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1.
Nat Rev Genet ; 21(4): 255-272, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32042148

RESUMO

Adeno-associated virus (AAV) vector-mediated gene delivery was recently approved for the treatment of inherited blindness and spinal muscular atrophy, and long-term therapeutic effects have been achieved for other rare diseases, including haemophilia and Duchenne muscular dystrophy. However, current research indicates that the genetic modification of AAV vectors may further facilitate the success of AAV gene therapy. Vector engineering can increase AAV transduction efficiency (by optimizing the transgene cassette), vector tropism (using capsid engineering) and the ability of the capsid and transgene to avoid the host immune response (by genetically modifying these components), as well as optimize the large-scale production of AAV.


Assuntos
Dependovirus/genética , Terapia Genética , Vetores Genéticos , Imunidade Adaptativa , Engenharia Genética , Vetores Genéticos/imunologia , Imunidade Inata
2.
Nat Commun ; 11(1): 524, 2020 Jan 27.
Artigo em Inglês | MEDLINE | ID: mdl-31988324

RESUMO

Viruses naturally engage innate immunity, induce antigen presentation, and mediate CD8 T cell priming against foreign antigens. Polioviruses can provide a context optimal for generating antigen-specific CD8 T cells, as they have natural tropism for dendritic cells, preeminent inducers of CD8 T cell immunity; elicit Th1-promoting inflammation; and lack interference with innate or adaptive immunity. However, notorious genetic instability and underlying neuropathogenicity has hampered poliovirus-based vector applications. Here we devised a strategy based on the polio:rhinovirus chimera PVSRIPO, devoid of viral neuropathogenicity after intracerebral inoculation in human subjects, for stable expression of exogenous antigens. PVSRIPO vectors infect, activate, and induce epitope presentation in DCs in vitro; they recruit and activate DCs with Th1-dominant cytokine profiles at the injection site in vivo. They efficiently prime tumor antigen-specific CD8 T cells in vivo, induce CD8 T cell migration to the tumor site, delay tumor growth and enhance survival in murine tumor models.


Assuntos
Linfócitos T CD8-Positivos/fisiologia , Células Dendríticas/imunologia , Poliovirus/imunologia , Animais , Vacinas Anticâncer , Vetores Genéticos/imunologia , Glioma/imunologia , Células HEK293 , Células HeLa , Humanos , Imunidade Inata , Imunoterapia/métodos , Interferon Tipo I/imunologia , Melanoma/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Poliovirus/genética
3.
Int J Mol Sci ; 20(18)2019 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-31510028

RESUMO

Vaccination has had great success in combating diseases, especially infectious diseases. However, traditional vaccination strategies are ineffective for several life-threatening diseases, including acquired immunodeficiency syndrome (AIDS), tuberculosis, malaria, and cancer. Viral vaccine vectors represent a promising strategy because they can efficiently deliver foreign genes and enhance antigen presentation in vivo. However, several limitations, including pre-existing immunity and packaging capacity, block the application of viral vectors. Cytomegalovirus (CMV) has been demonstrated as a new type of viral vector with additional advantages. CMV could systematically elicit and maintain high frequencies of effector memory T cells through the "memory inflation" mechanism. Studies have shown that CMV can be genetically modified to induce distinct patterns of CD8+ T-cell responses, while some unconventional CD8+ T-cell responses are rarely induced through conventional vaccine strategies. CMV has been used as a vaccine vector to deliver many disease-specific antigens, and the efficacy of these vaccines was tested in different animal models. Promising results demonstrated that the robust and unconventional T-cell responses elicited by the CMV-based vaccine vector are essential to control these diseases. These accumulated data and evidence strongly suggest that a CMV-based vaccine vector represents a promising approach to develop novel prophylactic and therapeutic vaccines against some epidemic pathogens and tumors.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Citomegalovirus/imunologia , Vetores Genéticos/imunologia , Neoplasias/imunologia , Tuberculose/imunologia , Vacinas Virais/imunologia , Animais , Citomegalovirus/genética , Vetores Genéticos/genética , Humanos , Memória Imunológica/imunologia , Neoplasias/prevenção & controle , Tuberculose/prevenção & controle , Vacinação/métodos , Vacinas Virais/administração & dosagem
4.
Gene Ther ; 26(9): 399-406, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31467408

