RESUMO
CD4+ T cells are central mediators of adaptive and innate immune responses and constitute a major reservoir for human immunodeficiency virus (HIV) in vivo. Detailed investigations of resting human CD4+ T cells have been precluded by the absence of efficient approaches for genetic manipulation limiting our understanding of HIV replication and restricting efforts to find a cure. Here we report a method for rapid, efficient, activation-neutral gene editing of resting, polyclonal human CD4+ T cells using optimized cell cultivation and nucleofection conditions of Cas9-guide RNA ribonucleoprotein complexes. Up to six genes, including HIV dependency and restriction factors, were knocked out individually or simultaneously and functionally characterized. Moreover, we demonstrate the knock in of double-stranded DNA donor templates into different endogenous loci, enabling the study of the physiological interplay of cellular and viral components at single-cell resolution. Together, this technique allows improved molecular and functional characterizations of HIV biology and general immune functions in resting CD4+ T cells.
Assuntos
Linfócitos T CD4-Positivos/fisiologia , Sistemas CRISPR-Cas/genética , Edição de Genes/métodos , Infecções por HIV/genética , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/virologia , Proteína 9 Associada à CRISPR/genética , Movimento Celular/genética , Células Cultivadas , DNA , Técnicas de Inativação de Genes , Infecções por HIV/metabolismo , Humanos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas de Resistência a Myxovirus/genética , Proteínas de Resistência a Myxovirus/metabolismo , RNA Guia de Cinetoplastídeos , Proteína 1 com Domínio SAM e Domínio HD/genética , Transgenes , Fatores de Poliadenilação e Clivagem de mRNA/genética , Fatores de Poliadenilação e Clivagem de mRNA/metabolismoRESUMO
Mammalian cells release different types of vesicles, collectively termed extracellular vesicles (EVs). EVs contain cellular microRNAs (miRNAs) with an apparent potential to deliver their miRNA cargo to recipient cells to affect the stability of individual mRNAs and the cells' transcriptome. The extent to which miRNAs are exported via the EV route and whether they contribute to cell-cell communication are controversial. To address these issues, we defined multiple properties of EVs and analyzed their capacity to deliver packaged miRNAs into target cells to exert biological functions. We applied well-defined approaches to produce and characterize purified EVs with or without specific viral miRNAs. We found that only a small fraction of EVs carried miRNAs. EVs readily bound to different target cell types, but EVs did not fuse detectably with cellular membranes to deliver their cargo. We engineered EVs to be fusogenic and document their capacity to deliver functional messenger RNAs. Engineered fusogenic EVs, however, did not detectably alter the functionality of cells exposed to miRNA-carrying EVs. These results suggest that EV-borne miRNAs do not act as effectors of cell-to-cell communication.
Assuntos
Comunicação Celular/genética , Vesículas Extracelulares/genética , MicroRNAs/genética , Transcriptoma/genética , Animais , Citometria de Fluxo , Células HEK293 , Humanos , Luciferases/genética , Plasmídeos/genética , RNA Mensageiro/genética , TransfecçãoRESUMO
The emergence of more transmissible or aggressive variants of SARS-CoV-2 requires the development of antiviral medication that is quickly adjustable to evolving viral escape mutations. Here we report the synthesis of chemically stabilized small interfering RNA (siRNA) against SARS-CoV-2. The siRNA can be further modified with receptor ligands such as peptides using CuI -catalysed click-chemistry. We demonstrate that optimized siRNAs can reduce viral loads and virus-induced cytotoxicity by up to five orders of magnitude in cell lines challenged with SARS-CoV-2. Furthermore, we show that an ACE2-binding peptide-conjugated siRNA is able to reduce virus replication and virus-induced apoptosis in 3D mucociliary lung microtissues. The adjustment of the siRNA sequence allows a rapid adaptation of their antiviral activity against different variants of concern. The ability to conjugate the siRNA via click-chemistry to receptor ligands facilitates the construction of targeted siRNAs for a flexible antiviral defence strategy.
