia , Vacinas de DNA/administração & dosagem , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , COVID-19/genética , COVID-19/virologia , Feminino , Furões , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Domínios Proteicos , Ratos Sprague-DawleyRESUMO
Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide, and several molecular pathways that underlie the molecular tumorigenesis of HNSCC have been identified. Among them, amplification or overexpression of ΔNp63 isoforms is observed in the majority of HNSCCs. Here, we unveiled a ΔNp63-dependent transcriptional program able to regulate the metabolism and the signaling of hyaluronic acid (HA), the major component of the extracellular matrix (ECM). We found that ∆Np63 is capable of sustaining the production of HA levels in cell culture and in vivo by regulating the expression of the HA synthase HAS3 and two hyaluronidase genes, HYAL-1 and HYAL-3. In addition, ∆Np63 directly regulates the expression of CD44, the major HA cell membrane receptor. By controlling this transcriptional program, ∆Np63 sustains the epithelial growth factor receptor (EGF-R) activation and the expression of ABCC1 multidrug transporter gene, thus contributing to tumor cell proliferation and chemoresistance. Importantly, p63 expression is positively correlated with CD44, HAS3, and ABCC1 expression in squamous cell carcinoma datasets and p63-HA pathway is a negative prognostic factor of HNSCC patient survival. Altogether, our data shed light on a ∆Np63-dependent pathway functionally important to the regulation of HNSCC progression.
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Biomarcadores Tumorais/metabolismo , Carcinogênese/genética , Carcinoma de Células Escamosas/metabolismo , Neoplasias de Cabeça e Pescoço/metabolismo , Ácido Hialurônico/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Biomarcadores Tumorais/genética , Carcinogênese/metabolismo , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Neoplasias de Cabeça e Pescoço/genética , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Receptores de Hialuronatos/genética , Receptores de Hialuronatos/metabolismo , Hialuronan Sintases/genética , Hialuronan Sintases/metabolismo , Ácido Hialurônico/genética , Hialuronoglucosaminidase/genética , Hialuronoglucosaminidase/metabolismo , Transdução de Sinais , Fatores de Transcrição/genética , Ativação Transcricional , Proteínas Supressoras de Tumor/genéticaRESUMO
Olaparib (AZD-2281, Ku-0059436) is an orally bioavailable and well-tolerated poly(ADP-ribose) polymerase (PARP) inhibitor currently under investigation in patients with solid tumors. To study the clinical potential of olaparib as a single-agent for the treatment of acute myeloid leukemia (AML) patients, we analyzed the in vitro sensitivity of AML cell lines and primary blasts. Clinically achievable concentrations of olaparib were able to induce cell death in the majority of primary AML case samples (88%) and tested cell lines. At these concentrations, olaparib preferentially killed leukemic blasts sparing normal lymphocytes derived from the same patient and did not substantially affect the viability of normal bone marrow and CD34-enriched peripheral blood cells obtained from healthy donors. Most primary AML analyzed were characterized by low BRCA1 mRNA level and undetectable protein expression that likely contributed to explain their sensitivity to olaparib. Noteworthy, while PARP1 over-expression was detected in blasts not responsive to olaparib, phosphorylation of the histone H2AFX (γH2AX) was associated with drug sensitivity. As to genetic features of tested cases the highest sensitivity was shown by a patient carrying a 11q23 deletion. The high sensitivity of AML blasts and the identification of biomarkers potentially able to predict response and/or resistance may foster further investigation of olaparib monotherapy for AML patients unfit to conventional chemotherapy.
