RESUMO
The approval of the first oncolytic virus for the treatment of metastatic melanoma and the compiling evidence that the use of oncolytic viruses can enhance cancer immunotherapies targeted against various immune checkpoint proteins has attracted great interest in the field of cancer virotherapy. We have developed a novel platform for clinically relevant enveloped viruses that can direct the virus-induced immune response against tumor antigens. By physically attaching tumor-specific peptides onto the viral envelope of vaccinia virus and herpes simplex virus 1 (HSV-1), we were able to induce a strong T cell-specific immune response toward these tumor antigens. These therapeutic peptides could be attached onto the viral envelope by using a cell-penetrating peptide sequence derived from human immunodeficiency virus Tat N-terminally fused to the tumor-specific peptides or, alternatively, therapeutic peptides could be conjugated with cholesterol for the attachment of the peptides onto the viral envelope. We used two mouse models of melanoma termed B16.OVA and B16-F10 for testing the efficacy of OVA SIINFEKL-peptide-coated viruses and gp100-Trp2-peptide-coated viruses, respectively, and show that by coating the viral envelope with therapeutic peptides, the anti-tumor immunity and the number of tumor-specific CD8+ T cells in the tumor microenvironment can be significantly enhanced.
Assuntos
Vacinas Anticâncer/química , Peptídeos/metabolismo , Células A549 , Animais , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Vacinas Anticâncer/imunologia , Vacinas Anticâncer/uso terapêutico , Linhagem Celular Tumoral , Chlorocebus aethiops , Herpesvirus Humano 1/metabolismo , Humanos , Melanoma/imunologia , Melanoma/terapia , Camundongos , Terapia Viral Oncolítica/métodos , Vírus Oncolíticos , Peptídeos/imunologia , Vaccinia virus/metabolismo , Células Vero , Proteínas do Envelope Viral/metabolismoRESUMO
Reassortment of their segmented genomes allows influenza A viruses (IAV) to gain new characteristics, which potentially enable them to cross the species barrier and infect new hosts. Improved replication was observed for reassortants of the strictly avian IAV A/FPV/Rostock/34 (FPV, H7N1) containing the NS segment from A/Goose/Guangdong/1/1996 (GD, H5N1), but not for reassortants containing the NS segment of A/Mallard/NL/12/2000 (MA, H7N3). The NS1 of GD and MA differ only in 8 aa positions. Here, we show that efficient replication of FPV-NSMA-derived mutants was linked to the presence of a single substitution (D74N) and more prominently to a triple substitution (P3S+R41K+D74N) in the NS1MA protein. The substitution(s) led to (i) increased virus titres, (ii) larger plaque sizes and (iii) increased levels and faster kinetics of viral mRNA and protein accumulation in mammalian cells. Interestingly, the NS1 substitutions did not affect viral growth characteristics in avian cells. Furthermore, we show that an FPV mutant with N74 in the NS1 (already possessing S3+K41) is able to replicate and cause disease in mice, demonstrating a key role of NS1 in the adaptation of avian IAV to mammalian hosts. Our data suggest that (i) adaptation to mammalian hosts does not necessarily compromise replication in the natural (avian) host and (ii) very few genetic changes may pave the way for zoonotic transmission. The study reinforces the need for close surveillance and characterization of circulating avian IAV to identify genetic signatures that indicate a potential risk for efficient transmission of avian strains to mammalian hosts.
