RESUMO
Vaccines and therapeutics using in vitro transcribed mRNA hold enormous potential for human and veterinary medicine. Transfection agents are widely considered to be necessary to protect mRNA and enhance transfection, but they add expense and raise concerns regarding quality control and safety. We found that such complex mRNA delivery systems can be avoided when transfecting epithelial cells by aerosolizing the mRNA into micron-sized droplets. In an equine in vivo model, we demonstrated that the translation of mRNA into a functional protein did not depend on the addition of a polyethylenimine (PEI)-derived transfection agent. We were able to safely and effectively transfect the bronchial epithelium of foals using naked mRNA (i.e., mRNA formulated in a sodium citrate buffer without a delivery vehicle). Endoscopic examination of the bronchial tree and histology of mucosal biopsies indicated no gross or microscopic adverse effects of the transfection. Our data suggest that mRNA administered by an atomization device eliminates the need for chemical transfection agents, which can reduce the cost and the safety risks of delivering mRNA to the respiratory tract of animals and humans.
Assuntos
Cavalos , Sprays Nasais , RNA Mensageiro/administração & dosagem , Mucosa Respiratória , Animais , Animais Recém-Nascidos , Células Cultivadas , Portadores de Fármacos/administração & dosagem , Portadores de Fármacos/efeitos adversos , Portadores de Fármacos/farmacocinética , Sistemas de Liberação de Medicamentos/efeitos adversos , Sistemas de Liberação de Medicamentos/métodos , Sistemas de Liberação de Medicamentos/veterinária , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Feminino , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Nebulizadores e Vaporizadores/veterinária , Polietilenoimina/administração & dosagem , Polietilenoimina/química , RNA Mensageiro/efeitos adversos , RNA Mensageiro/farmacocinética , Mucosa Respiratória/efeitos dos fármacos , Mucosa Respiratória/metabolismo , Transcrição Gênica , Transfecção/métodos , Transfecção/veterinária , Vacinas de DNA/administração & dosagem , Vacinas de DNA/efeitos adversos , Vacinas de DNA/farmacocinéticaRESUMO
PURPOSE: Anthrax is a lethal bacterial disease caused by gram-positive bacterium Bacillus anthracis and vaccination is a desirable method to prevent anthrax infections. In the present study, DNA vaccine encoding a protective antigen of Bacillus anthracis was prepared and we investigated the influence of DNA electrotransfer in the skin on the induced immune response and biodistribution. METHODS AND RESULTS: The tdTomato reporter gene for the whole animal in vivo imaging was used to assess gene transfer efficiency into the skin as a function of electrical parameters. Compared to that with 25 V, the transgene expression of red fluorescent protein increased significantly when a voltage of 90 V was used. Delivery of DNA vaccines expressing Bacillus anthracis protective antigen domain 4 (PAD4) with an applied voltage of 90 V induced robust PA-D4-specific antibody responses. In addition, the in vivo fate of anthrax DNA vaccine was studied after intradermal administration into the mouse. DNA plasmids remained at the skin injection site for an appropriate period of time after immunization. Intradermal administration of DNA vaccine resulted in detection in various organs (viz., lung, heart, kidney, spleen, brain, and liver), although the levels were significantly reduced. CONCLUSION: Our results offer important insights into how anthrax DNA vaccine delivery by intradermal electroporation affects the immune response and biodistribution of DNA vaccine. Therefore, it may provide valuable information for the development of effective DNA vaccines against anthrax infection.
Assuntos
Vacinas contra Antraz/administração & dosagem , Vacinas de DNA/administração & dosagem , Animais , Vacinas contra Antraz/farmacocinética , Anticorpos Antibacterianos/sangue , Antígenos de Bactérias/imunologia , Bacillus anthracis/imunologia , Toxinas Bacterianas/imunologia , Eletroporação , Feminino , Expressão Gênica , Genes Reporter , Imunoglobulina G/sangue , Injeções Intradérmicas , Proteínas Luminescentes/genética , Camundongos Endogâmicos BALB C , Plasmídeos , Pele/metabolismo , Distribuição Tecidual , Vacinas de DNA/farmacocinética , Proteína Vermelha FluorescenteRESUMO
ChAd3-EBO-Z is an investigational adenovirus-based vaccine for the prevention of Ebola virus disease. Two nonclinical studies were performed to evaluate the biodistribution, local tolerance and potential local and systemic toxic effects of this vaccine. In the biodistribution study, rats received a single intramuscular injection of either ChAd3-EBO-Z or saline. Enlargement of the draining lymph nodes, starting on day 2, was noticed in ChAd3-EBO-Z-treated rats, indicating that an immune response had taken place. Viral DNA was mainly found at the injection sites and in the draining lymph nodes, from where it progressively disappeared during the observation period, while it was found only transiently and occasionally in other organs. In the repeated-dose toxicity study, either ChAd3-EBO-Z or saline was administered intramuscularly to rabbits on two occasions with a 2-week interval. General health status, rectal temperature, local tolerance, ophthalmology, hematology, coagulation and blood chemistry parameters were monitored. Macroscopic and microscopic evaluations were performed. Treatment-related changes included a transient increase in neutrophil count, C-reactive protein and fibrinogen levels, and a transient decrease in platelet count. As expected, microscopic observations 3 days after the second injection were related to the elicited inflammatory reaction, and these inflammatory responses had almost completely disappeared 29 days after the second immunization. In conclusion, the vaccine was locally and systemically well-tolerated and the viral vector was partially or totally cleared from the organs where it disseminated, supporting the clinical development of the vaccine.