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Proteína BRCA1/metabolismo , Biomarcadores Tumorais/metabolismo , Inibidores Enzimáticos/farmacologia , Histonas/metabolismo , Leucemia Mieloide Aguda/tratamento farmacológico , Ftalazinas/farmacologia , Piperazinas/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases , Proteína BRCA1/genética , Biomarcadores Tumorais/genética , Deleção Cromossômica , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HL-60 , Histonas/genética , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Masculino , Poli(ADP-Ribose) Polimerase-1 , Poli(ADP-Ribose) Polimerases/genética , Poli(ADP-Ribose) Polimerases/metabolismo , Células U937RESUMO
The COVID-19 pandemic, once a global crisis, is now largely under control, a testament to the extraordinary global efforts involving vaccination and public health measures. However, the relentless evolution of SARS-CoV-2, leading to the emergence of new variants, continues to underscore the importance of remaining vigilant and adaptable. Monoclonal antibodies (mAbs) have stood out as a powerful and immediate therapeutic response to COVID-19. Despite the success of mAbs, the evolution of SARS-CoV-2 continues to pose challenges and the available antibodies are no longer effective. New variants require the ongoing development of effective antibodies. In the present study, we describe the generation and characterization of neutralizing mAbs against the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein by combining plasmid DNA and recombinant protein vaccination. By integrating genetic immunization for rapid antibody production and the potent immune stimulation enabled by protein vaccination, we produced a rich pool of antibodies, each with unique binding and neutralizing specificities, tested with the ELISA, BLI and FACS assays and the pseudovirus assay, respectively. Here, we present a panel of mAbs effective against the SARS-CoV-2 variants up to Omicron BA.1 and BA.5, with the flexibility to target emerging variants. This approach ensures the preparedness principle is in place to address SARS-CoV-2 actual and future infections.
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Tumor-associated antigens (TAAs) represent attractive targets in the development of anti-cancer vaccines. The filamentous bacteriophage is a safe and versatile delivery nanosystem, and recombinant bacteriophages expressing TAA-derived peptides at a high density on the viral coat proteins improve TAA immunogenicity, triggering effective in vivo anti-tumor responses. To enhance the efficacy of the bacteriophage as an anti-tumor vaccine, we designed and generated phage particles expressing a CD8+ peptide derived from the human cancer germline antigen NY-ESO-1 decorated with the immunologically active lipid alpha-GalactosylCeramide (α-GalCer), a potent activator of invariant natural killer T (iNKT) cells. The immune response to phage expressing the human TAA NY-ESO-1 and delivering α-GalCer, namely fdNY-ESO-1/α-GalCer, was analyzed either in vitro or in vivo, using an HLA-A2 transgenic mouse model (HHK). By using NY-ESO-1-specific TCR-engineered T cells and iNKT hybridoma cells, we observed the efficacy of the fdNY-ESO-1/α-GalCer co-delivery strategy at inducing activation of both the cell subsets. Moreover, in vivo administration of fdNY-ESO-1 decorated with α-GalCer lipid in the absence of adjuvants strongly enhances the expansion of NY-ESO-1-specific CD8+ T cells in HHK mice. In conclusion, the filamentous bacteriophage delivering TAA-derived peptides and the α-GalCer lipid may represent a novel and promising anti-tumor vaccination strategy.
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Proteínas de Membrana , Neoplasias , Humanos , Camundongos , Animais , Proteínas de Membrana/metabolismo , Linfócitos T CD8-Positivos , Galactosilceramidas/metabolismo , Antígenos de Neoplasias , Peptídeos , Camundongos Transgênicos , Anticorpos/metabolismoRESUMO
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiologic agent of the COVID-19 pandemic, has been shown to infect a wide range of animal species, especially mammals, and besides human-to-human transmission, human-to-animal transmission has also been observed in some wild animals and pets, especially in cats. It has been demonstrated that cats are permissive to COVID-19 and are susceptible to airborne infections. Given the high transmissibility potential of SARS-CoV-2 to different host species and the close contact between humans and animals, it is crucial to find mechanisms to prevent the transmission chain and reduce the risk of spillover to susceptible species. Here, we show results from a clinical trial conducted in domestic cats to assess safety and immunogenicity of a linear DNA (linDNA) vaccine encoding the receptor-binding domain (RBD) from SARS-CoV-2 (Lin-COVID-eVax). Lin-COVID-eVax proved to be safe, with no significant adverse events, and was able to elicit both RBD-specific antibodies and T cells. Also, the linDNA vaccine induced neutralizing antibody titers against ancestral SARS-CoV-2 virus and its variants. These findings demonstrate the safety and immunogenicity of a genetic vaccine against COVID-19 administered to cats and strongly support the development of vaccines for preventing viral spread in susceptible species, especially those in close contact with humans.
