RESUMO
BACKGROUND: Chitinase-like proteins (CLPs) play a key role in immunosuppression under inflammatory conditions such as cancer. CLPs are enzymatically inactive and become neutralized upon binding of their natural ligand chitin, potentially reducing CLP-driven immunosuppression. We investigated the efficacy of chitin treatment in the context of triple-negative breast cancer (TNBC) using complementary mouse models. We also evaluated the immunomodulatory influence of chitin on immune checkpoint blockade (ICB) and compared its efficacy as general CLP blocker with blockade of a single CLP, i.e. chitinase 3-like 1 (CHI3L1). METHODS: Female BALB/c mice were intraductally injected with luciferase-expressing 4T1 or 66cl4 cells and systemically treated with chitin in combination with or without anti-programmed death (PD)-1 ICB. For single CLP blockade, tumor-bearing mice were treated with anti-CHI3L1 antibodies. Metastatic progression was monitored through bioluminescence imaging. Immune cell changes in primary tumors and lymphoid organs (i.e. axillary lymph nodes and spleen) were investigated through flow cytometry, immunohistochemistry, cytokine profiling and RNA-sequencing. CHI3L1-stimulated RAW264.7 macrophages were subjected to 2D lymphatic endothelial cell adhesion and 3D lymphatic integration in vitro assays for studying macrophage-mediated lymphatic remodeling. RESULTS: Chitin significantly reduced primary tumor progression in the 4T1-based model by decreasing the high production of CLPs that originate from tumor-associated neutrophils (TANs) and Stat3 signaling, prominently affecting the CHI3L1 and CHI3L3 primary tumor levels. It reduced immunosuppressive cell types and increased anti-tumorigenic T-cells in primary tumors as well as axillary lymph nodes. Chitin also significantly reduced CHI3L3 primary tumor levels and immunosuppression in the 66cl4-based model. Compared to anti-CHI3L1, chitin enhanced primary tumor growth reduction and anti-tumorigenicity. Both treatments equally inhibited lymphatic adhesion and integration of macrophages, thereby hampering lymphatic tumor cell spreading. Upon ICB combination therapy, chitin alleviated anti-PD-1 resistance in both TNBC models, providing a significant add-on reduction in primary tumor and lung metastatic growth compared to chitin monotherapy. These add-on effects occurred through additional increase in CD8α+ T-cell infiltration and activation in primary tumor and lymphoid organs. CONCLUSIONS: Chitin, as a general CLP blocker, reduces CLP production, enhances anti-tumor immunity as well as ICB responses, supporting its potential clinical relevance in immunosuppressed TNBC patients.
Assuntos
Quitina , Quitinases , Neoplasias de Mama Triplo Negativas , Animais , Feminino , Humanos , Camundongos , Linhagem Celular Tumoral , Quitina/farmacologia , Quitina/uso terapêutico , Quitinases/uso terapêutico , Terapia de Imunossupressão , Metástase Linfática , Proteínas/uso terapêutico , Neoplasias de Mama Triplo Negativas/patologiaRESUMO
Poly(I:C) is a synthetic analogue of dsRNA capable of activating both TLR3 and RLRs, such as MDA-5 and RIG-I, as pathogen recognition receptors. While poly(I:C) is known to provoke a robust type I IFN, type III IFN, and Th1 cytokine response, its therapeutic use as a vaccine adjuvant is limited due to its vulnerability to nucleases and poor uptake by immune cells. is encapsulated poly(I:C) into lipid nanoparticles (LNPs) containing an ionizable cationic lipid that can electrostatically interact with poly(I:C). LNP-formulated poly(I:C) triggered both lysosomal TLR3 and cytoplasmic RLRs, in vitro and in vivo, whereas poly(I:C) in an unformulated soluble form only triggered endosomal-localized TLR3. Administration of LNP-formulated poly(I:C) in mouse models led to efficient translocation to lymphoid tissue and concurrent innate immune activation following intramuscular (IM) administration, resulting in a significant increase in innate immune activation compared to unformulated soluble poly(I:C). When used as an adjuvant for recombinant full-length SARS-CoV-2 spike protein, LNP-formulated poly(I:C) elicited potent anti-spike antibody titers, surpassing those of unformulated soluble poly(I:C) by orders of magnitude and offered complete protection against a SARS-CoV-2 viral challenge in vivo, and serum from these mice are capable of significantly reducing viral infection in vitro.