Assuntos
Adenoviridae/genética , Vacinas contra Ebola/farmacocinética , Ebolavirus/imunologia , Vetores Genéticos , Animais , Vacinas contra Ebola/administração & dosagem , Vacinas contra Ebola/toxicidade , Feminino , Esquemas de Imunização , Imunogenicidade da Vacina , Injeções Intramusculares , Masculino , Coelhos , Ratos Sprague-Dawley , Distribuição Tecidual , Vacinas de DNA/administração & dosagem , Vacinas de DNA/farmacocinética , Vacinas de DNA/toxicidadeRESUMO
DNA vaccines, the third-generation vaccines, were extensively studied. The attenuated Salmonella choleraesuis (S. choleraesuis) was widely focused as a carrier to deliver DNA vaccines in the chromosome-plasmid balanced-lethal system. The efficacy of inhibin DNA vaccine delivered by attenuated S. choleraesuis was proved in mice and cows in our previous studies. In this study, the efficacy of inhibin DNA vaccine was confirmed in rhesus monkeys. To further study the biodistribution and safety, the mice were immunized under laboratory conditions. The results of the rhesus monkeys showed the plasma IgA and IgG titres against inhibin were elevated, and the oestradiol (E2 ) and progesterone (P4 ) levels were increased with immunizing inhibin DNA vaccine. The biodistribution and safety assessment displayed the body weight, pathological change and haematology indexes where there is no significant difference between vaccinated mice and control. And the genomics analysis showed there was no integration of the inhibin gene into the mouse genome 2 months after immunization. This study indicated the inhibin DNA vaccine delivered by attenuated S. choleraesuis was safe. And this vaccine was a potential means to improve their reproductive traits in primates and other animals.
Assuntos
Portadores de Fármacos , Imunoterapia/métodos , Infertilidade/terapia , Inibinas/imunologia , Salmonella arizonae/genética , Vacinas de DNA/efeitos adversos , Vacinas de DNA/imunologia , Animais , Estradiol/sangue , Imunidade Humoral , Imunoglobulina A/sangue , Imunoglobulina G/sangue , Imunoterapia/efeitos adversos , Inibinas/genética , Macaca mulatta , Camundongos , Progesterona/sangue , Vacinas Atenuadas/administração & dosagem , Vacinas Atenuadas/imunologia , Vacinas Atenuadas/farmacocinética , Vacinas de DNA/administração & dosagem , Vacinas de DNA/farmacocinética , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/imunologiaRESUMO
DNA vaccination holds the potential to treat or prevent nearly any immunogenic disease, including cancer. To date, these vaccines have demonstrated limited immunogenicity in vivo due to the absence of a suitable delivery system which can protect DNA from degradation and improve transfection efficiencies in vivo. Recently, microneedles have been described as a novel physical delivery technology to enhance DNA vaccine immunogenicity. Of these devices, dissolvable microneedles promise a safe, pain-free delivery system which may simultaneously improve DNA stability within a solid matrix and increase DNA delivery compared to solid arrays. However, to date little work has directly compared the suitability of different dissolvable matrices for formulation of DNA-loaded microneedles. Therefore, the current study examined the ability of 4 polymers to formulate mechanically robust, functional DNA loaded dissolvable microneedles. Additionally, complexation of DNA to a cationic delivery peptide, RALA, prior to incorporation into the dissolvable matrix was explored as a means to improve transfection efficacies following release from the polymer matrix. Our data demonstrates that DNA is degraded following incorporation into PVP, but not PVA matrices. The complexation of DNA to RALA prior to incorporation into polymers resulted in higher recovery from dissolvable matrices, and increased transfection efficiencies in vitro. Additionally, RALA/DNA nanoparticles released from dissolvable PVA matrices demonstrated up to 10-fold higher transfection efficiencies than the corresponding complexes released from PVP matrices, indicating that PVA is a superior polymer for this microneedle application.