Assuntos
COVID-19 , SARS-CoV-2 , Antivirais/farmacologia , Humanos , Ligantes , RNA Interferente Pequeno/farmacologia , SARS-CoV-2/genética , Replicação ViralRESUMO
Poxviruses use a complex strategy to escape immune control, by expressing immunomodulatory proteins that could limit their use as vaccine vectors. To test the role of poxvirus NF-κB pathway inhibitors A52, B15, and K7 in immunity, we deleted their genes in an NYVAC (New York vaccinia virus) strain that expresses HIV-1 clade C antigens. After infection of mice, ablation of the A52R, B15R, and K7R genes increased dendritic cell, natural killer cell, and neutrophil migration as well as chemokine/cytokine expression. Revertant viruses with these genes confirmed their role in inhibiting the innate immune system. To different extents, enhanced innate immune responses correlated with increased HIV Pol- and Gag-specific polyfunctional CD8 T cell and HIV Env-specific IgG responses induced by single-, double-, and triple-deletion mutants. These poxvirus proteins thus influence innate and adaptive cell-mediated and humoral immunity, and their ablation offers alternatives for design of vaccine vectors that regulate immune responses distinctly.IMPORTANCE Poxvirus vectors are used in clinical trials as candidate vaccines for several pathogens, yet how these vectors influence the immune system is unknown. We developed distinct poxvirus vectors that express heterologous antigens but lack different inhibitors of the central host-cell signaling pathway. Using mice, we studied the capacity of these viruses to induce innate and adaptive immune responses and showed that these vectors can distinctly regulate the magnitude and quality of these responses. These findings provide important insights into the mechanism of poxvirus-induced immune response and alternative strategies for vaccine vector design.
Assuntos
Interações Hospedeiro-Patógeno , Evasão da Resposta Imune , Subunidade p50 de NF-kappa B/antagonistas & inibidores , Vaccinia virus/imunologia , Vaccinia virus/patogenicidade , Proteínas Virais/metabolismo , Animais , Movimento Celular , Citocinas/biossíntese , Células Dendríticas/imunologia , Modelos Animais de Doenças , Deleção de Genes , Células Matadoras Naturais/imunologia , Camundongos , Neutrófilos/imunologia , Vacínia/patologia , Vacínia/virologia , Proteínas Virais/genéticaRESUMO
Neutrophils are antigen-transporting cells that generate vaccinia virus (VACV)-specific T-cell responses, yet how VACV modulates neutrophil recruitment and its significance in the immune response are unknown. We generated an attenuated VACV strain that expresses HIV-1 clade C antigens but lacks three specific viral genes (A52R, K7R, and B15R). We found that these genes act together to inhibit the NFκB signaling pathway. Triple ablation in modified virus restored NFκB function in macrophages. After virus infection of mice, NFκB pathway activation led to expression of several cytokines/chemokines that increased the migration of neutrophil populations (Nα and Nß) to the infection site. Nß cells displayed features of antigen-presenting cells and activated virus-specific CD8 T cells. Enhanced neutrophil trafficking to the infection site correlated with an increased T-cell response to HIV vector-delivered antigens. These results identify a mechanism for poxvirus-induced immune response and alternatives for vaccine vector design.