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DNA integrity is a key issue in gene therapy and genetic vaccine approaches based on plasmid DNA. In contrast to messenger RNA that requires a controlled cold chain for efficacy, DNA molecules are considered to be more stable. In this study, we challenged this concept by characterizing the immunological response induced by a plasmid DNA vaccine delivered using electroporation. As a model, we used COVID-eVax, a plasmid DNA-based vaccine that targets the receptor binding domain (RBD) of the SARS-CoV-2 spike protein. Increased nicked DNA was produced by using either an accelerated stability protocol or a lyophilization protocol. Surprisingly, the immune response induced in vivo was only minimally affected by the percentage of open circular DNA. This result suggests that plasmid DNA vaccines, such as COVID-eVax that have recently completed a phase I clinical trial, retain their efficacy upon storage at higher temperatures, and this feature may facilitate their use in low-/middle-income countries.
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Background: Few data are available about the durability of the response, the induction of neutralizing antibodies, and the cellular response upon the third dose of the anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine in hemato-oncological patients. Objective: To investigate the antibody and cellular response to the BNT162b2 vaccine in patients with hematological malignancy. Methods: We measured SARS-CoV-2 anti-spike antibodies, anti-Omicron neutralizing antibodies, and T-cell responses 1 month after the third dose of vaccine in 93 fragile patients with hematological malignancy (FHM), 51 fragile not oncological subjects (FNO) aged 80-92, and 47 employees of the hospital (healthcare workers, (HW), aged 23-66 years. Blood samples were collected at day 0 (T0), 21 (T1), 35 (T2), 84 (T3), 168 (T4), 351 (T pre-3D), and 381 (T post-3D) after the first dose of vaccine. Serum IgG antibodies against S1/S2 antigens of SARS-CoV-2 spike protein were measured at every time point. Neutralizing antibodies were measured at T2, T3 (anti-Alpha), T4 (anti-Delta), and T post-3D (anti-Omicron). T cell response was assessed at T post-3D. Results: An increase in anti-S1/S2 antigen antibodies compared to T0 was observed in the three groups at T post-3D. After the third vaccine dose, the median antibody level of FHM subjects was higher than after the second dose and above the putative protection threshold, although lower than in the other groups. The neutralizing activity of antibodies against the Omicron variant of the virus was tested at T2 and T post-3D. 42.3% of FHM, 80,0% of FNO, and 90,0% of HW had anti-Omicron neutralizing antibodies at T post-3D. To get more insight into the breadth of antibody responses, we analyzed neutralizing capacity against BA.4/BA.5, BF.7, BQ.1, XBB.1.5 since also for the Omicron variants, different mutations have been reported especially for the spike protein. The memory T-cell response was lower in FHM than in FNO and HW cohorts. Data on breakthrough infections and deaths suggested that the positivity threshold of the test is protective after the third dose of the vaccine in all cohorts. Conclusion: FHM have a relevant response to the BNT162b2 vaccine, with increasing antibody levels after the third dose coupled with, although low, a T-cell response. FHM need repeated vaccine doses to attain a protective immunological response.
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COVID-19 , Neoplasias Hematológicas , Glicoproteína da Espícula de Coronavírus , Humanos , Vacinas contra COVID-19 , Vacina BNT162 , COVID-19/prevenção & controle , SARS-CoV-2 , Anticorpos Neutralizantes , Anticorpos AntiviraisRESUMO
Immune checkpoint inhibitors (ICI) based on anti-CTLA-4 (αCTLA-4) and anti-PD1 (αPD1) are being tested in combination with different therapeutic approaches including other immunotherapies such as neoantigen cancer vaccines (NCV). Here we explored, in two cancer murine models, different therapeutic combinations of ICI with personalized DNA vaccines expressing neoantigens and delivered by electroporation (EP). Anti-cancer efficacy was evaluated using vaccines with or without CD4 epitopes. Therapeutic DNA vaccines showed synergistic effects in different therapeutic protocols including established large tumors. Flow cytometry (FC) was utilized to measure CD8, CD4, Treg, and switched B cells as well as neoantigen-specific immune responses, which were also measured by IFN-γ ELIspot. Immune responses were augmented in combination with αCTLA4 but not with αPD1 in the MC38 tumor-bearing mice, significantly impacting tumor growth. Similarly, neoantigen-specific T cell immune responses were enhanced in combined treatment with αCTLA-4 in the CT26 tumor model where large tumors regressed in all mice, while monotherapy with αCTLA-4 was less efficacious. In line with previous evidence, we observed an increased switched B cells in the spleen of mice treated with αCTLA-4 alone or in combination with NCV. These results support the use of NCV delivered by DNA-EP with αCTLA-4 and suggest a new combined therapy for clinical testing.