Assuntos
Lipossomos , Nanopartículas , Poli I-C , Glicoproteína da Espícula de Coronavírus , Receptor 3 Toll-Like , Animais , Camundongos , Humanos , Receptor 3 Toll-Like/genética , Receptor 3 Toll-Like/metabolismo , Adjuvantes Imunológicos/farmacologiaRESUMO
Self-amplifying RNA vaccines may induce equivalent or more potent immune responses at lower doses compared to non-replicating mRNA vaccines via amplified antigen expression. In this paper, we demonstrate that 1 µg of an LNP-formulated dual-antigen self-amplifying RNA vaccine (ZIP1642), encoding both the S-RBD and N antigen, elicits considerably higher neutralizing antibody titers against Wuhan-like Beta B.1.351 and Delta B.1.617.2 SARS-CoV-2 variants compared to those of convalescent patients. In addition, ZIP1642 vaccination in mice expanded both S- and N-specific CD3+CD4+ and CD3+CD8+ T cells and caused a Th1 shifted cytokine response. We demonstrate that the induction of such dual antigen-targeted cell-mediated immune response may provide better protection against variants displaying highly mutated Spike proteins, as infectious viral loads of both Wuhan-like and Beta variants were decreased after challenge of ZIP1642 vaccinated hamsters. Supported by these results, we encourage redirecting focus toward the induction of multiple antigen-targeted cell-mediated immunity in addition to neutralizing antibody responses to bypass waning antibody responses and attenuate infectious breakthrough and disease severity of future SARS-CoV-2 variants.
Assuntos
COVID-19 , Vacinas Virais , Animais , Anticorpos Neutralizantes , Anticorpos Antivirais , Linfócitos T CD8-Positivos , COVID-19/prevenção & controle , Vacinas contra COVID-19 , Cricetinae , Humanos , Imunidade Celular , Imunidade Humoral , Camundongos , Camundongos Endogâmicos BALB C , RNA , SARS-CoV-2/genética , Vacinação , Vacinas Sintéticas , Vacinas de mRNARESUMO
Synthetic mRNAs are an appealing platform with multiple biomedical applications ranging from protein replacement therapy to vaccination. In comparison with conventional mRNA, synthetic self-amplifying mRNAs (sa-mRNAs) are gaining interest because of their higher and longer-lasting expression. However, sa-mRNAs also elicit an innate immune response, which may complicate their clinical application. Approaches to reduce the innate immunity of sa-mRNAs have not been studied in detail. Here we investigated, in vivo, the effect of several innate immune inhibitors and a novel cellulose-based mRNA purification approach on the type I interferon (IFN) response and the translation and vaccination efficacy of our formerly developed sa-mRNA vaccine against Zika virus. Among the investigated inhibitors, we found that corticosteroids and especially topical application of clobetasol at the sa-mRNA injection site was the most efficient in suppressing the type I IFN response and increasing the translation of sa-mRNA. However, clobetasol prevented formation of antibodies against sa-mRNA-encoded antigens and should therefore be avoided in a vaccination context. Residual dsRNA by-products of the in vitro transcription reaction are known inducers of immediate type I IFN responses. We additionally demonstrate a drastic reduction of these dsRNA by-products upon cellulose-based purification, reducing the innate immune response and improving sa-mRNA vaccination efficacy.