Assuntos
Linfócitos T CD8-Positivos/imunologia , HIV-1/imunologia , Doenças do Sistema Imunitário , Transtornos Leucocíticos , NF-kappa B/metabolismo , Animais , Células Apresentadoras de Antígenos/imunologia , Linhagem Celular , Deleção de Genes , Genes Virais , Antígenos HIV/imunologia , Humanos , Ativação Linfocitária/imunologia , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Modelos Biológicos , Infiltração de Neutrófilos , Especificidade da Espécie , Vacínia/imunologia , Vacínia/virologia , Vaccinia virus/genéticaRESUMO
UNLABELLED: The generation of vaccines against HIV/AIDS able to induce long-lasting protective immunity remains a major goal in the HIV field. The modest efficacy (31.2%) against HIV infection observed in the RV144 phase III clinical trial highlighted the need for further improvement of HIV vaccine candidates, formulation, and vaccine regimen. In this study, we have generated two novel NYVAC vectors, expressing HIV-1 clade C gp140(ZM96) (NYVAC-gp140) or Gag(ZM96)-Pol-Nef(CN54) (NYVAC-Gag-Pol-Nef), and defined their virological and immunological characteristics in cultured cells and in mice. The insertion of HIV genes does not affect the replication capacity of NYVAC recombinants in primary chicken embryo fibroblast cells, HIV sequences remain stable after multiple passages, and HIV antigens are correctly expressed and released from cells, with Env as a trimer (NYVAC-gp140), while in NYVAC-Gag-Pol-Nef-infected cells Gag-induced virus-like particles (VLPs) are abundant. Electron microscopy revealed that VLPs accumulated with time at the cell surface, with no interference with NYVAC morphogenesis. Both vectors trigger specific innate responses in human cells and show an attenuation profile in immunocompromised adult BALB/c and newborn CD1 mice after intracranial inoculation. Analysis of the immune responses elicited in mice after homologous NYVAC prime/NYVAC boost immunization shows that recombinant viruses induced polyfunctional Env-specific CD4 or Gag-specific CD8 T cell responses. Antibody responses against gp140 and p17/p24 were elicited. Our findings showed important insights into virus-host cell interactions of NYVAC vectors expressing HIV antigens, with the activation of specific immune parameters which will help to unravel potential correlates of protection against HIV in human clinical trials with these vectors. IMPORTANCE: We have generated two novel NYVAC-based HIV vaccine candidates expressing HIV-1 clade C trimeric soluble gp140 (ZM96) and Gag(ZM96)-Pol-Nef(CN54) as VLPs. These vectors are stable and express high levels of both HIV-1 antigens. Gag-induced VLPs do not interfere with NYVAC morphogenesis, are highly attenuated in immunocompromised and newborn mice after intracranial inoculation, trigger specific innate immune responses in human cells, and activate T (Env-specific CD4 and Gag-specific CD8) and B cell immune responses to the HIV antigens, leading to high antibody titers against gp140. For these reasons, these vectors can be considered vaccine candidates against HIV/AIDS and currently are being tested in macaques and humans.
Assuntos
Vacinas contra a AIDS/imunologia , Vacinação/métodos , Vacinas de Partículas Semelhantes a Vírus/imunologia , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologia , Produtos do Gene gag do Vírus da Imunodeficiência Humana/imunologia , Produtos do Gene nef do Vírus da Imunodeficiência Humana/imunologia , Vacinas contra a AIDS/administração & dosagem , Vacinas contra a AIDS/genética , Animais , Linfócitos T CD8-Positivos/imunologia , Células Cultivadas , Galinhas , Anticorpos Anti-HIV/sangue , Camundongos , Microscopia Eletrônica de Transmissão , Vacinas de Partículas Semelhantes a Vírus/administração & dosagem , Vacinas de Partículas Semelhantes a Vírus/genética , Vacinas de Partículas Semelhantes a Vírus/ultraestrutura , Produtos do Gene env do Vírus da Imunodeficiência Humana/genética , Produtos do Gene gag do Vírus da Imunodeficiência Humana/genética , Produtos do Gene nef do Vírus da Imunodeficiência Humana/genéticaRESUMO
Vaccinia viruses (VACVs) with distinct early promoters have been developed to enhance antigen expression and improve antigen-specific CD8 T-cell responses. It has not been demonstrated how the length of the spacer between the coding region of the gene and its regulatory early promoter motif influences antigen expression, and whether the timing of gene expression can modify the antigen-specific CD4 T-cell response. We generated several recombinant VACVs based on the attenuated modified vaccinia Ankara (MVA) strain, which express GFP or the Leishmania LACK antigen under the control of an optimized promoter, using different spacer lengths. Longer spacer length increased GFP and LACK early expression, which correlated with an enhanced LACK-specific memory CD4 and CD8 T-cell response. These results show the importance of promoter spacer length for early antigen expression by VACV and provide alternative strategies for the design of poxvirus-based vaccines.
