RESUMO
This article reports on an American Society of Pharmacology and Therapeutics, Division of Drug Metabolism and Disposition symposium held at Experimental Biology on April 2, 2022, in Philadelphia. As of July 2022, over 500 million people have been infected with SARS-CoV-2 (the virus causing COVID-19) and over 12 billion vaccine doses have been administered. Clinically significant interactions between viral infections and hepatic drug metabolism were first recognized over 40 years ago during a cluster of pediatric theophylline toxicity cases attributed to reduced hepatic drug metabolism amid an influenza B outbreak. Today, a substantive body of research supports that the activated innate immune response generally decreases hepatic cytochrome P450 activity. The interactions extend to drug transporters and other organs and have the potential to impact drug absorption, distribution, metabolism, and excretion (ADME). Based on this knowledge, altered ADME is predicted with SARS-CoV-2 infection or vaccination. The report begins with a clinical case exploring the possibility of SARS-CoV-2 vaccination increasing clozapine levels. This is followed by discussions of how SARS-CoV-2 infection or vaccines alter the metabolism and disposition of complex drugs, such as nanoparticles and biologics and small molecule therapies. The review concludes with a discussion of the effects of viral infections on placental amino acid transport and their potential to impact fetal development. The session improved our understanding of the impact of emerging viral infections and vaccine technologies on drug metabolism and disposition, which will help mitigate drug toxicity and improve drug and vaccine safety and effectiveness. SIGNIFICANCE STATEMENT: Altered pharmacokinetics of small molecule and complex molecule drugs and fetal brain distribution of amino acids following SARS-CoV-2 infection or immunization are possible. The proposed mechanisms involve decreased liver cytochrome P450 metabolism of small molecules, enhanced innate immune system metabolism of complex molecules, and altered placental and fetal blood-brain barrier amino acid transport, respectively. Future research is needed to understand the effects of these interactions on adverse drug responses, drug and vaccine safety, and effectiveness and fetal neurodevelopment.
Assuntos
Vacinas contra COVID-19 , COVID-19 , Criança , Feminino , Humanos , Gravidez , COVID-19/prevenção & controle , Vacinas contra COVID-19/efeitos adversos , Placenta , SARS-CoV-2 , VacinasRESUMO
Landmark studies describing the effect of microbial infection on the expression and activity of hepatic CYP3A used bacterial lipopolysaccharide as a model antigen. Our efforts to determine whether these findings were translatable to viral infections led us to observations suggesting that engagement of integrin receptors is key in the initiation of processes responsible for changes in hepatic CYP3A4 during infection and inflammation. Studies outlined in this article were designed to evaluate whether engagement of integrins, receptors commonly used by a variety of microbes to enter cellular targets, is vital in the regulation of CYP3A in the presence and absence of virus infection. Mice infected with a recombinant adenovirus (AdlacZ) experienced a 70% reduction in hepatic CYP3A catalytic activity. Infection with a mutant virus with integrin-binding arginine-glycine-aspartic acid (RGD) sequences deleted from the penton base protein of the virus capsid (AdΔRGD) did not alter CYP3A activity. CYP3A mRNA and protein levels in AdlacZ-treated animals were also suppressed, whereas those of mice given AdΔRGD were not significantly different from uninfected control mice. Silencing of the integrinß-subunit reverted adenovirus-mediated CYP3A4 suppression in vitro. Silencing of theα-subunit did not. Suppression of integrin subunits had a profound effect on nuclear receptors pregnane X receptor and constitutive androstane receptor, whereas retinoid X receptorαwas largely unaffected. To our knowledge, this is the first time that extracellular receptors, like integrins, have been indicated in the regulation of CYP3A. This finding has several implications owing to the important role of integrins in normal physiologic process and in many disease states.