Assuntos
Imunidade Inata/genética , RNA Mensageiro/genética , Vacinação , Infecção por Zika virus/tratamento farmacológico , Corticosteroides/química , Celulose/química , Clobetasol/farmacologia , Regulação da Expressão Gênica/genética , Humanos , Imunidade Inata/efeitos dos fármacos , Imunidade Inata/imunologia , Interferon Tipo I/genética , Interferon Tipo I/imunologia , Biossíntese de Proteínas/efeitos dos fármacos , Biossíntese de Proteínas/imunologia , RNA Mensageiro/síntese química , RNA Mensageiro/química , RNA Mensageiro/farmacologia , Zika virus/efeitos dos fármacos , Zika virus/patogenicidade , Infecção por Zika virus/imunologia , Infecção por Zika virus/virologiaRESUMO
Synthetic mRNA therapeutics have the potential to revolutionize healthcare, as they enable patients to produce therapeutic proteins inside their own bodies. However, convenient methods that allow external control over the timing and magnitude of protein production after in vivo delivery of synthetic mRNA are lacking. In this study, we validate the in vivo utility of a synthetic self-amplifying mRNA (RNA replicon) whose expression can be turned off using a genetic switch that responds to oral administration of trimethoprim (TMP), a US Food and Drug Administration (FDA)-approved small-molecule drug. After intramuscular electroporation, the engineered RNA replicon exhibited dose-dependent and reversible expression of its encoded protein upon TMP administration. The TMP serum level needed for maximal downregulation of protein translation was approximately 45-fold below that used in humans for therapeutic purposes. To demonstrate the therapeutic potential of the technology, we injected mice with a TMP-responsive RNA replicon encoding erythropoietin (EPO) and successfully controlled the timing and magnitude of EPO production as well as changes in hematocrit. This work demonstrates the feasibility of controlling mRNA kinetics in vivo, thereby broadly expanding the clinical versatility of mRNA therapeutics.
Assuntos
Eritropoetina/metabolismo , Antagonistas do Ácido Fólico/administração & dosagem , Biossíntese de Proteínas , RNA Mensageiro/metabolismo , Replicon , Trimetoprima/administração & dosagem , Animais , Eletroporação , Eritropoetina/genética , Feminino , Terapia Genética , Injeções Intramusculares , Camundongos , Camundongos Endogâmicos BALB C , RNA Mensageiro/genéticaRESUMO
Small-molecular Toll-like receptor 7/8 (TLR7/8) agonists hold promise as immune modulators for a variety of immune therapeutic purposes including cancer therapy or vaccination. However, due to their rapid systemic distribution causing difficult-to-control inflammatory off-target effects, their application is still problematic, in particular systemically. To address this problem, we designed and robustly fabricated pH-responsive nanogels serving as versatile immunodrug nanocarriers for safe delivery of TLR7/8-stimulating imidazoquinolines after intravenous administration. To this aim, a primary amine-reactive methacrylamide monomer bearing a pendant squaric ester amide is introduced, which is polymerized under controlled RAFT polymerization conditions. Corresponding PEG-derived squaric ester amide block copolymers self-assemble into precursor micelles in polar protic solvents. Their cores are amine-reactive and can sequentially be transformed by acid-sensitive cross-linkers, dyes, and imidazoquinolines. Remaining squaric ester amides are hydrophilized affording fully hydrophilic nanogels with profound stability in human plasma but stimuli-responsive degradation upon exposure to endolysosomal pH conditions. The immunomodulatory behavior of the imidazoquinolines alone or conjugated to the nanogels was demonstrated by macrophages in vitro. In vivo, however, we observed a remarkable impact of the nanogel: After intravenous injection, a spatially controlled immunostimulatory activity was evident in the spleen, whereas systemic off-target inflammatory responses triggered by the small-molecular imidazoquinoline analogue were absent. These findings underline the potential of squaric ester-based, pH-degradable nanogels as a promising platform to permit intravenous administration routes of small-molecular TLR7/8 agonists and, thus, the opportunity to explore their adjuvant potency for systemic vaccination or cancer immunotherapy purposes.