Assuntos
Antígenos de Protozoários/genética , Expressão Gênica , Vetores Genéticos/genética , Leishmaniose Visceral/imunologia , Regiões Promotoras Genéticas , Proteínas de Protozoários/genética , Vaccinia virus/genética , Animais , Antígenos de Protozoários/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Vetores Genéticos/imunologia , Humanos , Imunização , Leishmania infantum/genética , Leishmania infantum/imunologia , Leishmaniose Visceral/parasitologia , Leishmaniose Visceral/prevenção & controle , Masculino , Camundongos Endogâmicos BALB C , Proteínas de Protozoários/imunologia , Vaccinia virus/imunologiaRESUMO
Replication-competent poxvirus vectors with an attenuation phenotype and with a high immunogenic capacity of the foreign expressed antigen are being pursued as novel vaccine vectors against different pathogens. In this investigation, we have examined the replication and immunogenic characteristics of two vaccinia virus (VACV) mutants, M65 and M101. These mutants were generated after 65 and 101 serial passages of persistently infected Friend erythroleukemia (FEL) cells. In cultured cells of different origins, the mutants are replication competent and have growth kinetics similar to or slightly reduced in comparison with those of the parental Western Reserve (WR) virus strain. In normal and immune-suppressed infected mice, the mutants showed different levels of attenuation and pathogenicity in comparison with WR and modified vaccinia Ankara (MVA) strains. Wide genome analysis after deep sequencing revealed selected genomic deletions and mutations in a number of viral open reading frames (ORFs). Mice immunized in a DNA prime/mutant boost regimen with viral vectors expressing the LACK (Leishmania homologue for receptors of activated C kinase) antigen of Leishmania infantum showed protection or a delay in the onset of cutaneous leishmaniasis. Protection was similar to that triggered by MVA-LACK. In immunized mice, both polyfunctional CD4(+) and CD8(+) T cells with an effector memory phenotype were activated by the two mutants, but the DNA-LACK/M65-LACK protocol preferentially induced CD4(+) whereas DNA-LACK/M101-LACK preferentially induced CD8(+) T cell responses. Altogether, our findings showed the adaptive changes of the WR genome during long-term virus-host cell interaction and how the replication competency of M65 and M101 mutants confers distinct biological properties and immunogenicity in mice compared to those of the MVA strain. These mutants could have applicability for understanding VACV biology and as potential vaccine vectors against pathogens and tumors.