Assuntos
Portadores de Fármacos , Sistemas de Liberação de Medicamentos/instrumentação , Agulhas , Polímeros , Vacinação/instrumentação , Vacinas de DNA/administração & dosagem , Animais , Masculino , Camundongos Endogâmicos C57BL , Vacinas de DNA/farmacocinéticaRESUMO
Cyclophosphamide (CY) is a DNA alkylating agent, which is widely used with other chemotherapy drugs in the treatment of various types of cancer. It can be used not only as a chemotherapeutic but also as an immunomodulatory agent to inhibit IL-10 expression and T regulatory cells (Tregs). Fibroblast activation protein α (FAPα) is expressed in cancer-associated fibroblasts in the tumor microenvironment. Immunotherapy based on FAPα, as a tumor stromal antigen, typically induces specific immune response targeting the tumor microenvironment. This study evaluated the efficacy of a previously unreported CY combination strategy to enhance the limited anti-tumor effect of a DNA vaccine targeting FAPα. The results suggested CY administration could promote the percentage of splenic CD8+ T cells and decrease the proportion of CD4 + CD25 + Foxp3+ Tregs in spleen. In tumor tissues, levels of immunosuppressive cytokines including IL-10 and CXCL-12 were also reduced. Meanwhile, the CY combination did not impair the FAPα-specific immunity induced by the DNA vaccine and further reduced tumor stromal factors. Most importantly, FAP-vaccinated mice also treated with CY chemotherapy showed a marked suppression of tumor growth (inhibition ratio =80%) and a prolongation of survival time. Thus, the combination of FAPα immunotherapy and chemotherapy with CY offers new insights into improving cancer therapies.
Assuntos
Vacinas Anticâncer/farmacologia , Ciclofosfamida/farmacocinética , Gelatinases/farmacologia , Imunidade Celular/efeitos dos fármacos , Neoplasias Mamárias Experimentais/terapia , Proteínas de Membrana/farmacologia , Serina Endopeptidases/farmacologia , Vacinas de DNA/farmacocinética , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/patologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/patologia , Vacinas Anticâncer/imunologia , Endopeptidases , Feminino , Gelatinases/imunologia , Neoplasias Mamárias Experimentais/imunologia , Neoplasias Mamárias Experimentais/patologia , Proteínas de Membrana/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Serina Endopeptidases/imunologia , Vacinas de DNA/imunologiaRESUMO
PURPOSE: In order to enhance cellular uptake and to facilitate transdermal delivery of DNA vaccine, polyamidoamine (PAMAM) dendrimers conjugated with HIV transactivator of transcription (TAT) was developed. METHODS: First, the plasmid DNA (pIRES-H5/GFP) nanoparticle was formulated using PAMAM dendrimer and TAT peptide and then characterized for surface charge, particle size, DNA encapsulation and protection of the pIRES-H5/GFP DNA plasmid to enzymatic digestion. Subsequently, the potency of the TAT-conjugated dendrimer for gene delivery was evaluated through in vitro transfection into Vero cells followed by gene expression analysis including western blotting, fluorescent microscopy and PCR. The effect of the TAT peptide on cellular uptake of DNA vaccine was studied by qRT-PCR and flow cytometry. Finally, the ability of TAT-conjugated PAMAM dendrimer for transdermal delivery of the DNA plasmid was assessed through artificial membranes followed by qRT-PCR and flow cytometry. RESULTS: TAT-conjugated PAMAM dendrimer showed the ability to form a compact and nanometre-sized polyplexes with the plasmid DNA, having the size range of 105 to 115 nm and a positive charge of +42 to +45 mV over the N/P ratio of 6:1(+/-). In vitro transfection analysis into Vero cells confirms the high potency of TAT-conjugated PAMAM dendrimer to enhance the cellular uptake of DNA vaccine. The permeability value assay through artificial membranes reveals that TAT-conjugated PAMAM has more capacity for transdermal delivery of the DNA compared to unmodified PAMAM dendrimer (P<0.05). CONCLUSIONS: The findings of this study suggest that TAT-conjugated PAMAM dendrimer is a promising non-viral vector for transdermal use.This article is open to POST-PUBLICATION REVIEW. Registered readers (see "For Readers") may comment by clicking on ABSTRACT on the issue's contents page.
Assuntos
Dendrímeros/química , Dendrímeros/farmacocinética , Técnicas de Transferência de Genes , Peptídeos/química , Vacinas de DNA/química , Vacinas de DNA/farmacocinética , Produtos do Gene tat do Vírus da Imunodeficiência Humana/química , Animais , Chlorocebus aethiops , Peptídeos/farmacocinética , Poliaminas/química , Vacinas de DNA/genética , Células Vero , Produtos do Gene tat do Vírus da Imunodeficiência Humana/farmacocinéticaRESUMO
We determined the efficacy and safety of chitosan (CS)-coated poly(lactic-co-glycolic) acid (PLGA) nanoparticles (NPs) as a delivery system for a vaccine to protect chickens against Newcastle disease virus (NDV). The newly constructed vaccine contained DNA (the F gene) of NDV. The Newcastle disease virus (NDV) F gene deoxyribonucleic acid (DNA) plasmid (pFDNA)-CS/PLGA-NPs were spherical (diameter =699.1 ± 5.21 nm [mean ± standard deviation]) and smooth, with an encapsulation efficiency of 98.1% and a Zeta potential of +6.35 mV. An in vitro release assay indicated that CS controlled the burst release of plasmid DNA, such that up to 67.4% of the entire quantity of plasmid DNA was steadily released from the pFDNA-CS/PLGA-NPs. An in vitro expression assay indicated that the expression of nanoparticles (NPs) was maintained in the NPs. In an immunization test with specific pathogen-free chickens, the pFDNA-CS/PLGA-NPs induced stronger cellular, humoral, and mucosal immune responses than the plasmid DNA vaccine alone. The pFDNA-CS/PLGA-NPs did not harm 293T cells in an in vitro assay and did not harm chickens in an in vivo assay. Overall, the results indicated that CS-coated PLGA NPs can serve as an efficient and safe mucosal immune delivery system for NDV DNA vaccine.