RESUMO

Differences between mouse and human hearts pose a significant limitation to the value of small animal models when predicting vector behavior following recombinant adeno-associated viral (rAAV) vector-mediated cardiac gene therapy. Hence, sheep have been adopted as a preclinical animal, as they better model the anatomy and cardiac physiological processes of humans. There is, however, no comprehensive data on the shedding profile of rAAV in sheep following intracoronary delivery, so as to understand biosafety risks in future preclinical and clinical applications. In this study, sheep received intracoronary delivery of rAAV serotypes 2/6 (2 × 1012 vg), 2/8, and 2/9 (1 × 1013 vg) at doses previously administered in preclinical and clinical trials. This was followed by assessment over 96 h to examine vector shedding in urine, feces, nasal mucus, and saliva samples. Vector genomes were detected via real-time quantitative PCR in urine and feces up to 48 and 72 h post vector delivery, respectively. Of these results, functional vector particles were only detected via a highly sensitive infectious replication assay in feces samples up to 48 h following vector delivery. We conclude that rAAV-mediated gene transfer into sheep hearts results in low-grade shedding of non-functional vector particles for all excreta samples, except in the case of feces, where functional vector particles are present up to 48 h following vector delivery. These results may be used to inform containment and decontamination guidelines for large animal dealings, and to understand the biosafety risks associated with future preclinical and clinical uses of rAAV.


Assuntos
Dependovirus/genética , Vetores Genéticos , Eliminação de Partículas Virais , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Cateterismo , Vasos Coronários , Dependovirus/imunologia , Dependovirus/fisiologia , Vetores Genéticos/administração & dosagem , Vetores Genéticos/imunologia , Células HeLa , Humanos , Injeções Intra-Arteriais , Masculino , Infecções por Parvoviridae/imunologia , Infecções por Parvoviridae/urina , Infecções por Parvoviridae/virologia , Reação em Cadeia da Polimerase em Tempo Real , Ovinos , Replicação Viral
5.
Tuberculosis (Edinb) ; 117: 24-30, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31378264

RESUMO

The aim of this study was to determine the reliability of lactic acid bacteria (LAB) as heterologous hosts for the expression of MPB70 and MPB83, two Mycobacterium bovis antigens that possess diagnostics and immunogenic properties, respectively. We therefore generated recombinant cells of Lactococcus lactis and Lactobacillus plantarum that carried hybrid genes encoding MPB70 and MPB83 fused to signal peptides that are specifically recognized by LAB. Only L. lactis was able to secrete MPB70 using the L. lactis signal peptide Usp45, and to produce MPB83 as an immunogenic membrane protein following its expression with the signal peptide of the L. plantarum lipoprotein prsA. Inactivated cells of MPB83-expressing L. lactis cultures enhanced NF-κB activation in macrophages. Our results show that L. lactis is a reliable host for the secretion and functional expression of antigens that are naturally produced by M. bovis, the causative agent of bovine tuberculosis (bTB). This represents the first step on a long process to establishing whether recombinant LAB could serve as a food-grade platform for potential diagnostic tools and/or vaccine interventions for use against bTB, a chronic disease that primarily affects cattle but also humans and a wide range of domestic and wild animals.


Assuntos
Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Lactobacillales/metabolismo , Proteínas de Membrana/metabolismo , Mycobacterium bovis/imunologia , Antígenos de Bactérias/genética , Proteínas de Bactérias/genética , Células Cultivadas , DNA Bacteriano/biossíntese , Expressão Gênica , Vetores Genéticos/imunologia , Humanos , Lactobacillales/genética , Lactobacillus plantarum/genética , Lactobacillus plantarum/metabolismo , Lactococcus lactis/genética , Lactococcus lactis/metabolismo , Macrófagos/metabolismo , Macrófagos/microbiologia , Proteínas de Membrana/genética , NF-kappa B/metabolismo , Recombinação Genética
6.
Emerg Microbes Infect ; 8(1): 1086-1097, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31339465

RESUMO

In the last few decades, Ebola virus (EBOV) has emerged periodically and infected people in Africa, resulting in an extremely high mortality rate. With no available prophylaxis or cure so far, a highly effective Ebola vaccine is urgently needed. In this study, we developed a novel chimpanzee adenovirus-based prime-boost vaccine by exploiting two recombinant replication-deficient chimpanzee adenoviral vectors, AdC7 and AdC68, which express glycoproteins (GP) of the EBOV strain identified in the 2014 outbreak. Our results indicated that a single immunization using AdC7 or AdC68 could stimulate potent EBOV-specific antibody responses, whereas the AdC7 prime-AdC68 boost regimen induced much stronger and sustained humoral and cellular immune responses in both mice and rhesus monkeys, compared with AdC7 or AdC68 single vaccination or the AdC68 prime-AdC7 boost regimen. This prime-boost vaccine could also protect mice from the simulated infection with EBOV-like particle (EBOVLP) in biosafety level 2 (BSL-2) laboratories, and antibodies from the prime-boost immunized rhesus macaques could passively provide protection against EBOVLP infection. Altogether, our results show that the AdC7 prime-AdC68 boost vaccine is a promising candidate for further development to combat EBOV infections.