Assuntos
Vetores Genéticos/efeitos adversos , Leishmaniose Cutânea/prevenção & controle , Vacinas Atenuadas/administração & dosagem , Vacinas/administração & dosagem , Vaccinia virus/genética , Replicação Viral , Animais , Antígenos de Protozoários/genética , Antígenos de Protozoários/imunologia , Antígenos de Protozoários/metabolismo , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular , Embrião de Galinha , Feminino , Fibroblastos/virologia , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Vetores Genéticos/imunologia , Células HeLa , Humanos , Imunização , Rim/citologia , Rim/virologia , Leishmaniose Cutânea/imunologia , Camundongos , Mutação , Proteínas de Protozoários/genética , Proteínas de Protozoários/imunologia , Proteínas de Protozoários/metabolismo , Inoculações Seriadas , Vacinas/genética , Vacinas/imunologia , Vacinas Atenuadas/genética , Vacinas Atenuadas/imunologia , Vaccinia virus/classificação , Vaccinia virus/imunologia , Vaccinia virus/fisiologiaRESUMO
Resting CD4 T cells resist productive HIV-1 infection. The HIV-2/simian immunodeficiency virus protein viral accessory protein X (Vpx) renders these cells permissive to infection, presumably by alleviating blocks at cytoplasmic reverse transcription and subsequent nuclear import of reverse-transcription/pre-integration complexes (RTC/PICs). Here, spatial analyses using quantitative virus imaging techniques reveal that HIV-1 capsids containing RTC/PICs are readily imported into the nucleus, recruit the host dependency factor CPSF6, and translocate to nuclear speckles in resting CD4 T cells. Reverse transcription, however, remains incomplete, impeding proviral integration and viral gene expression. Vpx or pharmacological inhibition of the deoxynucleotide triphosphohydrolase (dNTPase) activity of the restriction factor SAM domain and HD domain-containing protein 1 (SAMHD1) increases levels of nuclear reverse-transcribed cDNA and facilitates HIV-1 integration. Nuclear import and intranuclear transport of viral complexes therefore do not pose important blocks to HIV-1 in resting CD4 T cells, and the limitation to reverse transcription by SAMHD1's dNTPase activity constitutes the main pre-integration block to infection.
Assuntos
Infecções por HIV , Soropositividade para HIV , HIV-1 , Proteínas Monoméricas de Ligação ao GTP , Animais , Humanos , HIV-1/genética , Linfócitos T CD4-Positivos/metabolismo , Proteína 1 com Domínio SAM e Domínio HD/metabolismo , HIV-2/genética , Proteínas Virais Reguladoras e Acessórias/metabolismo , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Células HEK293RESUMO
Immune cell phenotyping frequently detects lineage-unrelated receptors. Here, we report that surface receptors can be transferred from primary macrophages to CD4 T cells and identify the Fcγ receptor CD32 as driver and cargo of this trogocytotic transfer. Filamentous CD32+ nanoprotrusions deposit distinct plasma membrane patches onto target T cells. Transferred receptors confer cell migration and adhesion properties, and macrophage-derived membrane patches render resting CD4 T cells susceptible to infection by serving as hotspots for HIV-1 binding. Antibodies that recognize T cell epitopes enhance CD32-mediated trogocytosis. Such autoreactive anti-HIV-1 envelope antibodies can be found in the blood of HIV-1 patients and, consistently, the percentage of CD32+ CD4 T cells is increased in their blood. This CD32-mediated, antigen-independent cell communication mode transiently expands the receptor repertoire and functionality of immune cells. HIV-1 hijacks this mechanism by triggering the generation of trogocytosis-promoting autoantibodies to gain access to immune cells critical to its persistence.
Assuntos
Infecções por HIV , Soropositividade para HIV , HIV-1 , Humanos , Linfócitos T CD4-Positivos , Receptores de IgG/metabolismo , Autoanticorpos/metabolismo , TrogocitoseRESUMO
Here we describe the design and strength of a new synthetic late-early optimized (LEO) vaccinia virus (VACV) promoter used as a transcriptional regulator of GFP expression during modified vaccinia Ankara infection. In contrast to the described synthetic VACV promoter (pS), LEO induced significantly higher levels of GFP expression in vitro within the first hour after infection, which correlated with an enhancement in the GFP-specific CD8 T-cell response detected in vivo, demonstrating its potential use in future vaccines.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Desenho de Fármacos , Proteínas de Fluorescência Verde/imunologia , Regiões Promotoras Genéticas/genética , Vaccinia virus/genética , Vacínia/imunologia , Vacinas Virais/genética , Animais , Sequência de Bases , Linfócitos T CD8-Positivos/metabolismo , Citocinas/metabolismo , Proteínas de Fluorescência Verde/genética , Camundongos , Vacínia/prevenção & controle , Vaccinia virus/imunologia , Vaccinia virus/fisiologiaRESUMO
Infection-neutralizing antibody responses after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection or coronavirus disease 2019 vaccination are an essential component of antiviral immunity. Antibody-mediated protection is challenged by the emergence of SARS-CoV-2 variants of concern (VoCs) with immune escape properties, such as omicron (B.1.1.529), which is rapidly spreading worldwide. Here we report neutralizing antibody dynamics in a longitudinal cohort of coronavirus disease 2019 convalescent and infection-naive individuals vaccinated with mRNA BNT162b2 by quantifying SARS-CoV-2 spike protein antibodies and determining their avidity and neutralization capacity in serum. Using live-virus neutralization assays, we show that a superior infection-neutralizing capacity against all VoCs, including omicron, developed after either two vaccinations in convalescents or a third vaccination or breakthrough infection of twice-vaccinated, naive individuals. These three consecutive spike antigen exposures resulted in an increasing neutralization capacity per anti-spike antibody unit and were paralleled by stepwise increases in antibody avidity. We conclude that an infection-plus-vaccination-induced hybrid immunity or a triple immunization can induce high-quality antibodies with superior neutralization capacity against VoCs, including omicron.