Assuntos
Quitosana/química , DNA Viral/farmacocinética , Portadores de Fármacos/química , Ácido Láctico/química , Nanopartículas/química , Vírus da Doença de Newcastle/genética , Ácido Poliglicólico/química , Vacinas de DNA/farmacocinética , Animais , Galinhas , Quitosana/farmacocinética , DNA Viral/genética , DNA Viral/imunologia , DNA Viral/metabolismo , Portadores de Fármacos/farmacocinética , Imunoglobulina A/análise , Ácido Láctico/farmacocinética , Linfócitos/imunologia , Doença de Newcastle/prevenção & controle , Doença de Newcastle/virologia , Ácido Poliglicólico/farmacocinética , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Lágrimas/química , Vacinas de DNA/administração & dosagem , Vacinas de DNA/genética , Vacinas de DNA/imunologiaRESUMO
We have previously shown that the combination of MIDGE-Th1 DNA vectors with the cationic lipid SAINT-18 increases the immune response to the encoded antigen in mice. Here, we report on experiments to further optimize and characterize this approach. We evaluated different formulations of MIDGE-Th1 vectors with SAINT-18 by assessing their influence on the transfection efficiency in cell culture and on the immune response in mice. We found that high amounts of SAINT-18 in formulations with a w/w ratio MIDGE Th1/SAINT-18 of 1:4.8 are beneficial for cell transfection in vitro. In contrast, the formulation of HBsAg-encoding MIDGE-Th1 DNA vectors with the lowest amount of SAINT-18 (w/w ratio MIDGE Th1/SAINT-18 of 1:0.5) resulted in the highest serum IgG1 and IgG2a levels after intradermal immunization of mice. Consequently, latter formulation was selected for a comparative biodistribution study in rats. Following intradermal administration of both naked and formulated MIDGE-Th1 DNA, the vectors localized primarily at the site of injection. Vector DNA levels decreased substantially over the two months duration of the study. When administered in combination with SAINT-18, the vectors were found in significantly higher amounts in draining lymph nodes in comparison to administration of naked MIDGE-Th1 DNA. We propose that the high immune responses induced by MIDGE-Th1/SAINT-18 lipoplexes are mediated by enhanced transfection of cells in vivo, resulting in stronger antigen expression and presentation. Importantly, the combination of MIDGE-Th1 vectors with SAINT-18 was well tolerated in mice and rats and is expected to be safe in human clinical applications.
Assuntos
Vetores Genéticos/química , Compostos de Piridínio/química , Vacinas de DNA/imunologia , Animais , Anticorpos Antivirais/sangue , Cátions , Feminino , Vetores Genéticos/farmacocinética , Antígenos de Superfície da Hepatite B/imunologia , Imunoglobulina G/sangue , Linfonodos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Compostos de Piridínio/farmacocinética , Ratos , Ratos Wistar , Células Th1/imunologia , Distribuição Tecidual , Transfecção , Vacinas de DNA/farmacocinéticaRESUMO
There is no clinically available cancer immunotherapy that exploits Langerhans cells (LCs), the epidermal precursors of dendritic cells (DCs) that are the natural agent of antigen delivery. We developed a DNA formulation with a polymer and obtained synthetic 'pathogen-like' nanoparticles that preferentially targeted LCs in epidermal cultures. These nanoparticles applied topically under a patch-elicited robust immune responses in human subjects. To demonstrate the mechanism of action of this novel vaccination strategy in live animals, we assembled a high-resolution two-photon laser scanning-microscope. Nanoparticles applied on the native skin poorly penetrated and poorly induced LC motility. The combination of nanoparticle administration and skin treatment was essential both for efficient loading the vaccine into the epidermis and for potent activation of the LCs to migrate into the lymph nodes. LCs in the epidermis picked up nanoparticles and accumulated them in the nuclear region demonstrating an effective nuclear DNA delivery in vivo. Tissue distribution studies revealed that the majority of the DNA was targeted to the lymph nodes. Preclinical toxicity of the LC-targeting DNA vaccine was limited to mild and transient local erythema caused by the skin treatment. This novel, clinically proven LC-targeting DNA vaccine platform technology broadens the options on DC-targeting vaccines to generate therapeutic immunity against cancer.