Assuntos
Adenoviridae/imunologia , Ebolavirus/imunologia , Doença pelo Vírus Ebola/imunologia , Adenoviridae/genética , Animais , Anticorpos Antivirais/imunologia , Modelos Animais de Doenças , Ebolavirus/genética , Feminino , Vetores Genéticos/genética , Vetores Genéticos/imunologia , Doença pelo Vírus Ebola/prevenção & controle , Doença pelo Vírus Ebola/virologia , Humanos , Imunidade Celular , Imunização Secundária , Macaca mulatta , Masculino , Camundongos , Camundongos Endogâmicos BALB C
7.
PLoS One ; 14(6): e0215031, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31163034

RESUMO

Many mRNA-based vaccines have been investigated for their specific potential to activate dendritic cells (DCs), the highly-specialized antigen-presenting cells of the immune system that play a key role in inducing effective CD4+ and CD8+ T-cell responses. In this paper we report a new vaccine/gene delivery platform that demonstrates the benefits of using a self-amplifying ("replicon") mRNA that is protected in a viral-protein capsid. Purified capsid protein from the plant virus Cowpea Chlorotic Mottle Virus (CCMV) is used to in vitro assemble monodisperse virus-like particles (VLPs) containing reporter proteins (e.g., Luciferase or eYFP) or the tandem-repeat model antigen SIINFEKL in RNA gene form, coupled to the RNA-dependent RNA polymerase from the Nodamura insect virus. Incubation of immature DCs with these VLPs results in increased activation of maturation markers - CD80, CD86 and MHC-II - and enhanced RNA replication levels, relative to incubation with unpackaged replicon mRNA. Higher RNA uptake/replication and enhanced DC activation were detected in a dose-dependent manner when the CCMV-VLPs were pre-incubated with anti-CCMV antibodies. In all experiments the expression of maturation markers correlates with the RNA levels of the DCs. Overall, these studies demonstrate that: VLP protection enhances mRNA uptake by DCs; coupling replicons to the gene of interest increases RNA and protein levels in the cell; and the presence of anti-VLP antibodies enhances mRNA levels and activation of DCs in vitro. Finally, preliminary in vivo experiments involving mouse vaccinations with SIINFEKL-replicon VLPs indicate a small but significant increase in antigen-specific T cells that are doubly positive for IFN and TFN induction.


Assuntos
Bromovirus/metabolismo , Proteínas do Capsídeo/genética , Células Dendríticas/imunologia , RNA Mensageiro/administração & dosagem , Vacinas de Partículas Semelhantes a Vírus/genética , Animais , Bromovirus/genética , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Linhagem Celular , Cricetinae , Células Dendríticas/virologia , Feminino , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Vetores Genéticos/imunologia , Camundongos , RNA Mensageiro/imunologia , Análise de Célula Única , Montagem de Vírus
8.
J Biosci ; 44(2)2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31180054

RESUMO

This study was conducted to determine whether exposure to particulate matter 2.5 (PM2.5) affects the immune tolerance of neonatal mice via the regulation of PD-L1 expression. One-week-old BALB/c mice were exposed to PM2.5 for 8 days. From day 8 to day 18, the mice were treated with 5 µg house dust mite (HDM) (i. n.) every two days. Adenovirus-carried PD-L1 overexpression vectors were infected into mice via nasal inhalation 6 days after exposure to PM2.5. Airway hyperresponsiveness (AHR) was examined in mice 19 days after exposure to PM2.5, and the related parameters of airway inflammation were studied on day 22. Co-exposure to PM2.5 and HDM reduced PD-L1 expression but greatly increased infiltration of inflammatory cells, which was reversed by PD-L1 overexpression. Co-exposure to PM2.5 and HDM also elevated serum IL-4, IL-5 and IL-13 levels and reduced TGF-ß level. Exposure to PM2.5 alone slightly increased the numbers of dendritic cells (DCs) but reduced the numbers of antigen-presenting cells expressing PD-L1 and Treg cells. Therefore, early exposure to PM2.5 reduced PD-L1 expression in the lungs of neonatal mice, which interfered with immune tolerance establishment and subsequently resulted in allergic airway inflammation.


Assuntos
Antígeno B7-H1/imunologia , Células Dendríticas/efeitos dos fármacos , Tolerância Imunológica/efeitos dos fármacos , Pulmão/efeitos dos fármacos , Material Particulado/administração & dosagem , Hipersensibilidade Respiratória/imunologia , Adenoviridae/genética , Adenoviridae/imunologia , Administração por Inalação , Animais , Animais Recém-Nascidos , Antígeno B7-H1/antagonistas & inibidores , Antígeno B7-H1/genética , Células Dendríticas/imunologia , Células Dendríticas/patologia , Regulação da Expressão Gênica , Vetores Genéticos/administração & dosagem , Vetores Genéticos/química , Vetores Genéticos/imunologia , Interleucina-13/genética , Interleucina-13/imunologia , Interleucina-4/genética , Interleucina-4/imunologia , Interleucina-5/genética , Interleucina-5/imunologia , Pulmão/imunologia , Pulmão/patologia , Camundongos , Camundongos Endogâmicos BALB C , Pyroglyphidae/química , Pyroglyphidae/imunologia , Hipersensibilidade Respiratória/induzido quimicamente , Hipersensibilidade Respiratória/genética , Hipersensibilidade Respiratória/patologia , Transdução de Sinais , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/patologia , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/imunologia
9.
Gene Ther ; 26(6): 264-276, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31110296