Assuntos
Citocromo P-450 CYP3A/metabolismo , Cadeias beta de Integrinas/metabolismo , Fígado/enzimologia , Fígado/metabolismo , Adenoviridae/metabolismo , Animais , Receptor Constitutivo de Androstano , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Receptor de Pregnano X , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores de Esteroides , Receptor X Retinoide alfa/metabolismoRESUMO
The severity and longevity of the current Ebola outbreak highlight the need for a fast-acting yet long-lasting vaccine for at-risk populations (medical personnel and rural villagers) where repeated prime-boost regimens are not feasible. While recombinant adenovirus (rAd)-based vaccines have conferred full protection against multiple strains of Ebola after a single immunization, their efficacy is impaired by pre-existing immunity (PEI) to adenovirus. To address this important issue, a panel of formulations was evaluated by an in vitro assay for their ability to protect rAd from neutralization. An amphiphilic polymer (F16, FW â¼39,000) significantly improved transgene expression in the presence of anti-Ad neutralizing antibodies (NAB) at concentrations of 5 times the 50% neutralizing dose (ND50). In vivo performance of rAd in F16 was compared with unformulated virus, virus modified with poly(ethylene) glycol (PEG), and virus incorporated into poly(lactic-co-glycolic) acid (PLGA) polymeric beads. Histochemical analysis of lung tissue revealed that F16 promoted strong levels of transgene expression in naive mice and those that were exposed to adenovirus in the nasal cavity 28 days prior to immunization. Multiparameter flow cytometry revealed that F16 induced significantly more polyfunctional antigen-specific CD8+ T cells simultaneously producing IFN-γ, IL-2, and TNF-α than other test formulations. These effects were not compromised by PEI. Data from formulations that provided partial protection from challenge consistently identified specific immunological requirements necessary for protection. This approach may be useful for development of formulations for other vaccine platforms that also employ ubiquitous pathogens as carriers like the influenza virus.
Assuntos
Infecções por Adenoviridae/imunologia , Adenoviridae/imunologia , Vacinas contra Ebola/administração & dosagem , Ebolavirus/imunologia , Doença pelo Vírus Ebola/prevenção & controle , Imunidade Inata , Sprays Nasais , Adenoviridae/genética , Animais , Células Cultivadas , Vacinas contra Ebola/síntese química , Vacinas contra Ebola/imunologia , Vetores Genéticos/imunologia , Células HEK293 , Células HeLa , Doença pelo Vírus Ebola/imunologia , Humanos , Imunização Secundária , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Transgenes/imunologia , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/química , Vacinas Sintéticas/imunologiaRESUMO
As the Ebola outbreak in West Africa continues and cases appear in the United States and other countries, the need for long-lasting vaccines to preserve global health is imminent. Here, we evaluate the long-term efficacy of a respiratory and sublingual (SL) adenovirus-based vaccine in non-human primates in two phases. In the first, a single respiratory dose of 1.4×10(9) infectious virus particles (ivp)/kg of Ad-CAGoptZGP induced strong Ebola glycoprotein (GP) specific CD8+ and CD4+ T cell responses and Ebola GP-specific antibodies in systemic and mucosal compartments and was partially (67%) protective from challenge 62 days after immunization. The same dose given by the SL route induced Ebola GP-specific CD8+ T cell responses similar to that of intramuscular (IM) injection, however, the Ebola GP-specific antibody response was low. All primates succumbed to infection. Three primates were then given the vaccine in a formulation that improved the immune response to Ebola in rodents. Three primates were immunized with 2.0×10(10) ivp/kg of vaccine by the SL route. Diverse populations of polyfunctional Ebola GP-specific CD4+ and CD8+ T cells and significant anti-Ebola GP antibodies were present in samples collected 150 days after respiratory immunization. The formulated vaccine was fully protective against challenge 21 weeks after immunization. While diverse populations of Ebola GP-specific CD4+ T cells were produced after SL immunization, antibodies were not neutralizing and the vaccine was unprotective. To our knowledge, this is the first time that durable protection from a single dose respiratory adenovirus-based Ebola vaccine has been demonstrated in primates.