Assuntos
Vacina BNT162 , COVID-19 , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Vacina BNT162/imunologia , COVID-19/imunologia , COVID-19/prevenção & controle , Humanos , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , VacinaçãoRESUMO
Neutrophils are innate immune cells involved in the elimination of pathogens and can also induce adaptive immune responses. Nα and Nß neutrophils have been described with distinct in vitro capacity to generate antigen-specific CD8 T-cell responses. However, how these cell types exert their role in vivo and how manipulation of Nß/Nα ratio influences vaccine-mediated immune responses are not known. In this study, we find that these neutrophil subtypes show distinct migratory and motility patterns and different ability to interact with CD8 T cells in the spleen following vaccinia virus (VACV) infection. Moreover, after analysis of adhesion, inflammatory, and migration markers, we observe that Nß neutrophils overexpress the α4ß1 integrin compared to Nα. Finally, by inhibiting α4ß1 integrin, we increase the Nß/Nα ratio and enhance CD8 T-cell responses to HIV VACV-delivered antigens. These findings provide significant advancements in the comprehension of neutrophil-based control of adaptive immune system and their relevance in vaccine design.
RESUMO
There is an urgent need for the development of potent vaccination regimens that are able to induce specific T and B cell responses against human immunodeficiency virus type 1 (HIV-1). Here, we describe the generation and characterization of a fusion antigen comprised of the HIV-1 envelope GP120 glycoprotein from clade C (GP120C) fused at its C-terminus, with the modified vaccinia virus (VACV) 14K protein (A27L gene) (termed GP120C14K). The design is directed toward improving the immunogenicity of the GP120C protein through its oligomerization facilitated by the fused VACV 14K protein that results in hexamer-like structures. Two different immunogens were generated: a recombinant GP120C14K fusion protein (purified from a stable CHO-K1 cell line) and a recombinant modified vaccinia virus Ankara (MVA) poxvirus vector expressing the GP120C14K fusion protein (termed MVA-GP120C14K). The GP120C14K fusion protein is recognized by broadly neutralizing antibodies (bNAbs) against HIV-1. In a murine model, a heterologous prime/boost immunization regimen with MVA-GP120C14K prime followed by adjuvanted GP120C14K protein boost generated stronger and polyfunctional HIV-1 Env-specific CD8 T cell responses when compared with the delivery of the monomeric GP120C form. Furthermore, the immunization protocol MVA-GP120C14K/GP120C14K elicited higher HIV-1 Env-specific T follicular helper cells, germinal center B cells and antibody responses than monomeric GP120. In addition, a similar MVA-GP120C14K prime/GP120C14K protein boost regimen performed in rabbits triggered high HIV-1-Env-specific IgG binding antibody titers that were capable of neutralizing HIV-1 pseudoviruses. The extent of HIV-1 neutralization was comparable to that elicited by the current standard GP140 SOSIP trimers from clades B and C when immunized as MVA-SOSIP prime/SOSIP protein boost regimen. Overall, the novel fusion antigen and the corresponding immunization scheme provided in this report can therefore be considered as potential vaccine strategies against HIV-1.