Assuntos
Adjuvantes Imunológicos/química , Ésteres/química , Nanogéis/química , Receptor 7 Toll-Like/agonistas , Receptor 8 Toll-Like/agonistas , Animais , Portadores de Fármacos/química , Liberação Controlada de Fármacos , Humanos , Concentração de Íons de Hidrogênio , Imunoterapia , Camundongos Endogâmicos BALB C , Micelas , Imagem Óptica , Polimerização , Polímeros/químicaRESUMO
The search for vaccines that protect from severe morbidity and mortality because of infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes coronavirus disease 2019 (COVID-19) is a race against the clock and the virus. Here we describe an amphiphilic imidazoquinoline (IMDQ-PEG-CHOL) TLR7/8 adjuvant, consisting of an imidazoquinoline conjugated to the chain end of a cholesterol-poly(ethylene glycol) macromolecular amphiphile. It is water-soluble and exhibits massive translocation to lymph nodes upon local administration through binding to albumin, affording localized innate immune activation and reduction in systemic inflammation. The adjuvanticity of IMDQ-PEG-CHOL was validated in a licensed vaccine setting (quadrivalent influenza vaccine) and an experimental trimeric recombinant SARS-CoV-2 spike protein vaccine, showing robust IgG2a and IgG1 antibody titers in mice that could neutralize viral infection in vitro and in vivo in a mouse model.
Assuntos
Adjuvantes Imunológicos/uso terapêutico , Vacinas contra COVID-19/uso terapêutico , COVID-19/prevenção & controle , Imidazóis/uso terapêutico , Imunidade Inata/efeitos dos fármacos , Quinolinas/uso terapêutico , Animais , Vacinas contra COVID-19/imunologia , Colesterol/análogos & derivados , Colesterol/imunologia , Colesterol/uso terapêutico , Feminino , Humanos , Imidazóis/imunologia , Vírus da Influenza A Subtipo H1N1/efeitos dos fármacos , Vacinas contra Influenza/imunologia , Vacinas contra Influenza/uso terapêutico , Influenza Humana/prevenção & controle , Glicoproteínas de Membrana/agonistas , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Polietilenoglicóis/uso terapêutico , Quinolinas/imunologia , Proteínas Recombinantes/imunologia , SARS-CoV-2/efeitos dos fármacos , Glicoproteína da Espícula de Coronavírus/imunologia , Tensoativos/uso terapêutico , Receptor 7 Toll-Like/agonistas , Receptor 8 Toll-Like/agonistasRESUMO
Synthetic immune-stimulatory drugs such as agonists of the Toll-like receptors (TLR) 7/8 are potent activators of antigen-presenting cells (APCs), however, they also induce severe side effects due to leakage from the site of injection into systemic circulation. Here, we report on the design and synthesis of an amphiphilic polymer-prodrug conjugate of an imidazoquinoline TLR7/8 agonist that in aqueous medium forms vesicular structures of 200 nm. The conjugate contains an endosomal enzyme-responsive linker enabling degradation of the vesicles and release of the TLR7/8 agonist in native form after endocytosis, which results in high in vitro TLR agonist activity. In a mouse model, locally administered vesicles provoke significantly more potent and long-lasting immune stimulation in terms of interferon expression at the injection site and in draining lymphoid tissue compared to a nonamphiphilic control and the native TLR agonist. Moreover, the vesicles induce robust activation of dendritic cells in the draining lymph node in vivo.