Assuntos
Células de Langerhans , Linfonodos/efeitos dos fármacos , Vacinas de DNA/administração & dosagem , Administração Tópica , Animais , Movimento Celular , Sistemas de Liberação de Medicamentos , Células Epidérmicas , Epiderme/efeitos dos fármacos , Imunoterapia/métodos , Camundongos Transgênicos , Microscopia Confocal/instrumentação , Microscopia Confocal/métodos , Nanopartículas/administração & dosagem , Coelhos , Distribuição Tecidual , Vacinas de DNA/farmacocinéticaRESUMO
This report shows that a nanovector composed of peptide-based nanofibrous hydrogel can condense DNA to result in strong immune responses against HIV. This nanovector can strongly activate both humoral and cellular immune responses to a balanced level rarely reported in previous studies, which is crucial for HIV prevention and therapy. In addition, this nanovector shows good biosafety in vitro and in vivo. Detailed characterizations show that the nanofibrous structure of the hydrogel is critical for the dramatically improved immune responses compared to existing materials. This peptide-based nanofibrous hydrogel shows great potential for efficacious HIV DNA vaccines and can be potentially used for delivering other vaccines and drugs.
Assuntos
Vacinas contra a AIDS , Infecções por HIV/prevenção & controle , Hidrogéis , Nanofibras/química , Vacinas de DNA , Vacinas contra a AIDS/química , Vacinas contra a AIDS/imunologia , Vacinas contra a AIDS/farmacologia , Animais , Infecções por HIV/imunologia , Humanos , Hidrogéis/química , Hidrogéis/farmacologia , Imunidade Celular/efeitos dos fármacos , Imunidade Humoral/efeitos dos fármacos , Camundongos , Vacinas de DNA/química , Vacinas de DNA/imunologia , Vacinas de DNA/farmacocinéticaRESUMO
Hepatitis B virus (HBV) persistence is facilitated by exhaustion of CD8 T cells that express the inhibitory receptor programmed cell death-1 (PD-1). Improvement of the HBV-specific T cell function has been obtained in vitro by inhibiting the PD-1/PD-ligand 1 (PD-L1) interaction. In this study, we examined whether in vivo blockade of the PD-1 pathway enhances virus-specific T cell immunity and leads to the resolution of chronic hepadnaviral infection in the woodchuck model. The woodchuck PD-1 was first cloned, characterized, and its expression patterns on T cells from woodchucks with acute or chronic woodchuck hepatitis virus (WHV) infection were investigated. Woodchucks chronically infected with WHV received a combination therapy with nucleoside analogue entecavir (ETV), therapeutic DNA vaccination and woodchuck PD-L1 antibody treatment. The gain of T cell function and the suppression of WHV replication by this therapy were evaluated. We could show that PD-1 expression on CD8 T cells was correlated with WHV viral loads during WHV infection. ETV treatment significantly decreased PD-1 expression on CD8 T cells in chronic carriers. In vivo blockade of PD-1/PD-L1 pathway on CD8 T cells, in combination with ETV treatment and DNA vaccination, potently enhanced the function of virus-specific T cells. Moreover, the combination therapy potently suppressed WHV replication, leading to sustained immunological control of viral infection, anti-WHs antibody development and complete viral clearance in some woodchucks. Our results provide a new approach to improve T cell function in chronic hepatitis B infection, which may be used to design new immunotherapeutic strategies in patients.
Assuntos
Antivirais/farmacologia , Antígeno B7-H1/imunologia , Linfócitos T CD8-Positivos/imunologia , Guanina/análogos & derivados , Vírus da Hepatite B da Marmota/fisiologia , Hepatite B Crônica/terapia , Vacinas Virais/farmacologia , Replicação Viral/imunologia , Animais , Antígeno B7-H1/antagonistas & inibidores , Linfócitos T CD8-Positivos/patologia , Guanina/farmacologia , Hepatite B Crônica/imunologia , Hepatite B Crônica/patologia , Marmota , Camundongos , Receptor de Morte Celular Programada 1/imunologia , Vacinação , Vacinas de DNA/imunologia , Vacinas de DNA/farmacocinética , Vacinas Virais/imunologia , Replicação Viral/efeitos dos fármacosRESUMO
BACKGROUND: Gene vaccines offer attractive rationales for prophylactic as well as therapeutic treatments of type I allergies. DNA and mRNA vaccines have been shown to prevent from allergic sensitization and to counterbalance established allergic immune reactions. Recent advances in gene vaccine manipulation offer additional opportunities for modulation of T helper cell profiles by specific targeting of cellular compartments. METHODS: DNA vaccines encoding the major birch pollen allergen Bet v 1.0101 were equipped with different leader sequences to shuttle the antigen to lysosomes (LIMP-II), to trigger cellular secretion (hTPA), or to induce proteasomal degradation via forced ubiquitination (ubi). Mice were pre-vaccinated with these constructs and the protective efficacy was tested by subcutaneous Th2-promoting challenges, followed by allergen inhalation. IgG antibody subclass distribution and allergen-specific IgE as well as cytokine profiles from re-stimulated splenocytes and from BALFs were assessed. The cellular composition of BALFs, and lung resistance and compliance were determined. RESULTS: Immunization with all targeting variants protected from allergic sensitization, i.e. IgE induction, airway hyperresponsiveness, lung inflammation, and systemic and local Th2 cytokine expression. Surprisingly, protection did not clearly correlate with the induction of a systemic Th1 cytokine profile, but rather with proliferating CD4+ CD25+ FoxP3+ T regulatory cells in splenocyte cultures. Targeting the allergen to proteasomal or lysosomal degradation severely down-regulated antibody induction after vaccination, while T cell responses remained unaffected. Although secretion of antigen promoted the highest numbers of Th1 cells, this vaccine type was the least efficient in suppressing the establishment of an allergic immune response. CONCLUSION: This comparative analysis highlights the modulatory effect of antigen targeting on the resulting immune response, with a special emphasis on prophylactic anti-allergy DNA vaccination. Targeting the antigen to proteasomal or lysosomal degradation reduces the availability of native allergen, thereby rendering the vaccine hypoallergenic without compromising efficacy, an important feature for a therapeutic setting.