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The COVID-19 pandemic is entering a new era with the approval of many SARS-CoV-2 vaccines. In spite of the restoration of an almost normal way of life thanks to the immune protection elicited by these innovative vaccines, we are still facing high viral circulation, with a significant number of deaths. To further explore alternative vaccination platforms, we developed COVID-eVax-a genetic vaccine based on plasmid DNA encoding the RBD domain of the SARS-CoV-2 spike protein. Here, we describe the correlation between immune responses and the evolution of viral infection in ferrets infected with the live virus. We demonstrate COVID-eVax immunogenicity as means of antibody response and, above all, a significant T-cell response, thus proving the critical role of T-cell immunity, in addition to the neutralizing antibody activity, in controlling viral spread.
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BACKGROUND: DNA-based vaccines represent a simple, safe and promising strategy for harnessing the immune system to fight infectious diseases as well as various forms of cancer and thus are considered an important tool in the cancer immunotherapy toolbox. Nonetheless, the manufacture of plasmid DNA vaccines has several drawbacks, including long lead times and the need to remove impurities from bacterial cultures. Here we report the development of polymerase chain reaction (PCR)-produced amplicon expression vectors as DNA vaccines and their in vivo application to elicit antigen-specific immune responses in animal cancer models. METHODS: Plasmid DNA and amplicon expression was assessed both in vitro, by Hela cells transfection, and in vivo, by evaluating luciferase expression in wild-type mice through optical imaging. Immunogenicity induced by DNA amplicons was assessed by vaccinating wild-type mice against a tumor-associated antigen, whereas the antitumoral effect of DNA amplicons was evaluated in a murine cancer model in combination with immune-checkpoint inhibitors (ICIs). RESULTS: Amplicons encoding tumor-associated-antigens, such as telomerase reverse transcriptase or neoantigens expressed by murine tumor cell lines, were able to elicit antigen-specific immune responses and proved to significantly impact tumor growth when administered in combination with ICIs. CONCLUSIONS: These results strongly support the further exploration of the use of PCR-based amplicons as an innovative immunotherapeutic approach to cancer treatment.
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Vacinas Anticâncer , Neoplasias , Vacinas de DNA , Animais , Antígenos de Neoplasias , DNA , Células HeLa , Humanos , Imunoterapia/métodos , Camundongos , Neoplasias/tratamento farmacológico , Neoplasias/terapiaRESUMO
Cancer is a heterogeneous disease and its treatment remains unsatisfactory with inconstant therapeutic responses. This variability could be related, at least in part, to different and highly personalized gut microbiota compositions. Different studies have shown an impact of microbiota on antitumor therapy. It has been demonstrated that some gut bacteria influences the development and differentiation of immune cells, suggesting that different microbiota compositions could affect the efficacy of the antitumor vaccine. Emerging data suggest that recognition of neoantigens for the generation of neoantigen cancer vaccines (NCVs) could have a key role in the activity of clinical immunotherapies. However, it is still unknown whether there is a crosstalk between microbiota and NCV. This study aimed to understand the possible mechanisms of interaction between gut microbiota and NCV delivered by DNA-electroporation (DNA-EP). We found that decreased microbiota diversity induced by prolonged antibiotic (ATB) treatment is associated with higher intratumor specific immune responses and consequently to a better antitumor effect induced by NCV delivered by DNA-EP.