Assuntos
Vírus da Influenza A/fisiologia , Proteínas Mutantes/genética , Mutação de Sentido Incorreto , Proteínas não Estruturais Virais/genética , Replicação Viral , Substituição de Aminoácidos , Animais , Linhagem Celular , Modelos Animais de Doenças , Humanos , Vírus da Influenza A/genética , Camundongos Endogâmicos C57BL , Infecções por Orthomyxoviridae/patologia , Infecções por Orthomyxoviridae/virologiaRESUMO
The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) posed a threat to public health and the global economy, necessitating the development of various vaccination strategies. Mutations in the SPIKE protein gene, a crucial component of mRNA and adenovirus-based vaccines, raised concerns about vaccine efficacy, prompting the need for rapid vaccine updates. To address this, we leveraged PeptiCRAd, an oncolytic vaccine based on tumor antigen decorated oncolytic adenoviruses, creating a vaccine platform called PeptiVAX. First, we identified multiple CD8 T-cell epitopes from highly conserved regions across coronaviruses, expanding the range of T-cell responses to non-SPIKE proteins. We designed short segments containing the predicted epitopes presented by common HLA-Is in the global population. Testing the immunogenicity, we characterized T-cell responses to candidate peptides in peripheral blood mononuclear cells (PBMCs) from pre-pandemic healthy donors and ICU patients. As a proof of concept in mice, we selected a peptide with epitopes predicted to bind to murine MHC-I haplotypes. Our technology successfully elicited peptide-specific T-cell responses, unaffected by the use of unarmed adenoviral vectors or adeno-based vaccines encoding SPIKE. In conclusion, PeptiVAX represents a fast and adaptable SARS-CoV-2 vaccine delivery system that broadens T-cell responses beyond the SPIKE protein, offering potential benefits for vaccine effectiveness.
Assuntos
COVID-19 , Vacinas Virais , Humanos , Camundongos , Animais , Vacinas contra COVID-19 , COVID-19/prevenção & controle , Glicoproteína da Espícula de Coronavírus/genética , Leucócitos Mononucleares , SARS-CoV-2 , Peptídeos/química , Epitopos de Linfócito TRESUMO
BACKGROUND: Intratumoral BCG therapy, one of the earliest immunotherapies, can lead to infiltration of immune cells into a treated tumor. However, an increase in the number of BCG-induced tumor-specific T cells in the tumor microenvironment could lead to enhanced therapeutic effects. METHODS: Here, we have developed a novel cancer vaccine platform based on BCG that can broaden BCG-induced immune responses to include tumor antigens. By physically attaching tumor-specific peptides onto the mycobacterial outer membrane, we were able to induce strong systemic and intratumoral T cell-specific immune responses toward the attached tumor antigens. These therapeutic peptides can be efficiently attached to the mycobacterial outer membrane using a poly-lysine sequence N-terminally fused to the tumor-specific peptides. RESULTS: Using two mouse models of melanoma and a mouse model of colorectal cancer, we observed that the antitumor immune responses of BCG could be improved by coating the BCG with tumor-specific peptides. In addition, by combining this novel cancer vaccine platform with anti-programmed death 1 (anti-PD-1) immune checkpoint inhibitor (ICI) therapy, the number of responders to anti-PD-1 immunotherapy was markedly increased. CONCLUSIONS: This study shows that intratumoral BCG immunotherapy can be improved by coating the bacteria with modified tumor-specific peptides. In addition, this improved BCG immunotherapy can be combined with ICI therapy to obtain enhanced tumor growth control. These results warrant clinical testing of this novel cancer vaccine platform.
Assuntos
Vacina BCG/uso terapêutico , Vacinas Anticâncer/uso terapêutico , Imunoterapia/métodos , Medicina de Precisão/métodos , Animais , Vacina BCG/farmacologia , Vacinas Anticâncer/farmacologia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Humanos , CamundongosRESUMO
Oncolytic viruses (OVs) have been shown to induce anti-cancer immunity and enhance cancer immunotherapies, such as immune checkpoint inhibitor therapies. OV therapies can be further improved by arming OVs with immunostimulatory molecules, including various cytokines or chemokines. Here, we have developed a novel adenovirus encoding two immunostimulatory molecules: cluster of differentiation 40 ligand (CD40L) and tumor necrosis factor receptor superfamily member 4 ligand (OX40L). This novel virus, designated VALO-D102, is designed to activate both innate and adaptive immune responses against tumors. CD40L affects the innate side by licensing antigen-presenting cells to drive CD8+ T cell responses, and OX40L increases clonal expansion and survival of CD8+ T cells and formation of a larger pool of memory T cells. VALO-D102 and its murine surrogate VALO-mD901, expressing murine OX40L and CD40L, were used in our previously developed PeptiCRAd cancer vaccine platform. Intratumoral administration of PeptiCRAd significantly increased tumor-specific T cell responses, reduced tumor growth, and induced systemic anti-cancer immunity in two mouse models of melanoma. In addition, PeptiCRAd therapy, in combination with anti-PD-1 immune checkpoint inhibitor therapy, significantly improved tumor growth control as compared to either monotherapy alone.