Assuntos
Antialérgicos/imunologia , Antialérgicos/farmacocinética , Hipersensibilidade/prevenção & controle , Vacinas de DNA/imunologia , Vacinas de DNA/farmacocinética , Alérgenos/genética , Alérgenos/imunologia , Animais , Betula , Citocinas/metabolismo , Feminino , Hipersensibilidade/imunologia , Imunoglobulina E/sangue , Imunoglobulina G/sangue , Leucócitos Mononucleares/imunologia , Pulmão/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Transporte Proteico , Baço/imunologiaRESUMO
The efficacy of DNA vaccines is highly dependent on the methods used for their delivery and the choice of delivery sites/targets for gene injection, pointing at the necessity of a strict control over the gene delivery process. Here, we have investigated the effect of the injection site on gene expression and immunogenicity in BALB/c mice, using as a model a weak gene immunogen, DNA encoding firefly luciferase (Luc) delivered by superficial or deep injection with subsequent electroporation (EP). Immunization was assessed by monitoring the in vivo expression of luciferase by 2D- and 3D-bioluminescence imaging (BLI) and by the end-point immunoassays. Anti-Luc antibodies were assessed by ELISA, and T-cell response by IFN-γ and IL-2 FluoroSpot in which mouse splenocytes were stimulated with Luc or a peptide representing its immunodominant CD8+ T-cell epitope GFQSMYTFV. Monitoring of immunization by BLI identified EP parameters supporting the highest Luc gene uptake and expression. Superficial injection of Luc DNA followed by optimal EP led to a low level Luc expression in the mouse skin, and triggered a CD8+ T-cell response characterized by the peptide-specific secretion of IFN-γ and IL-2, but no specific antibodies. Intramuscular gene delivery resulted in a several-fold higher Luc expression and anti-Luc antibody, but induced low IL-2 and virtually no specific IFN-γ. Photon flux from the sites of Luc gene injection was inversely proportional to the immune response against GFQSMYTFV (p<0.05). Thus, BLI permitted to control the accuracy of gene delivery and transfection with respect to the injection site as well as the parameters of electroporation. Further, it confirmed the critical role of the site of DNA administration for the type and magnitude of the vaccine-specific immune response. This argues for the use of luminescent reporters in the preclinical gene vaccine tests to monitor both gene delivery and the immune response development in live animals.
Assuntos
Imunização/métodos , Medições Luminescentes , Imagem Óptica , Vacinas de DNA/administração & dosagem , Vacinas de DNA/farmacocinética , Animais , Anticorpos/sangue , Ensaio de Imunoadsorção Enzimática , Feminino , Genes Reporter , Imunoensaio , Proteínas de Insetos/imunologia , Interferon gama/metabolismo , Interleucina-2/metabolismo , Luciferases de Vaga-Lume/biossíntese , Luciferases de Vaga-Lume/genética , Camundongos , Camundongos Endogâmicos BALB C , Linfócitos T/imunologiaRESUMO
ONRAB(®) is a recombinant human adenovirus type 5 (HAd5) with the rabies glycoprotein gene incorporated into its genome. ONRAB(®) has been used in Canada as an oral rabies vaccine in target wildlife species such as: red fox (Vulpes vulpes), raccoon (Procyon lotor), and striped skunk (Mepthis mephitis). We evaluated the safety of ONRAB(®) in non-target wildlife species likely to contact the vaccine baits during oral rabies vaccine campaigns in the United States. We investigated the effects of oral inoculation of high titer ONRAB(®), approximately ten times the dose given to target species, in wood rats (Neotoma spp.), eastern cottontail rabbits (Sylvilagus floridanus), Virginia opossums (Didelphis virginiana), eastern wild turkeys (Meleagris gallopavo silvestri), and fox squirrels (Sciurus niger). We performed real-time polymerase chain reaction (PCR) on fecal swabs, oral swabs, and tissues, including lung, liver, kidney, small intestine, large intestine, and when appropriate nasal turbinates, to detect ONRAB(®) DNA from inoculated animals. By seven days post-inoculation, turkeys, opossums, and cottontails had all stopped shedding ONRAB(®) DNA. One wood rat and one fox squirrel still had detectable levels of ONRAB(®) DNA in fecal swabs 14 days post-inoculation. Real-time PCR analysis of the tissues revealed some ONRAB(®) DNA persisting in certain tissues; however, there were no significant gross or histologic lesions associated with ONRAB(®) in any of the species studied. Our results suggest that many non-target species are not likely to be impacted by the distribution of ONRAB(®) as part of oral rabies vaccination programs in the United States.