Assuntos
Vacinas contra a AIDS/imunologia , Anticorpos Anti-HIV/biossíntese , Proteína gp120 do Envelope de HIV/imunologia , HIV-1/imunologia , Proteínas Recombinantes de Fusão/imunologia , Linfócitos T/imunologia , Vaccinia virus/imunologia , Proteínas Virais/imunologia , Animais , Anticorpos Neutralizantes/biossíntese , Células CHO , Cricetulus , Feminino , Humanos , Imunização , Camundongos , Camundongos Endogâmicos BALB C , Coelhos , Proteínas Recombinantes de Fusão/químicaRESUMO
Despite the effectiveness of classic treatments and available diagnostic tools, cancer continues to be a leading world health problem, with devastating cancer-related death rates. Advances in oncolytic virotherapy have shown promise as potentially effective treatment options in the fight against cancer. The poxviruses have many features that make them an attractive platform for the development of oncolytic vectors, with some candidates currently in clinical trials. Here, we report the design and generation of a new oncolytic vector based on the vaccinia virus Western Reserve (WR) strain. We show that the WR-Δ4 virus, with the combined deletion of four specific viral genes that act on metabolic, proliferation, and signaling pathways (A48R, B18R, C11R, and J2R), has effective anti-tumor capabilities in vivo. In WR-Δ4-infected mice, we observed strong viral attenuation, reduced virus dissemination, and efficient tumor cell growth control in the B16F10 syngeneic melanoma model, with enhanced neutrophil migration and activation of tumor antigen-specific immune responses. This approach provides an alternative strategy toward ongoing efforts to develop an optimal oncolytic poxvirus vector.
RESUMO
The goal of successful anti-tumoural immunity is the development of long-term protective immunity to prevent relapse. Infiltration of tumours with CD8+ T cells with a resident memory (Trm) phenotype correlates with improved survival. However, the interplay of circulating CD8+ T cells and Trm cells remains poorly explored in tumour immunity. Using different vaccination strategies that fine-tune the generation of Trm cells or circulating memory T cells, here we show that, while both subsets are sufficient for anti-tumour immunity, the presence of Trm cells improves anti-tumour efficacy. Transferred central memory T cells (Tcm) generate Trm cells following viral infection or tumour challenge. Anti-PD-1 treatment promotes infiltration of transferred Tcm cells within tumours, improving anti-tumour immunity. Moreover, Batf3-dependent dendritic cells are essential for reactivation of circulating memory anti-tumour response. Our findings show the plasticity, collaboration and requirements for reactivation of memory CD8+ T cells subsets needed for optimal tumour vaccination and immunotherapy.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Memória Imunológica/imunologia , Linfócitos do Interstício Tumoral/imunologia , Melanoma Experimental/imunologia , Subpopulações de Linfócitos T/imunologia , Vacínia/imunologia , Adaptação Fisiológica/imunologia , Animais , Antineoplásicos Imunológicos/imunologia , Antineoplásicos Imunológicos/farmacologia , Fatores de Transcrição de Zíper de Leucina Básica/genética , Linfócitos T CD8-Positivos/efeitos dos fármacos , Vacinas Anticâncer/imunologia , Linhagem Celular Tumoral , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Linfócitos do Interstício Tumoral/efeitos dos fármacos , Camundongos , Camundongos Knockout , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Proteínas Repressoras/genética , Subpopulações de Linfócitos T/efeitos dos fármacos , Vaccinia virusRESUMO
Vaccines against the preerythrocytic stages of malaria are appealing because the parasite can be eliminated before disease onset and because they offer the unique possibility of targeting the parasite with both antibodies and T cells. Although the role of CD8+ T cells in preerythrocytic malaria stages is well documented, a highly effective T cell-inducing vaccine remains to be advanced. Here we report the development of a prime-boost immunization regimen with the Plasmodium falciparum circumsporozoite protein (PfCS) fused to the oligomer-forming vaccinia virus A27 protein and a modified vaccinia virus Ankara (MVA) vector expressing PfCS. This protocol induced polyfunctional CD8+ T cells with an effector memory phenotype and high PfCS antibody levels. These immune responses correlated with inhibition of liver-stage parasitemia in 80% and sterile protection in 40% of mice challenged with a transgenic P. berghei parasite line that expressed PfCS. Our findings underscore the potential of T and B cell immunization strategies for improving protective effectiveness against malaria.