Assuntos
Adenoviridae/imunologia , Vacinas contra Ebola/administração & dosagem , Ebolavirus/imunologia , Doença pelo Vírus Ebola/prevenção & controle , Vacinas Sintéticas/administração & dosagem , Adenoviridae/genética , Animais , Células Cultivadas , Chlorocebus aethiops , Células HEK293 , Doença pelo Vírus Ebola/imunologia , Humanos , Macaca fascicularis , Masculino , Vacinação/métodos , Vacinas Sintéticas/genética , Células VeroRESUMO
HC-04 cells were evaluated as an in vitro model for mechanistic study of changes in the function of hepatic CYP3A during virus infection. Similar to in vivo observations, infection with a first generation recombinant adenovirus significantly inhibited CYP3A4 catalytic activity in an isoform-specific manner. Virus (MOI 100) significantly reduced expression of the retinoid X receptor (RXR) by 30% 96 hours after infection. Cytoplasmic concentrations of the pregnane X receptor (PXR) were reduced by 50%, whereas the amount of the constitutive androstane receptor (CAR) in the nuclear fraction doubled with respect to uninfected controls. Hepatocyte nuclear factor 4α (HNF-4α) and peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) were also reduced by â¼70% during infection. Virus suppressed CYP3A4 activity in the presence of the PXR agonist rifampicin and did not affect CYP3A4 activity in the presence of the CAR agonist CITCO [6-(4-chlorophenyl) imidazo[2,1-b][1,3]thiazole-5-carbaldehyde-O-(3,4-dichlorobenzyl)oxime], suggesting that virus-induced modification of PXR may be responsible for observed changes in hepatic CYP3A4. The HC-04 cell line is easy to maintain, and CYP3A4 in these cells was responsive to known inducers and suppressors. Dexamethasone (200 µM) and phenobarbital (500 µM) increased activity by 230 and 124%, whereas ketoconazole (10 µM) and lipopolysaccharide (LPS) (10 µg/ml) reduced activity by 90 and 92%, respectively. This suggests that HC-04 cells can be a valuable tool for mechanistic study of drug metabolism during infection and for routine toxicological screening of novel compounds prior to use in the clinic.
Assuntos
Infecções por Adenovirus Humanos/enzimologia , Citocromo P-450 CYP3A/metabolismo , Fígado/enzimologia , Sequência de Bases , Western Blotting , Linhagem Celular , Primers do DNA , Humanos , Técnicas In Vitro , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Marburg and Ebola hemorrhagic fevers have been described as the most virulent viral diseases known to man due to associative lethality rates of up to 90%. Death can occur within days to weeks of exposure and there is currently no licensed vaccine or therapeutic. Recent evidence suggests an important role for antiviral T cells in conferring protection, but little detailed analysis of this response as driven by a protective vaccine has been reported. We developed a synthetic polyvalent-filovirus DNA vaccine against Marburg marburgvirus (MARV), Zaire ebolavirus (ZEBOV), and Sudan ebolavirus (SUDV). Preclinical efficacy studies were performed in guinea pigs and mice using rodent-adapted viruses, whereas murine T-cell responses were extensively analyzed using a novel modified assay described herein. Vaccination was highly potent, elicited robust neutralizing antibodies, and completely protected against MARV and ZEBOV challenge. Comprehensive T-cell analysis revealed cytotoxic T lymphocytes (CTLs) of great magnitude, epitopic breadth, and Th1-type marker expression. This model provides an important preclinical tool for studying protective immune correlates that could be applied to existing platforms. Data herein support further evaluation of this enhanced gene-based approach in nonhuman primate studies for in depth analyses of T-cell epitopes in understanding protective efficacy.