Assuntos
Animais Selvagens/imunologia , Vacina Antirrábica/administração & dosagem , Vacina Antirrábica/efeitos adversos , Vacinas de DNA/farmacocinética , Administração Oral , Animais , Fezes , Lagomorpha/imunologia , Gambás/imunologia , Vacina Antirrábica/farmacocinética , Sciuridae/imunologia , Sigmodontinae/imunologia , Distribuição Tecidual , Perus/imunologia , Vacinas de DNA/administração & dosagem , Vacinas de DNA/efeitos adversosRESUMO
Chlamydia trachomatis is a bacterial sexually transmitted infection affecting millions of people worldwide. Previous vaccination attempts have employed the recombinant major outer membrane protein (MOMP) of C. trachomatis nonetheless, with limited success, perhaps, due to stability, degradation, and delivery issues. In this study we cloned C. trachomatis recombinant MOMP DNA (DMOMP) and encapsulated it in chitosan nanoparticles (DMCNP) using the complex coacervation technique. Physiochemical characterizations of DMCNP included transmission and scanning electron microcopy, Fourier transform infrared and ultraviolet-visible spectroscopy, and zeta potential. Encapsulated DMOMP was 167-250 nm, with a uniform spherical shape and homogenous morphology, and an encapsulation efficiency > 90%. A slow release pattern of encapsulated DMOMP, especially in acidic solution, was observed over 7 days. The zeta potential of DMCNP was ~8.80 mV, which indicated that it was highly stable. Toxicity studies of DMCNP (25-400 µg/mL) to Cos-7 cells using the MTT assay revealed minimal toxicity over 24-72 hours with >90% viable cells. Ultra-violet visible (UV-vis) spectra indicated encapsulated DMOMP protection by chitosan, whereas agarose gel electrophoresis verified its protection from enzymatic degradation. Expression of MOMP protein in DMCNP-transfected Cos-7 cells was demonstrated via Western blotting and immunofluorescence microscopy. Significantly, intramuscular injection of BALB/c mice with DMCNP confirmed the delivery of encapsulated DMOMP, and expression of the MOMP gene transcript in thigh muscles and spleens. Our data show that encapsulation of DMOMP in biodegradable chitosan nanoparticles imparts stability and protection from enzymatic digestion, and enhances delivery and expression of DMOMP in vitro and in mice. Further investigations of the nanoencapsulated DMCNP vaccine formulation against C. trachomatis in mice are warranted.
Assuntos
Quitosana/química , Chlamydia trachomatis/genética , Portadores de Fármacos/química , Porinas/genética , Vacinas de DNA/química , Animais , Células COS , Sobrevivência Celular/efeitos dos fármacos , Quitosana/farmacocinética , Quitosana/farmacologia , Chlorocebus aethiops , DNA Bacteriano/genética , Portadores de Fármacos/farmacocinética , Portadores de Fármacos/farmacologia , Estabilidade de Medicamentos , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Nanopartículas , Tamanho da Partícula , Porinas/metabolismo , RNA/análise , RNA/isolamento & purificação , Vacinas de DNA/genética , Vacinas de DNA/farmacocinética , Vacinas de DNA/farmacologiaRESUMO
BACKGROUND: A eukaryotic expression plasmid encoding glycoprotein C (gC) of Anatid herpesvirus 1 (AnHV-1) (pcDNA3.1-gC) was constructed and validated. The tissue distribution of chitosan/DNA complexes, liposome/DNA complexes and pcDNA3.1-gC alone were evaluated using a quantitative real-time PCR based TaqMan™ probe following intramuscular administration in ducklings. RESULTS: Compared with pcDNA3.1-gC alone, liposomes universally increased the plasmid DNA copy number at the injection sites, liver, spleen, heart, brain, bursa of Fabricius, and especially in the enteron (esophagus, duodenum, rectum, and cecum). Chitosan also universally increased the plasmid DNA copy number at the injection sites, liver, spleen, heart, brain and esophagus. Compared with lipoplex-gC, higher chitosan-gC plasmid DNA copy numbers were detected at the injection sites, liver, spleen, heart, brain and esophagus. In contrast, compared with lipoplex-gC, lower copy numbers of chitosan-gC plasmid DNA were detected in the duodenum, rectum and cecum. CONCLUSIONS: The results of this study demonstrated that chitosan and liposomes mediated rapid and extensive plasmid distribution in duck tissues, with low levels maintained from 1 d after DNA vaccination.