Assuntos
Imidazóis/farmacologia , Glicoproteínas de Membrana/agonistas , Pró-Fármacos/farmacologia , Quinolinas/farmacologia , Receptor 7 Toll-Like/agonistas , Receptor 8 Toll-Like/agonistas , beta-Galactosidase/imunologia , Animais , Imidazóis/química , Imidazóis/metabolismo , Imunidade Inata/efeitos dos fármacos , Imunidade Inata/imunologia , Glicoproteínas de Membrana/imunologia , Camundongos , Estrutura Molecular , Tamanho da Partícula , Polietilenoglicóis/química , Polietilenoglicóis/metabolismo , Polietilenoglicóis/farmacologia , Pró-Fármacos/química , Pró-Fármacos/metabolismo , Quinolinas/química , Quinolinas/metabolismo , Propriedades de Superfície , Receptor 7 Toll-Like/imunologia , Receptor 8 Toll-Like/imunologia , beta-Galactosidase/química , beta-Galactosidase/metabolismoRESUMO
Synthetic mRNA is an attractive vehicle for gene therapies because of its transient nature and improved safety profile over DNA. However, unlike DNA, broadly applicable methods to control expression from mRNA are lacking. Here we describe a platform for small-molecule-based regulation of expression from modified RNA (modRNA) and self-replicating RNA (replicon) delivered to mammalian cells. Specifically, we engineer small-molecule-responsive RNA binding proteins to control expression of proteins from RNA-encoded genetic circuits. Coupled with specific modRNA dosages or engineered elements from a replicon, including a subgenomic promoter library, we demonstrate the capability to externally regulate the timing and level of protein expression. These control mechanisms facilitate the construction of ON, OFF, and two-output switches, with potential therapeutic applications such as inducible cancer immunotherapies. These circuits, along with other synthetic networks that can be developed using these tools, will expand the utility of synthetic mRNA as a therapeutic modality.
Assuntos
Redes Reguladoras de Genes , Terapia Genética/métodos , Regiões Promotoras Genéticas , RNA Mensageiro/química , Proteínas de Ligação a RNA/química , RNA/química , Animais , Linhagem Celular , Cricetinae , DNA/química , Biblioteca Gênica , Engenharia Genética , Células HEK293 , Humanos , Imunoterapia , Camundongos , RNA Interferente Pequeno/metabolismo , Biologia SintéticaRESUMO
Uncontrolled systemic inflammatory immune triggering has hampered the clinical translation of several classes of small-molecule immunomodulators, such as imidazoquinoline TLR7/8 agonists for vaccine design and cancer immunotherapy. By taking advantage of the inherent serum-protein-binding property of lipid motifs and their tendency to accumulate in lymphoid tissue, we designed amphiphilic lipid-polymer conjugates that suppress systemic inflammation but provoke potent lymph-node immune activation. This work provides a rational basis for the design of lipid-polymer amphiphiles for optimized lymphoid targeting.
Assuntos
Imunidade Inata , Receptor 7 Toll-Like/agonistas , Receptor 8 Toll-Like/agonistas , Animais , Colesterol/química , Imidazóis/química , Imunidade Inata/efeitos dos fármacos , Fatores Imunológicos/química , Fatores Imunológicos/metabolismo , Fatores Imunológicos/farmacologia , Lipídeos/química , Linfonodos/efeitos dos fármacos , Linfonodos/imunologia , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , NF-kappa B/metabolismo , Polímeros/química , Quinolinas/química , Quinolinas/farmacologia , Células RAW 264.7 , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Receptor 7 Toll-Like/metabolismo , Receptor 8 Toll-Like/metabolismoRESUMO
Small molecule immuno-modulators such as agonists of Toll-like receptors (TLRs) are attractive compounds to stimulate innate immune cells toward potent antiviral and antitumor responses. However, small molecules rapidly enter the systemic circulation and cause "wasted inflammation". Hence, synthetic strategies to confine their radius of action to lymphoid tissue are of great relevance, to both enhance their efficacy and concomitantly limit toxicity. Here, we demonstrate that covalent conjugation of a small molecule TLR7/8 agonist immunomodulatory to a micelle-forming amphiphilic block copolymer greatly alters the pharmacokinetic profile, resulting in highly efficient lymphatic delivery. Moreover, we designed amphiphilic block copolymers in such a way to form thermodynamically stable micelles through π-π stacking between aromatic moieties, and we engineered the block copolymers to undergo an irreversible amphiphilic to hydrophilic transition in response to the acidic endosomal pH.