Assuntos
Doença do Vírus de Marburg/imunologia , Doença do Vírus de Marburg/prevenção & controle , Linfócitos T Citotóxicos/imunologia , Vacinas de DNA/imunologia , Animais , Linhagem Celular , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Humanos , Immunoblotting , Marburgvirus/imunologia , Marburgvirus/patogenicidade , Camundongos Endogâmicos C57BL , Vacinas de DNA/uso terapêutico , Vacinas Virais/imunologia , Vacinas Virais/uso terapêuticoRESUMO
COVID-19 vaccines consisting of mRNA lipid nanoparticles (LNPs) encoding the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein antigen protected millions of people from severe disease; however, they must be stored frozen prior to use. The objective of this study was to evaluate the compatibility and stability of mRNA LNPs within a polymer-based film matrix. An optimized formulation of polymer base, glycerol, surfactants, and PEGylated lipid that prevents damage to the LNP due to physical changes during the film-forming process (osmotic stress, surface tension, spatial stress, and water loss) was identified. Surfactants added to LNP stock prior to mixing with other film components contributed to this effect. Formulations prepared at pH ≥ 8.5 extended transfection efficiency beyond 4 weeks at 4°C when combined with known nucleic acid stabilizers. mRNA LNPs were most stable in films when manufactured in an environment of â¼50% relative humidity. The optimized formulation offers 16-week stability at 4°C.
RESUMO
Pre-existing immunity (PEI) to human adenovirus serotype 5 (Ad5) worldwide is the primary limitation to routine clinical use of Ad5-based vectors in immunization platforms. Using systemic and mucosal PEI induction models in rodents (mice and guinea pigs), we assessed the influence of PEI on the type of adaptive immune response elicited by an Ad5-based vaccine for Ebola with respect to immunization route. Splenocytes isolated from vaccinated animals revealed that immunization by the same route in which PEI was induced significantly compromised Ebola Zaire glycoprotein (ZGP)-specific IFN-γ+ CD8+ T cells and ZGP-specific multifunctional CD8+ T cell populations. ZGP-specific IgG1 antibody levels were also significantly reduced and a sharp increase in serum anti-Ad5 neutralizing antibody (NAB) titers were noted following immunization. These immune parameters correlated with poor survival after lethal challenge with rodent-adapted Ebola Zaire virus (ZEBOV). Although the number of IFN-γ+ CD8+ T cells was reduced in animals given the vaccine by a different route from that used for PEI induction, the multifunctional CD8+ T cell response was not compromised. Survival rates in these groups were higher than when PEI was induced by the same route as immunization. These results suggest that antigen-specific multifunctional CD8(+) T cell and Th2 type antibody responses compromised by PEI to Ad5 are required for protection from Ebola. They also illustrate that methods for induction of PEI used in preclinical studies must be carefully evaluated for successful development of novel Ad5-based vaccines.
Assuntos
Adenoviridae/imunologia , Vacinas contra Ebola/imunologia , Animais , Formação de Anticorpos/imunologia , Linfócitos T CD8-Positivos/imunologia , Ebolavirus/imunologia , Cobaias , Masculino , Camundongos , Linfócitos T/imunologiaRESUMO
Plasmids are essential source material for production of biological drugs, vaccines and vectors for gene therapy. They are commonly formulated as frozen solutions. Considering the cost associated with maintenance of cold chain conditions during storage and transport, there is a significant need for alternative methods for stabilization of plasmids at ambient temperature. The objective of these studies was to identify a film-based formulation that preserved transfection efficiency of plasmids at 25 °C. A model plasmid, pAAVlacZ, was used for these studies. Transfection efficiency and agarose gel electrophoresis were utilized to assess bioactivity and changes in physical conformation of plasmid during storage. An amino acid, capable of sustaining a positive charge while supporting an alkaline environment within the film matrix, preserved transfection efficiency for 9 months at 25 °C. Addition of sugar and a plasticizer to the formulation preserved the plasmid in an amorphous state and improved handling properties of the film. The manner in which excipients were incorporated into bulk formulations and environmental humidity in which films were stored significantly impacted transfection efficiency of plasmid in the rehydrated solution. Taken together, these results suggest that plasmids can be stored for extended periods of time without refrigeration within a film matrix.