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Vacinas Anticâncer , Microbiota , Neoplasias , Antígenos de Neoplasias , Eletroporação , Imunoterapia , Neoplasias/tratamento farmacológicoRESUMO
The endoplasmic reticulum aminopeptidase ERAP1 regulates innate and adaptive immune responses by trimming peptides for presentation by major histocompatibility complex (MHC) class I molecules. Previously, we have shown that genetic or pharmacological inhibition of ERAP1 on murine and human tumor cell lines perturbs the engagement of NK cell inhibitory receptors Ly49C/I and Killer-cell Immunoglobulin-like receptors (KIRs), respectively, by their specific ligands (MHC class I molecules), thus leading to NK cell killing. However, the effect of ERAP1 inhibition in tumor cells was highly variable, suggesting that its efficacy may depend on several factors, including MHC class I typing. To identify MHC class I alleles and KIRs that are more sensitive to ERAP1 depletion, we stably silenced ERAP1 expression in human HLA class I-negative B lymphoblastoid cell line 721.221 (referred to as 221) transfected with a panel of KIR ligands (i.e. HLA-B*51:01, -Cw3, -Cw4 and -Cw7), or HLA-A2 which does not bind any KIR, and tested their ability to induce NK cell degranulation and cytotoxicity. No change in HLA class I surface expression was detected in all 221 transfectant cells after ERAP1 depletion. In contrast, CD107a expression levels were significantly increased on NK cells stimulated with 221-B*51:01 cells lacking ERAP1, particularly in the KIR3DL1-positive NK cell subset. Consistently, genetic or pharmacological inhibition of ERAP1 impaired the recognition of HLA-B*51:01 by the YTS NK cell overexpressing KIR3DL1*001, suggesting that ERAP1 inhibition renders HLA-B*51:01 molecules less eligible for binding to KIR3DL1. Overall, these results identify HLA-B*51:01/KIR3DL1 as one of the most susceptible combinations for ERAP1 inhibition, suggesting that individuals carrying HLA-B*51:01-like antigens may be candidates for immunotherapy based on pharmacological inhibition of ERAP1.
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Aminopeptidases/metabolismo , Antígeno HLA-B51/metabolismo , Células Matadoras Naturais/enzimologia , Antígenos de Histocompatibilidade Menor/metabolismo , Neoplasias/enzimologia , Receptores KIR3DL1/metabolismo , Aminopeptidases/antagonistas & inibidores , Aminopeptidases/genética , Antineoplásicos/farmacologia , Degranulação Celular , Linhagem Celular , Técnicas de Cocultura , Citotoxicidade Imunológica , Inibidores Enzimáticos/farmacologia , Antígeno HLA-B51/genética , Humanos , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/imunologia , Antígenos de Histocompatibilidade Menor/genética , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/imunologia , Receptores KIR3DL1/genética , Transdução de SinaisRESUMO
COVID-19 is a highly infectious disease caused by a newly emerged coronavirus (SARS-CoV-2) that has rapidly progressed into a pandemic. This unprecedent emergency has stressed the significance of developing effective therapeutics to fight the current and future outbreaks. The receptor-binding domain (RBD) of the SARS-CoV-2 surface Spike protein is the main target for vaccines and represents a helpful "tool" to produce neutralizing antibodies or diagnostic kits. In this work, we provide a detailed characterization of the native RBD produced in three major model systems: Escherichia coli, insect and HEK-293 cells. Circular dichroism, gel filtration chromatography and thermal denaturation experiments indicated that recombinant SARS-CoV-2 RBD proteins are stable and correctly folded. In addition, their functionality and receptor-binding ability were further evaluated through ELISA, flow cytometry assays and bio-layer interferometry.