Assuntos
Linfonodos/efeitos dos fármacos , Polímeros/farmacologia , Tensoativos/farmacologia , Receptor 7 Toll-Like/agonistas , Receptor 8 Toll-Like/agonistas , Humanos , Concentração de Íons de Hidrogênio , Linfonodos/imunologia , Micelas , Modelos Moleculares , Estrutura Molecular , Polímeros/química , Tensoativos/química , Termodinâmica , Receptor 7 Toll-Like/imunologia , Receptor 8 Toll-Like/imunologiaRESUMO
Synthetic mRNA is becoming increasingly popular as an alternative to pDNA-based gene therapy. Currently, multiple synthetic mRNA platforms have been developed. In this study we investigated the expression kinetics and the changes in mRNA encoding cytokine and chemokine levels following intradermal electroporation in pigs of pDNA, self-replicating mRNA, and modified and unmodified mRNA. The self-replicating mRNA tended to induce the highest protein expression, followed by pDNA, modified mRNA, and unmodified mRNA. Interestingly, the self-replicating mRNA was able to maintain its high expression levels during at least 12 days. In contrast, the expression of pDNA and the nonreplicating mRNAs dropped after respectively one and two days. Six days after intradermal electroporation a dose-dependent expression was observed for all vectors. Again, also at lower doses, the self-replicating mRNA tended to show the highest expression. All the mRNA vectors, including the modified mRNA, induced elevated levels of mRNA encoding cytokines and chemokines in the porcine skin after intradermal electroporation, while no such response was noticed after intradermal electroporation of the pDNA vector.
Assuntos
DNA Circular/administração & dosagem , Técnicas de Transferência de Genes , Imunidade/genética , RNA Mensageiro/administração & dosagem , Animais , Quimiocinas/imunologia , Quimiocinas/metabolismo , Citocinas/imunologia , Citocinas/metabolismo , DNA Circular/genética , DNA Circular/metabolismo , Eletroporação/métodos , Feminino , Terapia Genética/métodos , Vetores Genéticos/genética , Cinética , Camundongos Endogâmicos BALB C , Modelos Animais , Plasmídeos/administração & dosagem , Plasmídeos/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Pele/metabolismo , Sus scrofaRESUMO
In this study, a xenogeneic DNA vaccine encoding for human vascular endothelial growth factor receptor-2 (hVEGFR-2) was evaluated in two murine tumor models, the B16-F10 melanoma and the EO771 breast carcinoma model. The vaccine was administered by intradermal injection followed by electroporation. The immunogenicity and the biological efficacy of the vaccine were tested in (1) a prophylactic setting, (2) a therapeutic setting, and (3) a therapeutic setting combined with surgical removal of the primary tumor. The tumor growth, survival, and development of an immune response were followed. The cellular immune response was measured by a bioluminescence-based cytotoxicity assay with vascular endothelial growth factor-2 (VEGFR-2)-expressing target cells. Humoral immune responses were quantified by enzyme-linked immunosorbent assay (ELISA). Ex vivo bioluminescence imaging and immunohistological observation of organs were used to detect (micro)metastases. A cellular and humoral immune response was present in prophylactically and therapeutically vaccinated mice, in both tumor models. Nevertheless, survival in prophylactically vaccinated mice was only moderately increased, and no beneficial effect on survival in therapeutically vaccinated mice could be demonstrated. An influx of CD3+ cells and a slight decrease in VEGFR-2 were noticed in the tumors of vaccinated mice. Unexpectedly, the vaccine caused an increased quantity of early micrometastases in the liver. Lung metastases were not increased by the vaccine. These early liver micrometastases did however not grow into macroscopic metastases in either control or vaccinated mice when allowed to develop further after surgical removal of the primary tumor.