RESUMO

The prevalence of adeno-associated virus (AAV) has been investigated in bat populations, but little is known about the biological properties of this virus. In this study, four full-length bat AAV capsid genes were isolated in China, with their amino acid sequences sharing 61% identity with those of AAV2 on average. These capsid genes could package AAV particles in combination with AAV2 rep and ITRs, albeit at a lower efficiency. Bat AAVs could only slightly infect mouse liver but could transduce mouse muscle to some extent after systemic administration with a higher muscle/liver ratio than that of primate AAVs. Bat AAV 10HB showed moderate muscle transduction, similar to that of AAV2, during direct intramuscular injection and, compared with other AAV serotypes, was also relatively efficient in resisting human antibody neutralization after intramuscular injection. Evolutionary analysis revealed a number of codons in bat AAV capsid genes subject to positive selection, with sites corresponding to V259 and N691 in 10HB capsids being localized on the surface of the AAV2 capsid. Mutagenesis studies indicated that the positive selection in bat AAV capsids is driven by their tropism evolution in host species. Taken together, the results of this study indicate that bat AAV 10HB vector has the possible applications for muscular gene therapy, especially in the presence of human AAV neutralizing antibodies.


Assuntos
Anticorpos Neutralizantes/imunologia , Dependovirus/imunologia , Terapia Genética/métodos , Vetores Genéticos/imunologia , Evasão da Resposta Imune , Animais , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/imunologia , Quirópteros , Dependovirus/genética , Técnicas de Transferência de Genes/efeitos adversos , Terapia Genética/efeitos adversos , Vetores Genéticos/genética , Células HEK293 , Células HT29 , Humanos , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos SCID , Músculo Esquelético/metabolismo
10.
Cells ; 8(5)2019 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-31130710

RESUMO

Host immune responses play an important role in the outcome of infection with hepatitis C virus (HCV). They can lead to viral clearance and a positive outcome, or progression and severity of chronic disease. Extensive research in the past >25 years into understanding the immune responses against HCV have still resulted in many unanswered questions implicating a role for unknown factors and events. In our earlier studies, we made a surprising discovery that peptides derived from structural and non-structural proteins of HCV have substantial amino acid sequence homologies with various proteins of adenoviruses and that immunizing mice with a non-replicating, non-recombinant adenovirus vector leads to induction of a robust cross-reactive cellular and humoral response against various HCV antigens. In this work, we further demonstrate antibody cross-reactivity between Ad and HCV in vivo. We also extend this observation to show that recombinant adenoviruses containing antigens from unrelated pathogens also possess the ability to induce cross-reactive immune responses against HCV antigens along with the induction of transgene antigen-specific immunity. This cross-reactive immunity can (a) accommodate the making of dual-pathogen vaccines, (b) play an important role in the natural course of HCV infection and (c) provide a plausible answer to many unexplained questions regarding immunity to HCV.


Assuntos
Infecções por Adenoviridae/virologia , Adenoviridae/imunologia , Vacinas contra Adenovirus/imunologia , Reações Cruzadas/imunologia , Vetores Genéticos/imunologia , Hepacivirus/imunologia , Antígenos da Hepatite C/imunologia , Hepatite C/virologia , Imunidade Heteróloga/imunologia , Animais , Antígenos de Bactérias/imunologia , Células Cultivadas , Feminino , Antígenos HIV/imunologia , Imunidade Celular/imunologia , Imunidade Humoral/imunologia , Imunização/métodos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Baço/patologia , Vacinação/métodos
11.
PLoS Pathog ; 15(5): e1007710, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31145755

RESUMO

Modified vaccinia virus Ankara (MVA) is the leading poxvirus vector for development of vaccines against diverse infectious diseases. This distinction is based on high expression of proteins and good immunogenicity despite an inability to assemble infectious progeny in human cells, which together promote efficacy and safety. Nevertheless, the basis for the host-range restriction is unknown despite past systematic attempts to identify the relevant missing viral gene(s). The search for host-range factors is exacerbated by the large number of deletions, truncations and mutations that occurred during the long passage history of MVA in chicken embryo fibroblasts. By whole genome sequencing of a panel of recombinant host-range extended (HRE) MVAs generated by marker rescue with 40 kbp segments of vaccinia virus DNA, we identified serine protease inhibitor 1 (SPI-1) as one of several candidate host-range factors present in those viruses that gained the ability to replicate in human cells. Electron microscopy revealed that the interruption of morphogenesis in human cells infected with MVA occurred at a similar stage as that of a vaccinia virus strain WR SPI-1 deletion mutant. Moreover, the introduction of the SPI-1 gene into the MVA genome led to more than a 2-log enhancement of virus spread in human diploid MRC-5 cells, whereas deletion of the gene diminished the spread of HRE viruses by similar extents. Furthermore, MRC-5 cells stably expressing SPI-1 also enhanced replication of MVA. A role for additional host range genes was suggested by the restoration of MVA replication to a lower level relative to HRE viruses, particularly in other human cell lines. Although multiple sequence alignments revealed genetic changes in addition to SPI-1 common to the HRE MVAs, no evidence for their host-range function was found by analysis thus far. Our finding that SPI-1 is host range factor for MVA should simplify use of high throughput RNAi or CRISPR/Cas single gene methods to identify additional viral and human restriction elements.