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COVID-19/metabolismo , SARS-CoV-2/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , Animais , Linhagem Celular , Escherichia coli/genética , Expressão Gênica , Células HEK293 , Humanos , Insetos/citologia , Ligação Proteica , Desnaturação Proteica , Domínios Proteicos , Dobramento de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , SARS-CoV-2/química , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genéticaRESUMO
In this study, we explored whether Nutlin-3a, a well-known, nontoxic small-molecule compound antagonizing the inhibitory interaction of MDM2 with the tumor suppressor p53, may restore ligands for natural killer (NK) cell-activating receptors (NK-AR) on neuroblastoma cells to enhance the NK cell-mediated killing. Neuroblastoma cell lines were treated with Nutlin-3a, and the expression of ligands for NKG2D and DNAM-1 NK-ARs and the neuroblastoma susceptibility to NK cells were evaluated. Adoptive transfer of human NK cells in a xenograft neuroblastoma-bearing NSG murine model was assessed. Two data sets of neuroblastoma patients were explored to correlate p53 expression with ligand expression. Luciferase assays and chromatin immunoprecipitation analysis of p53 functional binding on PVR promoter were performed. Primary neuroblastoma cells were also treated with Nutlin-3a, and neuroblastoma spheroids obtained from one high-risk patient were assayed for NK-cell cytotoxicity. We provide evidence showing that the Nutlin-3a-dependent rescue of p53 function in neuroblastoma cells resulted in (i) increased surface expression of ligands for NK-ARs, thus rendering neuroblastoma cell lines significantly more susceptible to NK cell-mediated killing; (ii) shrinkage of human neuroblastoma tumor masses that correlated with overall survival upon adoptive transfer of NK cells in neuroblastoma-bearing mice; (iii) and increased expression of ligands in primary neuroblastoma cells and boosting of NK cell-mediated disaggregation of neuroblastoma spheroids. We also found that p53 was a direct transcription factor regulating the expression of PVR ligand recognized by DNAM-1. Our findings demonstrated an immunomodulatory role of Nutlin-3a, which might be prospectively used for a novel NK cell-based immunotherapy for neuroblastoma.
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Antígenos de Diferenciação de Linfócitos T/imunologia , Imidazóis/farmacologia , Células Matadoras Naturais/imunologia , Subfamília K de Receptores Semelhantes a Lectina de Células NK/imunologia , Neuroblastoma/tratamento farmacológico , Piperazinas/farmacologia , Proteína Supressora de Tumor p53/metabolismo , Animais , Antígenos de Diferenciação de Linfócitos T/biossíntese , Linhagem Celular Tumoral , Citotoxicidade Imunológica , Feminino , Humanos , Ligantes , Camundongos , Camundongos Endogâmicos NOD , Subfamília K de Receptores Semelhantes a Lectina de Células NK/biossíntese , Neuroblastoma/imunologia , Neuroblastoma/patologia , Receptores de Células Matadoras Naturais/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Endoplasmic reticulum aminopeptidases ERAP1 and ERAP2 have recently emerged as important players in regulating innate and adaptive immune responses by trimming peptide ligands for MHC class I molecules. Functional polymorphisms in ERAP1 and ERAP2 genes have been associated with predisposition to several diseases including autoimmune diseases, viral infections, and virally induced cancers. In this chapter, we describe two basic methods for monitoring peptide-trimming activity by ER aminopeptidases and screening potential chemical inhibitors.
Assuntos
Aminopeptidases/metabolismo , Apresentação de Antígeno/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Antígenos de Histocompatibilidade Menor/metabolismo , Biologia Molecular/métodos , Peptídeos/metabolismo , Aminopeptidases/antagonistas & inibidores , Fluorescência , Corantes Fluorescentes/metabolismo , Células HeLa , Humanos , Sefarose/química , Especificidade por SubstratoRESUMO
The endoplasmic reticulum (ER) aminopeptidases ERAP1 and ERAP2 are two multifunctional enzymes playing an important role in the biological processes requiring trimming of substrates, including the generation of major histocompatibility complex (MHC) class I binding peptides. In the absence of ERAP enzymes, the cells exhibit a different pool of peptides on their surface which can promote both NK and CD8+ T cell-mediated immune responses. The expression of ERAP1 and ERAP2 is frequently altered in tumors, as compared to their normal counterparts, but how this affects tumor growth and anti-tumor immune responses has been little investigated. This review will provide an overview of current knowledge on transcriptional and post-transcriptional regulations of ERAP enzymes, and will discuss the contribution of recent studies to our understanding of ERAP1 and ERAP2 role in cancer immunity.