Assuntos
Alphaherpesvirinae/genética , Sistemas de Liberação de Medicamentos/métodos , Infecções por Herpesviridae/veterinária , Doenças das Aves Domésticas/prevenção & controle , Vacinas de DNA/farmacocinética , Proteínas do Envelope Viral/farmacocinética , Proteínas Virais/farmacocinética , Alphaherpesvirinae/imunologia , Animais , Quitosana/química , Portadores de Fármacos/química , Patos , Infecções por Herpesviridae/prevenção & controle , Infecções por Herpesviridae/virologia , Lipossomos/química , Doenças das Aves Domésticas/virologia , Distribuição Tecidual , Vacinas de DNA/administração & dosagem , Vacinas de DNA/química , Vacinas de DNA/genética , Proteínas do Envelope Viral/administração & dosagem , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/genética , Proteínas Virais/administração & dosagem , Proteínas Virais/química , Proteínas Virais/genéticaRESUMO
DNA plasmid immunization is a novel approach of preventive and therapeutic vaccine. More than 100 DNA vaccines have been on preclinical or clinical phase trials, and four kinds of DNA vaccines for livestock have been approved by USDA, CFIA, and APVMA. Schistosomiasis is a worldwide parasitic disease, and vaccine immunization is supposed to be a promising approach to control the health crisis. On the basis of former preclinical studies, we further focused on the pharmacokinetics and risk evaluation of DNA vaccine in vivo. In the present study, enhanced green fluorescent protein (EGFP) report gene was fused with Schistosoma japonicum 23 kDa transmembrane protein antigen gene (Sj23) and constructed into DNA vaccine pVIVO2-Sj23.EGFP. After intramuscularly injecting 100 µg of purified DNA vaccine plasmid to immunizate BALB/c mice, we studied the tissue distribution of DNA plasmid and expressed Sj23.EGFP antigen, the persistence time of elicited antibodies, and the risk of DNA vaccine transferred into intestinal microorganisms. The results showed that DNA vaccine plasmid could be distributed into all tissues of the body after injection; however, only few organs including the injected muscle were detected DNA vaccine at postimmunization until the 100 days by PCR technology; the detection of green fluorescence protein displayed that DNA vaccine could be expressed in almost every tissue and organs; the ELISA assay indicated the immune antibody against Sj23 could persist over 70 days; and the DNA vaccine transferring intestinal flora results was negative. The results indicated that the DNA vaccine has systemic protection and long-lasting effectivity and is safe to intestinal flora.
Assuntos
Antígenos de Helmintos/imunologia , Proteínas de Helminto/imunologia , Proteínas de Membrana/imunologia , Schistosoma japonicum/imunologia , Vacinas de DNA/efeitos adversos , Vacinas de DNA/farmacocinética , Animais , Anticorpos Anti-Helmínticos/sangue , Antígenos de Helmintos/genética , Ensaio de Imunoadsorção Enzimática , Fluorescência , Trato Gastrointestinal/microbiologia , Transferência Genética Horizontal , Genes Reporter , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/genética , Proteínas de Helminto/genética , Injeções Intramusculares , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos BALB C , Plasmídeos/administração & dosagem , Plasmídeos/efeitos adversos , Plasmídeos/farmacocinética , Reação em Cadeia da Polimerase , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Schistosoma japonicum/genética , Fatores de Tempo , Distribuição Tecidual , Vacinas de DNA/administração & dosagem , Vacinas de DNA/imunologiaRESUMO
DNA-based vector systems have been widely studied as new modalities for the prevention and treatment of human diseases. As for all other medicinal products, safety is an important aspect in the evaluation of such products. In this chapter we reflect on the basic safety issues which have been raised with respect to preventive and therapeutic DNA vaccines, including insertional mutagenesis in case of chromosomal integration, possible formation of anti-DNA antibodies, induction of autoimmune responses and/or immunological tolerance. In addition, local reactions at the site of administration and adverse effects resulting from plasmid DNA spread to nontarget tissues are discussed. Most importantly, however, the benefit-risk profile of a medicinal product is crucial for a decision on providing marketing authorization or not. A product has an acceptable benefit-risk profile if the benefits of the product outweigh its risks for the treated patient.
Assuntos
Biolística/efeitos adversos , Segurança , Vacinação/efeitos adversos , Vacinação/instrumentação , Vacinas de DNA/administração & dosagem , Vacinas de DNA/imunologia , Animais , Autoimunidade/genética , Autoimunidade/imunologia , Humanos , Tolerância Imunológica/genética , Tolerância Imunológica/imunologia , Vacinas de DNA/efeitos adversos , Vacinas de DNA/genética , Vacinas de DNA/farmacocinéticaRESUMO
Previously, we showed that transcutaneous (TC) DNA immunization by applying plasmid DNA onto a mouse skin area wherein the hair follicles were induced into growth stage by plucking the hair using warm waxing induced strong and functional antigen-specific antibody responses. In the present study, using plasmids that encode ß-galactosidase gene or ovalbumin (OVA) gene, we showed that this mode of TC DNA immunization not only induced specific antibody responses, but also induced antigen-specific cytotoxic T lymphocyte responses. In fact, TC DNA immunization using a plasmid that encodes OVA gene prevented the growth of OVA-expressing B16-OVA tumor cells in the immunized mice. Moreover, we provided additional evidence supporting that hair follicles are essential for this mode of TC DNA immunization.