Assuntos
Especificidade de Hospedeiro/imunologia , Inibidores de Serino Proteinase/imunologia , Vírus Vaccinia/fisiologia , Vaccinia/virologia , Vacinas Virais/imunologia , Replicação Viral , Células A549 , Vetores Genéticos/imunologia , Humanos , Inibidores de Serino Proteinase/genética , Vaccinia/imunologia , Vaccinia/prevenção & controle
12.
Gastroenterol Clin North Am ; 48(2): 319-330, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31046978

RESUMO

Recombinant vectors based on a nonpathogenic parvovirus, the adeno-associated virus (AAV), have taken center stage in the past decade. The safety of AAV vectors in clinical trials and clinical efficacy in several human diseases are now well documented. Despite these achievements, it is increasingly clear that the full potential of AAV vectors composed of the naturally occurring capsids is unlikely to be realized. This article describes advances that have been made and challenges that remain in the optimal use of AAV vectors in human gene therapy applications.


Assuntos
Dependovirus/genética , Terapia Genética/métodos , Vetores Genéticos/uso terapêutico , Hepatopatias/terapia , Animais , Vetores Genéticos/administração & dosagem , Vetores Genéticos/imunologia , Humanos , Hepatopatias/imunologia , Camundongos , Modelos Animais , Sorogrupo
13.
J Cancer Res Ther ; 15(Supplement): S1-S10, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30900613

RESUMO

A new modality of targeting therapeutic drugs based on the use of bacteriophage (virus), as an emerging tool for specific targeting and for vaccine development, has been an area of interest for genetic and cancer research. The approach is based on genetic manipulation and modification in the chemical structure of a filamentous bacteriophage that facilitates its application not only for in vivo imaging but also for therapeutic purpose, as a gene delivery vehicle, as drug carriers, and also as an immunomodulatory agent. Filamentous bacteriophage on account of its high surface holding ability with adaptable genetic engineering properties can effectively be used in loading of chemical and genetic drugs specifically on to the targeted lesion location. Moreover, the specific peptides/proteins exhibited on the phage surface can be applied directly as self-navigating drug delivery nanovehicles. The present review article has been framed with an objective to summarize the importance of bacteriophage in phage cancer therapy and to understand the possible future prospective of this approach in developing new tools for biotechnological and genetic research, especially in phage -mediated cancer therapy. Importantly, the peptides or proteins emerging from the surface of a nano carrier will make the expense of such peptides economically more effective as compared to other immunological tools, and this seems to be a potential approach for developing a new nanodrug carrier platform.


Assuntos
Antineoplásicos/administração & dosagem , Portadores de Fármacos/química , Vetores Genéticos/genética , Inovirus/genética , Neoplasias/terapia , Animais , Engenharia Genética , Terapia Genética/métodos , Vetores Genéticos/química , Vetores Genéticos/imunologia , Humanos , Inovirus/química , Inovirus/imunologia , Nanopartículas , Neoplasias/genética , Neoplasias/imunologia
14.
PLoS One ; 14(3): e0212811, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30897117

RESUMO

Adeno-associated virus (AAV) vectors represent promising candidates for gene therapy; however, pre-existing neutralizing antibodies (NAb) may reduce AAV vector delivery efficiency. In this study, the presence of AAV NAb was investigated in cats, which serve as a larger and outbred animal model for the prediction of gene therapy outcomes in humans but also in cats.Serum/plasma samples from 230 client-owned Swiss cats and 20 specified pathogen-free cats were investigated for NAb to AAV1, AAV2, AAV5, AAV6, AAV7, AAV8 and AAV9 using in vitro transduction inhibition and a beta-galactosidase assay. NAb to all tested AAV serotypes were found. Of the client-owned cats, 53% had NAb to one or more of the AAV serotypes. NAb (≥1:10) were found at frequencies of 5% (AAV6) to 28% (AAV7). The highest titers were found against AAV7 (≥1:160). The NAb prevalence to AAV2, AAV7 and AAV9 differed geographically. Regarding titers ≥1:10 against single AAV serotypes, age, breed and sex of the cats were not associated with the NAb prevalence. Cats with titers ≥1:20 against AAV2 and titers ≥1:40 against AAV7 were significantly younger than cats with low/no titers, and purebred cats were significantly more likely than non-purebred cats to have NAb to AAV2 (≥1:40). Additionally, regarding NAb to all AAV combined, female cats were significantly more likely than male cats to have NAb titers ≥1:40. Preliminary data using AAV-DJ indicated that less pre-existing NAb to the hybrid AAV-DJ can be expected compared to the wild-type AAV serotypes. AAV NAb will need to be taken into account for future in vivo gene therapy studies in cats.