Assuntos
Neoplasias da Mama/genética , Melanoma/genética , Vacinas de DNA/imunologia , Animais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Metástase Neoplásica , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/imunologiaAssuntos
Vacinas contra COVID-19 , COVID-19 , Imunidade Adaptativa , Animais , Humanos , Camundongos , RNA , RNA Mensageiro/genética , SARS-CoV-2RESUMO
DNA vaccination holds great promise for the prevention and treatment of cancer and infectious diseases. However, the clinical ability of DNA vaccines is still controversial due to the limited immune response initially observed in humans. We hypothesized that electroporation of a plasmid encoding the HIV-1 Gag viral capsid protein would enhance cancer DNA vaccine potency. DNA electroporation used to deliver plasmids in vivo, induced type I interferons, thereby supporting the activation of innate immunity. The coadministration of ovalbumin (OVA) and HIV-1 Gag encoding plasmids modulated the adaptive immune response. This strategy favored antigen-specific Th1 immunity, delayed B16F10-OVA tumor growth and improved mouse survival in both prophylactic and therapeutic vaccination approaches. Similarly, a prophylactic DNA immunization against the melanoma-associated antigen gp100 was enhanced by the codelivery of the HIV-1 Gag plasmid. The adjuvant effect was not driven by the formation of HIV-1 Gag virus-like particles. This work highlights the ability of both electroporation and the HIV-1 Gag plasmid to stimulate innate immunity for enhancing cancer DNA vaccine immunogenicity and demonstrates interesting tracks for the design of new translational genetic adjuvants to overcome the current limitations of DNA vaccines in humans.
Assuntos
Vacinas Anticâncer/genética , Vacinas Anticâncer/imunologia , Plasmídeos/genética , Vacinas de DNA/genética , Vacinas de DNA/imunologia , Produtos do Gene gag do Vírus da Imunodeficiência Humana/genética , Produtos do Gene gag do Vírus da Imunodeficiência Humana/imunologia , Animais , Linhagem Celular , Proliferação de Células , Modelos Animais de Doenças , Humanos , Interferon Tipo I/biossíntese , Melanoma Experimental/imunologia , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , Melanoma Experimental/terapia , Camundongos , Plasmídeos/administração & dosagem , Modelos de Riscos Proporcionais , Células Th1/imunologia , Células Th1/metabolismo , Transfecção , Resultado do Tratamento , Carga Tumoral , Vacinas de Partículas Semelhantes a Vírus/genética , Vacinas de Partículas Semelhantes a Vírus/imunologia , Vacinas de Partículas Semelhantes a Vírus/ultraestruturaRESUMO
Breast tumorigenesis is classically studied in mice by inoculating tumor cells in the fat pad, the adipose compartment of the mammary gland. Alternatively, the mammary ducts, which constitute the luminal mammary gland compartment, also provide a suitable inoculation site to induce breast cancer in murine models. The microenvironments in these compartments influence tumor cell progression, yet this effect has not been investigated in an immunocompetent context. Here, we compared both mammary gland compartments as distinct inoculation sites, taking into account the immunological aspect by inoculating 4T1 tumor cells in immunocompetent mice. Following tumor cell inoculation in the adipose compartment of non-pretreated/naive, hormonally pretreated/naive and non-pretreated/lactating mice, the primary tumors developed similarly. However, a slower onset of primary tumor growth was found after inoculations in the luminal compartment of non-pretreated/lactating mice. Despite this difference in tumor development rate, metastasis to the liver and lungs was equally observed and was accompanied by lymphatic spreading of tumor cells and progressive splenomegaly with both inoculation types. Chitinase 3-like 1 (CHI3L1) and lipocalin 2 (LCN2) served as innovative biomarkers for disease progression showing increased levels in primary tumors and sera of the non-pretreated/lactating inoculation groups. A slower increase in circulating CHI3L1 but not LCN2 levels, was observed after inoculations in the luminal compartment which corroborated the slower tumor development at this inoculation site. Our results highlight the critical impact of different mammary gland compartments on tumor development in syngeneic murine models and support the use of novel tumor progression biomarkers in an immune-competent environment.