RESUMO
Because of the high coverage of international vaccination programs, most people worldwide have been vaccinated against common pathogens, leading to acquired pathogen-specific immunity with a robust memory T-cell repertoire. Although CD8+ antitumor cytotoxic T lymphocytes (CTL) are the preferred effectors of cancer immunotherapy, CD4+ T-cell help is also required for an optimal antitumor immune response to occur. Hence, we investigated whether the pathogen-related CD4+ T-cell memory populations could be reengaged to support the CTLs, converting a weak primary antitumor immune response into a stronger secondary one. To this end, we used our PeptiCRAd technology that consists of an oncolytic adenovirus coated with MHC-I-restricted tumor-specific peptides and developed it further by introducing pathogen-specific MHC-II-restricted peptides. Mice preimmunized with tetanus vaccine were challenged with B16.OVA tumors and treated with the newly developed hybrid TT-OVA-PeptiCRAd containing both tetanus toxoid- and tumor-specific peptides. Treatment with the hybrid PeptiCRAd significantly enhanced antitumor efficacy and induced TT-specific, CD40 ligand-expressing CD4+ T helper cells and maturation of antigen-presenting cells. Importantly, this approach could be extended to naturally occurring tumor peptides (both tumor-associated antigens and neoantigens), as well as to other pathogens beyond tetanus, highlighting the usefulness of this technique to take full advantage of CD4+ memory T-cell repertoires when designing immunotherapeutic treatment regimens. Finally, the antitumor effect was even more prominent when combined with the immune checkpoint inhibitor anti-PD-1, strengthening the rationale behind combination therapy with oncolytic viruses. SIGNIFICANCE: These findings establish a novel technology that enhances oncolytic cancer immunotherapy by capitalizing on pre-acquired immunity to pathogens to convert a weak antitumor immune response into a much stronger one.
Assuntos
Vacinas Anticâncer/administração & dosagem , Vacina contra Difteria, Tétano e Coqueluche/administração & dosagem , Memória Imunológica , Imunoterapia/métodos , Melanoma Experimental/terapia , Vacina Antipólio de Vírus Inativado/administração & dosagem , Adenoviridae/genética , Adenoviridae/imunologia , Animais , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/imunologia , Antineoplásicos Imunológicos/administração & dosagem , Linfócitos T CD4-Positivos/imunologia , Vacinas Anticâncer/imunologia , Linhagem Celular Tumoral/transplante , Vacina contra Difteria, Tétano e Coqueluche/imunologia , Feminino , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/imunologia , Antígenos de Histocompatibilidade Classe II/genética , Antígenos de Histocompatibilidade Classe II/imunologia , Humanos , Melanoma Experimental/imunologia , Melanoma Experimental/patologia , Camundongos , Vírus Oncolíticos/genética , Vírus Oncolíticos/imunologia , Vacina Antipólio de Vírus Inativado/imunologia , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Receptor de Morte Celular Programada 1/imunologia , Linfócitos T Citotóxicos/imunologia , Vacinas Combinadas/administração & dosagem , Vacinas Combinadas/imunologiaRESUMO
Messenger RNA (mRNA) and microRNA (miRNA)-based therapeutics have become attractive alternatives to DNA-based therapeutics due to recent advances in manufacture, scalability and cost. Also, RNA-based therapeutics are considered safe since there are no risk of inducing genomic changes as well as the potential adverse effects would be only temporary due to the transient nature of RNA-based therapeutics. However, efficient in vivo delivery of RNA-based therapeutics remains a challenge. We have developed a delivery platform for RNA-based therapeutics by exploiting the physicochemical properties of enveloped viruses. By physically attaching cationic liposome/RNA complexes onto the viral envelope of vaccinia virus, we were able to deliver mRNA, self-replicating RNA as well as miRNA inside target cells. Also, we showed that this platform, called viRNA platform, can efficiently deliver functional miRNA mimics into B16.OVA tumour in vivo.