Assuntos
Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Dependovirus/imunologia , Terapia Genética/efeitos adversos , Vetores Genéticos/imunologia , Fatores Etários , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/isolamento & purificação , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/isolamento & purificação , Doenças do Gato/genética , Doenças do Gato/terapia , Gatos , Linhagem Celular Tumoral , Dependovirus/genética , Feminino , Doenças Genéticas Inatas/genética , Doenças Genéticas Inatas/terapia , Terapia Genética/métodos , Vetores Genéticos/genética , Células HEK293 , Humanos , Masculino , Modelos Animais , Sorogrupo , Fatores Sexuais
15.
Front Immunol ; 10: 59, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30761131

RESUMO

Dengue fever has become a global threat, causing millions of infections every year. An effective vaccine against all four serotypes of dengue virus (DENV) has not been developed yet. Among the different vaccination strategies available today, DNA vaccines are safe and practical, but currently induce relatively weak immune responses in humans. In order to improve immunogenicity, antigens may be targeted to dendritic cells (DCs), the main antigen presenting cells and orchestrators of the adaptive immune response, inducing T and B cell activation. It was previously shown that a DNA vaccine encoding a fusion protein comprised of an antigen and a single-chain Fv antibody (scFv) specific for the DC endocytic receptor DEC205 induced strong immune responses to the targeted antigen. In this work, we evaluate this strategy to improve the immunogenicity of dengue virus (DENV) proteins. Plasmids encoding the scFv αDEC205, or an isotype control (scFv ISO), fused to the DENV2 envelope protein domain III (EDIII) were generated, and EDIII specific immune responses were evaluated in immunized mice. BALB/c mice were intramuscularly (i.m.) immunized three times with plasmid DNAs encoding either scDEC-EDIII or scISO-EDIII followed by electroporation. Analyses of the antibody responses indicated that EDIII fusion with scFv targeting the DEC205 receptor significantly enhanced serum anti-EDIII IgG titers that inhibited DENV2 infection. Similarly, mice immunized with the scDEC-EDIII plasmid developed a robust CD4+ T cell response to the targeted antigen, allowing the identification of two linear epitopes recognized by the BALB/c haplotype. Taken together, these results indicate that targeting DENV2 EDIII protein to DCs using a DNA vaccine encoding the scFv αDEC205 improves both antibody and CD4+ T cell responses. This strategy opens perspectives for the use of DNA vaccines that encode antigens targeted to DCs as a strategy to increase immunogenicity.


Assuntos
Anticorpos Antivirais/imunologia , Especificidade de Anticorpos/imunologia , Linfócitos T CD4-Positivos/imunologia , Células Dendríticas/imunologia , Vacinas contra Dengue/imunologia , Vírus da Dengue/imunologia , Vacinas de DNA/imunologia , Proteínas do Envelope Viral/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Linfócitos T CD4-Positivos/metabolismo , Citocinas/biossíntese , Células Dendríticas/metabolismo , Dengue/prevenção & controle , Vacinas contra Dengue/genética , Modelos Animais de Doenças , Epitopos/química , Epitopos/imunologia , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Vetores Genéticos/imunologia , Humanos , Imunização , Ativação Linfocitária/imunologia , Masculino , Camundongos , Peptídeos/química , Peptídeos/imunologia , Vacinas de DNA/administração & dosagem , Células Vero
16.
Vet Res ; 50(1): 12, 2019 Feb 11.
Artigo em Inglês | MEDLINE | ID: mdl-30744668

RESUMO

Infectious bronchitis virus (IBV) causes a major disease problem for the poultry industry worldwide. The currently used live-attenuated vaccines have the tendency to mutate and/or recombine with circulating field strains resulting in the emergence of vaccine-derived variant viruses. In order to circumvent these issues, and to develop a vaccine that is more relevant to Egypt and its neighboring countries, a recombinant avirulent Newcastle disease virus (rNDV) strain LaSota was constructed to express the codon-optimized S glycoprotein of the Egyptian IBV variant strain IBV/Ck/EG/CU/4/2014 belonging to GI-23 lineage, that is prevalent in Egypt and in the Middle East. A wild type and two modified versions of the IBV S protein were expressed individually by rNDV. A high level of S protein expression was detected in vitro by Western blot and immunofluorescence analyses. All rNDV-vectored IBV vaccine candidates were genetically stable, slightly attenuated and showed growth patterns comparable to that of parental rLaSota virus. Single-dose vaccination of 1-day-old SPF White Leghorn chicks with the rNDVs expressing IBV S protein provided significant protection against clinical disease after IBV challenge but did not show reduction in tracheal viral shedding. Single-dose vaccination also provided complete protection against virulent NDV challenge. However, prime-boost vaccination using rNDV expressing the wild type IBV S protein provided better protection, after IBV challenge, against clinical signs and significantly reduced tracheal viral shedding. These results indicate that the NDV-vectored IBV vaccines are promising bivalent vaccine candidates to control both infectious bronchitis and Newcastle disease in Egypt.