Assuntos
DNA Recombinante , Excipientes , Plasmídeos , Transfecção , Excipientes/química , Terapia Genética/métodosRESUMO
Sublingual (SL) delivery, a noninvasive immunization method that bypasses the intestinal tract for direct entry into the circulation, was evaluated with an adenovirus (Ad5)-based vaccine for Ebola. Mice and guinea pigs were immunized via the intramuscular (IM), nasal (IN), oral (PO) and SL routes. SL immunization elicited strong transgene expression in and attracted CD11c(+) antigen presenting cells to the mucosa. A SL dose of 1 × 108 infectious particles induced Ebola Zaire glycoprotein (ZGP)-specific IFN-γ⺠T cells in spleen, bronchoalveolar lavage, mesenteric lymph nodes and submandibular lymph nodes (SMLN) of naive mice in a manner similar to the same dose given IN. Ex vivo CFSE and in vivo cytotoxic T lymphocyte (CTL) assays confirmed that SL immunization elicits a notable population of effector memory CD8+ T cells and strong CTL responses in spleen and SMLN. SL immunization induced significant ZGP-specific Th1 and Th2 type responses unaffected by pre-existing immunity (PEI) that protected mice and guinea pigs from lethal challenge. SL delivery protected more mice with PEI to Ad5 than IM injection. SL immunization also reduced systemic anti-Ad5 T and B cell responses in naive mice and those with PEI, suggesting that secondary immunizations could be highly effective for both populations.
Assuntos
Antígenos Virais/administração & dosagem , Vacinas contra Ebola/administração & dosagem , Ebolavirus , Doença pelo Vírus Ebola/prevenção & controle , Adenoviridae/genética , Administração Sublingual , Animais , Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/metabolismo , Antígenos Virais/efeitos adversos , Antígenos Virais/uso terapêutico , Antígeno CD11c/metabolismo , Linhagem Celular , Vacinas contra Ebola/efeitos adversos , Vacinas contra Ebola/imunologia , Cobaias , Doença pelo Vírus Ebola/imunologia , Doença pelo Vírus Ebola/mortalidade , Humanos , Imunidade Celular , Imunização Secundária , Memória Imunológica , Masculino , Camundongos , Mucosa Bucal/citologia , Mucosa Bucal/imunologia , Mucosa Bucal/metabolismo , Mucosa Bucal/virologia , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/efeitos adversos , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/uso terapêutico , Análise de Sobrevida , TransgenesRESUMO
Thermostability of vaccines and biologic drugs are key to increasing global access to a variety of life-saving agents. In this report, we characterize interactions between a novel zwitterionic surfactant and adenovirus serotype 5 which allow the virus to remain stable at room temperature in a thin film matrix. Complexity of the adenovirus capsid and the polydispersity of the surfactant required use of a variety of techniques to achieve this goal. The CMC of the surfactant in Tris buffer (pH 6.5) was estimated to be 0.7-1.17 × 10-4 M by the pyrene 1:3 ratio method. TEM images depict micelle formation around virus capsids. An estimated Kd of the virus-surfactant interaction of 2.25 × 10-9 M was determined by isothermal titration calorimetry. Associated data suggest that this interaction may be thermodynamically favorable and entropically driven. A competitive saturation study and TEM images indicate that the surfactant also binds to hexon proteins on the virus capsid. Taken together, these data support the working hypothesis that the surfactant is capable of forming micelles in the solid and liquid state and that it forms a protective coating around the virus by binding to hexon proteins on the virus capsid during the film forming process.