Assuntos
Neoplasias da Mama/terapia , Sistemas de Liberação de Medicamentos , Terapia Genética , Melanoma Experimental/terapia , MicroRNAs/administração & dosagem , RNA Mensageiro/metabolismo , Vaccinia virus/genética , Células A549 , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Feminino , Humanos , Melanoma Experimental/genética , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/genética , RNA Mensageiro/genéticaRESUMO
Virus-based cancer vaccines are nowadays considered an interesting approach in the field of cancer immunotherapy, despite the observation that the majority of the immune responses they elicit are against the virus and not against the tumor. In contrast, targeting tumor associated antigens is effective, however the identification of these antigens remains challenging. Here, we describe ExtraCRAd, a multi-vaccination strategy focused on an oncolytic virus artificially wrapped with tumor cancer membranes carrying tumor antigens. We demonstrate that ExtraCRAd displays increased infectivity and oncolytic effect in vitro and in vivo. We show that this nanoparticle platform controls the growth of aggressive melanoma and lung tumors in vivo both in preventive and therapeutic setting, creating a highly specific anti-cancer immune response. In conclusion, ExtraCRAd might serve as the next generation of personalized cancer vaccines with enhanced features over standard vaccination regimens, representing an alternative way to target cancer.
Assuntos
Vacinas Anticâncer/administração & dosagem , Imunoterapia/métodos , Neoplasias/terapia , Vírus Oncolíticos/imunologia , Vacinação/métodos , Adenoviridae/imunologia , Animais , Antígenos de Neoplasias/imunologia , Vacinas Anticâncer/imunologia , Linhagem Celular Tumoral/citologia , Linhagem Celular Tumoral/imunologia , Linhagem Celular Tumoral/transplante , Membrana Celular/imunologia , Modelos Animais de Doenças , Feminino , Humanos , Injeções Intralesionais , Camundongos , Nanopartículas/administração & dosagem , Neoplasias/imunologia , Resultado do Tratamento , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The non-structural protein-1 (NS1) of many influenza A strains, especially those of avian origin, contains an SH3 ligand motif, which binds tightly to the cellular adaptor proteins Crk (Chicken tumor virus number 10 (CT10) regulator of kinase) and Crk-like adapter protein (CrkL). This interaction has been shown to potentiate NS1-induced activation of the phosphatidylinositol 3-kinase (PI3K), but additional effects on the host cell physiology may exist. Here we show that NS1 can induce an efficient translocation of Crk proteins from the cytoplasm into the nucleus, which results in an altered pattern of nuclear protein tyrosine phosphorylation. This was not observed using NS1 proteins deficient in SH3 binding or engineered to be exclusively cytoplasmic, indicating a physical role for NS1 as a carrier in the nuclear translocation of Crk. These data further emphasize the role of Crk proteins as host cell interaction partners of NS1, and highlight the potential for host cell manipulation gained by a viral protein simply via acquiring a short SH3 binding motif.
Assuntos
Proteínas Proto-Oncogênicas c-crk/metabolismo , Proteínas não Estruturais Virais/metabolismo , Transporte Ativo do Núcleo Celular , Linhagem Celular , Interações Hospedeiro-Patógeno , Humanos , Vírus da Influenza A/fisiologia , Influenza Humana/metabolismo , Influenza Humana/virologia , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Ligação Proteica , Proteínas não Estruturais Virais/química , Domínios de Homologia de srcRESUMO
The non-structural protein-1 (NS1) of influenza A virus binds the p85ß subunit of phosphoinositide 3-kinase (PI3K) to induce PI3K activity in the infected cells. Some virus strains encode NS1 containing a motif that binds tightly to the SH3 domain of the cellular adapter proteins Crk and CrkL to potentiate NS1-induced PI3K activation. Here we show that this potentiation involves reorganization of the natural CrkL-p85ß complex into a novel trimeric complex where NS1 serves as a bridging factor. Of note, NS1 proteins that lack the SH3 binding capacity can also associate with CrkL, but in a less stable trimeric complex mediated by p85ß. The data presented here establish Crk proteins as general host cell cofactors of NS1, and show that the enhanced PI3K activation by SH3 binding-competent NS1 variants is mediated by a more efficient tethering of Crk proteins to the NS1-PI3K complex.