Assuntos
Galinhas , Infecções por Coronavirus/veterinária , Vírus da Bronquite Infecciosa/imunologia , Vírus da Doença de Newcastle/imunologia , Doenças das Aves Domésticas/prevenção & controle , Glicoproteína da Espícula de Coronavírus/imunologia , Vacinas Virais/imunologia , Animais , Linhagem Celular , Infecções por Coronavirus/prevenção & controle , Egito , Vetores Genéticos/imunologia , Vírus da Bronquite Infecciosa/genética , Vírus da Doença de Newcastle/genética , Glicoproteína da Espícula de Coronavírus/genética , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Vacinas Virais/administração & dosagem , Vacinas Virais/genética
17.
J Virol ; 93(7)2019 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-30674625

RESUMO

Hepatitis C is a liver disease caused by the hepatitis C virus (HCV) affecting 71 million people worldwide with no licensed vaccines that prevent infection. Here, we have generated four novel alphavirus-based DNA-launched self-amplifying RNA replicon (DREP) vaccines expressing either structural core-E1-E2 or nonstructural p7-NS2-NS3 HCV proteins of genotype 1a placed under the control of an alphavirus promoter, with or without an alphaviral translational enhancer (grouped as DREP-HCV or DREP-e-HCV, respectively). DREP vectors are known to induce cross-priming and further stimulation of immune responses through apoptosis, and here we demonstrate that they efficiently trigger apoptosis-related proteins in transfected cells. Immunization of mice with the DREP vaccines as the priming immunization followed by a heterologous boost with a recombinant modified vaccinia virus Ankara (MVA) vector expressing the nearly full-length genome of HCV (MVA-HCV) induced potent and long-lasting HCV-specific CD4+ and CD8+ T cell immune responses that were significantly stronger than those of a homologous MVA-HCV prime/boost immunization, with the DREP-e-HCV/MVA-HCV combination the most immunogenic regimen. HCV-specific CD4+ and CD8+ T cell responses were highly polyfunctional, had an effector memory phenotype, and were mainly directed against E1-E2 and NS2-NS3, respectively. Additionally, DREP/MVA-HCV immunization regimens induced higher antibody levels against HCV E2 protein than homologous MVA-HCV immunization. Collectively, these results provided an immunization protocol against HCV by inducing high levels of HCV-specific T cell responses as well as humoral responses. These findings reinforce the combined use of DREP-based vectors and MVA-HCV as promising prophylactic and therapeutic vaccines against HCV.IMPORTANCE HCV represents a global health problem as more than 71 million people are chronically infected worldwide. Direct-acting antiviral agents can cure HCV infection in most patients, but due to the high cost of these agents and the emergence of resistant mutants, they do not represent a feasible and affordable strategy to eradicate the virus. Therefore, a vaccine is an urgent goal that requires efforts to understand the correlates of protection for HCV clearance. Here, we describe for the first time the generation of novel vaccines against HCV based on alphavirus DNA replicons expressing HCV antigens. We demonstrate that potent T cell immune responses, as well as humoral immune responses, against HCV can be achieved in mice by using a combined heterologous prime/boost immunization protocol consisting of the administration of alphavirus replicon DNA vectors as the priming immunization followed by a boost with a recombinant modified vaccinia virus Ankara vector expressing HCV antigens.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Hepacivirus/imunologia , Hepatite C/imunologia , Replicon/imunologia , Vírus Vaccinia/imunologia , Vacinas Virais/imunologia , Alphavirus/imunologia , Animais , Anticorpos Antivirais/imunologia , DNA/imunologia , Vetores Genéticos/imunologia , Imunização/métodos , Camundongos , RNA/imunologia , Vacinação/métodos , Vacinas de DNA/imunologia , Proteínas não Estruturais Virais/imunologia
18.
Int J Mol Sci ; 20(2)2019 Jan 14.
Artigo em Inglês | MEDLINE | ID: mdl-30646574

RESUMO

Natural killer (NK) cells are innate immune cells that can be activated rapidly to target abnormal and virus-infected cells without prior sensitization. With significant advancements in cell biology technologies, many NK cell lines have been established. Among these cell lines, NK-92 cells are not only the most widely used but have also been approved for clinical applications. Additionally, chimeric antigen receptor-modified NK-92 cells (CAR-NK-92 cells) have shown strong antitumor effects. In this review, we summarize established human NK cell lines and their biological characteristics, and highlight the applications of NK-92 cells and CAR-NK-92 cells in tumor immunotherapy.