Assuntos
Anticorpos Antiprotozoários/imunologia , Linfócitos T CD8-Positivos/imunologia , Vacinas Antimaláricas/imunologia , Malária Falciparum/imunologia , Proteínas de Protozoários/imunologia , Animais , Malária Falciparum/prevenção & controle , Camundongos , Vacinas de DNA/imunologiaRESUMO
After Edward Jenner established human vaccination over 200 years ago, attenuated poxviruses became key players to contain the deadliest virus of its own family: Variola virus (VARV), the causative agent of smallpox. Cowpox virus (CPXV) and horsepox virus (HSPV) were extensively used to this end, passaged in cattle and humans until the appearance of vaccinia virus (VACV), which was used in the final campaigns aimed to eradicate the disease, an endeavor that was accomplished by the World Health Organization (WHO) in 1980. Ever since, naturally evolved strains used for vaccination were introduced into research laboratories where VACV and other poxviruses with improved safety profiles were generated. Recombinant DNA technology along with the DNA genome features of this virus family allowed the generation of vaccines against heterologous diseases, and the specific insertion and deletion of poxvirus genes generated an even broader spectrum of modified viruses with new properties that increase their immunogenicity and safety profile as vaccine vectors. In this review, we highlight the evolution of poxvirus vaccines, from first generation to the current status, pointing out how different vaccines have emerged and approaches that are being followed up in the development of more rational vaccines against a wide range of diseases.
Assuntos
Vacina Antivariólica/história , Vacina Antivariólica/isolamento & purificação , Varíola/prevenção & controle , Animais , História do Século XVIII , História do Século XIX , História do Século XX , História do Século XXI , Humanos , Poxviridae/imunologia , Poxviridae/isolamento & purificação , Vacina Antivariólica/imunologia , Vacinas Atenuadas/história , Vacinas Atenuadas/imunologia , Vacinas Atenuadas/isolamento & purificação , Vacinas Sintéticas/história , Vacinas Sintéticas/imunologia , Vacinas Sintéticas/isolamento & purificaçãoRESUMO
Heterologous vaccination based on priming with a plasmid DNA vector and boosting with an attenuated vaccinia virus MVA recombinant, with both vectors expressing the Leishmania infantum LACK antigen (DNA-LACK and MVA-LACK), has shown efficacy conferring protection in murine and canine models against cutaneus and visceral leishmaniasis, but the immune parameters of protection remain ill defined. Here we performed by flow cytometry an in depth analysis of the T cell populations induced in BALB/c mice during the vaccination protocol DNA-LACK/MVA-LACK, as well as after challenge with L. major parasites. In the adaptive response, there is a polyfunctional CD4(+) and CD8(+) T cell activation against LACK antigen. At the memory phase the heterologous vaccination induces high quality LACK-specific long-term CD4(+) and CD8(+) effector memory cells. After parasite challenge, there is a moderate boosting of LACK-specific CD4(+) and CD8(+) T cells. Anti-vector responses were largely CD8(+)-mediated. The immune parameters induced against LACK and triggered by the combined vaccination DNA/MVA protocol, like polyfunctionality of CD4(+) and CD8(+) T cells with an effector phenotype, could be relevant in protection against leishmaniasis.