Assuntos
Tecido Adiposo/patologia , Carcinogênese/patologia , Neoplasias Mamárias Experimentais/patologia , Neoplasias de Mama Triplo Negativas/patologia , Tecido Adiposo/metabolismo , Animais , Biomarcadores Tumorais/metabolismo , Carcinogênese/metabolismo , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Humanos , Lactação/metabolismo , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/secundário , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/secundário , Neoplasias Mamárias Experimentais/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Obesidade/metabolismo , Obesidade/patologia , Neoplasias de Mama Triplo Negativas/metabolismoRESUMO
Messenger RNA (mRNA) based vaccines have been introduced worldwide to combat the Covid-19 pandemic. These vaccines consist of non-amplifying mRNA formulated in lipid nanoparticles (LNPs). Consequently, LNPs are considered benchmark non-viral carriers for nucleic acid delivery. However, the formulation and manufacturing of these mRNA-LNP nanoparticles are expensive and time-consuming. Therefore, we used self-amplifying mRNA (saRNA) and synthesized novel polymers as alternative non-viral carrier platform to LNPs, which enable a simple, rapid, one-pot formulation of saRNA-polyplexes. Our novel polymer-based carrier platform consists of randomly concatenated ethylenimine and propylenimine comonomers, resulting in linear, poly(ethylenimine-ran-propylenimine) (L-PEIx-ran-PPIy) copolymers with controllable degrees of polymerization. Here we demonstrate in multiple cell lines, that our saRNA-polyplexes show comparable to higher in vitro saRNA transfection efficiencies and higher cell viabilities compared to formulations with Lipofectamine MessengerMAX™ (LFMM), a commercial, lipid-based carrier considered to be the in vitro gold standard carrier. This is especially true for our in vitro best performing saRNA-polyplexes with N/P 5, which are characterised with a size below 100 nm, a positive zeta potential, a near 100% encapsulation efficiency, a high retention capacity and the ability to protect the saRNA from degradation mediated by RNase A. Furthermore, an ex vivo hemolysis assay with pig red blood cells demonstrated that the saRNA-polyplexes exhibit negligible hemolytic activity. Finally, a bioluminescence-based in vivo study was performed over a 35-day period, and showed that the polymers result in a higher and prolonged bioluminescent signal compared to naked saRNA and L-PEI based polyplexes. Moreover, the polymers show different expression profiles compared to those of LNPs, with one of our new polymers (L-PPI250) demonstrating a higher sustained expression for at least 35 days after injection.
Assuntos
Polietilenoimina , RNA Mensageiro , Transfecção , Animais , Transfecção/métodos , Polietilenoimina/química , Humanos , RNA Mensageiro/genética , Camundongos , Polipropilenos/química , Polímeros/química , Portadores de Fármacos/química , SARS-CoV-2/efeitos dos fármacos , Nanopartículas/químicaRESUMO
This study reports on the immunogenicity and biodistribution of H5 hemagglutinin (HA)-based self-amplifying (sa) mRNA vaccines in mice. Four sa-mRNA vaccines encoding either a secreted full-length HA, a secreted HA head domain, a secreted HA stalk domain, or a full-length membrane-anchored HA were investigated. All vaccines elicited an adaptive immune response. However, the full-length HA sa-RNA vaccines demonstrated superior performance compared to head and stalk domain vaccines. The antibody titers positively correlated with the vaccine dose. Cellular immune responses and antigen-specific IgA antibodies in the lungs were also observed. The comparison of the sa-mRNA vaccines encoding the secreted and membrane-anchored full-length HA revealed that anchoring of the HA to the membrane significantly enhanced the antibody and cellular responses. In addition to the injection site, the intramuscularly injected sa-mRNA-LNPs were also detected in the draining lymph nodes, spleen, and to a lesser extent, in the lung, kidney, liver, and heart.