Assuntos
Imunoterapia , Células Matadoras Naturais/imunologia , Neoplasias/terapia , Receptores de Antígenos Quiméricos/imunologia , Linhagem Celular Tumoral , Citotoxicidade Imunológica/imunologia , Vetores Genéticos/imunologia , Vetores Genéticos/uso terapêutico , Humanos , Neoplasias/imunologia , Receptores de Antígenos Quiméricos/uso terapêutico
19.
J Virol ; 93(7)2019 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-30651356

RESUMO

Human respiratory syncytial virus (RSV) is a major pediatric respiratory pathogen. The attachment (G) and fusion (F) glycoproteins are major neutralization and protective antigens. RSV G is expressed as membrane-anchored (mG) and -secreted (sG) forms, both containing a central fractalkine-like CX3C motif. The CX3C motif and sG are thought to interfere with host immune responses and have been suggested to be omitted from a vaccine. We used a chimeric bovine/human parainfluenza virus type 3 (rB/HPIV3) vector to express RSV wild-type (wt) G and modified forms, including sG alone, mG alone, mutants with ablated CX3C, and G with enhanced packaging into vector virions. In hamsters, these viruses replicated to similar titers. When assayed with a complement-enhanced neutralization assay in Vero cells, sG did not reduce the serum RSV- or PIV3-neutralizing antibody (NAb) responses, whereas ablating CX3C drastically reduced the RSV NAb response. Protective efficacy against RSV challenge was not reduced by sG but was strongly dependent on the CX3C motif. In ciliated human airway epithelial (HAE) cells, NAbs induced by wt G, but not by wt F, completely blocked RSV infection in the absence of added complement. This activity was dependent on the integrity of the CX3C motif. In hamsters, the rB/HPIV3 expressing wt G conferred better protection against RSV challenge than that expressing wt F. Codon optimization of the wt G further increased its immunogenicity and protective efficacy. This study showed that ablation of the CX3C motif or sG in an RSV vaccine, as has been suggested previously, would be ill advised.IMPORTANCE Human RSV is the leading viral cause of severe pediatric respiratory illness. An RSV vaccine is not yet available. The RSV attachment protein G is an important protective and neutralization antigen. G contains a conserved fractalkine-like CX3C motif and is expressed in mG and sG forms. sG and the CX3C motif are thought to interfere with host immune responses, but this remains poorly characterized. Here, we used an attenuated chimeric bovine/human parainfluenza virus type 3 (rB/HPIV3) vector to express various modified forms of RSV G. We demonstrated that strong antibody and protective responses could be induced by G alone, and that this was highly dependent on the integrity of the CX3C motif. There was no evidence that sG or the CX3C motif impaired immune responses against RSV G or the rB/HPIV3 vector. rB/HPIV3 expressing wt RSV G provides a bivalent vaccine against RSV and HPIV3.


Assuntos
Vetores Genéticos/imunologia , Infecções por Vírus Respiratório Sincicial/imunologia , Vírus Sincicial Respiratório Humano/imunologia , Respirovirus/imunologia , Proteínas do Envelope Viral/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Bovinos , Criança , Feminino , Humanos , Macaca mulatta , Mesocricetus , Infecções por Vírus Respiratório Sincicial/virologia , Células Vero , Proteínas Virais de Fusão/imunologia , Vírion/imunologia , Replicação Viral/imunologia
20.
Viruses ; 11(2)2019 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-30691064

RESUMO

Decades ago, Friedmann and Roblin postulated several barriers to gene therapy, including tissue targeting, delivery across the blood⁻brain barrier (BBB), and host immune responses. These issues remain pertinent till today. Since then, several advances have been made in elucidating structures of adeno-associated virus (AAV) serotypes, antibody epitopes, and ways to modify antibody-binding sites. AAVs capsid has also been engineered to re-direct tissue tropism, reduce ubiquitination, and promote passage across the BBB. Furthermore, the use of high(er) dose recombinant AAV (rAAV) has been accompanied by a better understanding of immune responses in both experimental animals and early clinical trials, and novel work is being performed to modulate the immune response. While the immune responses to rAAV remains a major challenge in translating experimental drugs to approved medicine, and will likely require more than a single solution, we now better understand the hurdles to formulate and test experimental solutions to surmount them.


Assuntos
Dependovirus/imunologia , Terapia Genética , Vetores Genéticos/imunologia , Interações entre Hospedeiro e Microrganismos/imunologia , Imunidade Inata , Infecções por Parvoviridae/imunologia , Imunidade Adaptativa , Animais , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/imunologia , Ensaios Clínicos como Assunto , Humanos , Camundongos
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