Assuntos
Adenoviridae , Tensoativos , Adenoviridae/genética , Capsídeo , Proteínas do Capsídeo/genética , Micelas , Tensoativos/químicaRESUMO
BACKGROUND: Adeno-associated virus (AAV) vectors are stored and shipped frozen which poses logistic and economic barriers for global access to these therapeutics. To address this issue, we developed a method to stabilize AAV serotype 9 (AAV9) in a film matrix that can be stored at ambient temperature and administered by systemic injection. METHODS: AAV9 expressing the luciferase transgene was mixed with formulations, poured into molds and films dried under aseptic conditions. Films were packaged in individual particle-free bags with foil overlays and stored at various temperatures under controlled humidity. Recovery of AAV9 from films was determined by serial dilution of rehydrated film in media and infection of HeLa RC32 cells. Luciferase expression was compared to that of films rehydrated immediately after drying. Biodistribution of vector was determined by in vivo imaging and quantitative real-time PCR. Residual moisture in films was determined by Karl Fischer titration. RESULTS: AAV9 embedded within a film matrix and stored at 4 °C for 5 months retained 100% of initial titer. High and low viscosity formulations maintained 90 and 85% of initial titer after 6 months at 25 °C respectively. AAV was not detected after 4 months in a Standard Control Formulation under the same conditions. Biodistribution and transgene expression of AAV stored in film at 25 or 4 °C were as robust as vector stored at -80 °C in a Standard Control Formulation. CONCLUSIONS: These results suggest that storage of AAV in a film matrix facilitates easy transport of vector to remote sites without compromising in vivo performance.
Adeno-associated viruses (AAVs) are small viruses that are used to deliver medicines and vaccines. Prior to administration, they are stored in freezers set to very low temperatures and must be discarded if they thaw during transportation to clinics. AAV was embedded in a film to protect the virus during transportation and storage. The virus remained stable for 6 months at room temperature and during shipment from Texas to North Carolina. The ability to store and transport AAV without the need for complex packaging and temperature control will increase global access to vaccines and other medicines that use AAVs for delivery.
RESUMO
Clinically relevant doses of helper-dependent adenoviruses (HDAds) provoke the host response against capsid proteins in primates and rodents. To determine if PEGylation truly affects this, baboons and mice were given either HDAd or PEG-HDAd expressing beta-galactosidase at 5 × 10¹¹ or 3 × 10¹² virus particles per kilogram (vp/kg) by iv infusion. Serum cytokines and blood chemistries were assessed for 96 h. PEG-HDAd reduced IL-6 6-fold in mice and 3-fold in the primate. This vector reduced IL-12 by 50% in both animal models. PEGylation reduced serum transaminases by approximately 50% at each dose in the primate and the mouse. PEGylation did not alter hepatic transduction efficiency in the mouse but did reduce transduction efficiency in the liver and the spleen of primates. Unmodified and PEGylated virus suppressed hepatic CYP3A activity in both animal models. PEGylation doubled the half-life (t(½)) of the virus in the mouse and cut plasma clearance (CL) in half without affecting the half-life in primates. These results suggest that there are notable species-specific differences in the biodistribution of and response to PEG-modified vectors which may be linked to differences in binding properties to coagulation factors, receptor density and tissue architecture in the liver.
Assuntos
Adenoviridae/química , Adenoviridae/metabolismo , Polietilenoglicóis/química , Animais , Southern Blotting , Western Blotting , Fígado/metabolismo , Masculino , Camundongos , Papio , Reação em Cadeia da Polimerase , Transdução GenéticaRESUMO
OBJECTIVE: Infectious disease emergencies like the 2013-2016 Ebola epidemic and the 2009 influenza and current SARS-CoV-2 pandemics illustrate that vaccines are now given to diverse populations with preexisting pathologies requiring pharmacological management. Many natural biomolecules (steroid hormones, fatty acids, vitamins) and ~60% of prescribed medications are processed by hepatic cytochrome P450 (CYP) 3A4. The objective of this work was to determine the impact of infection and vaccines on drug metabolism. METHODS: The impact of an adenovirus-based vaccine expressing Ebola glycoprotein (AdEBO) and H1N1 and H3N2 influenza viruses on hepatic CYP 3A4 and associated nuclear receptors was evaluated in human hepatocytes (HC-04 cells) and in mice. RESULTS: CYP3A activity was suppressed by 55% in mice 24 h after administration of mouse-adapted H1N1, while Ë10% activity remained in HC-04 cells after infection with H1N1 and H3N2 due to global suppression of cellular translation capacity, indicated by reduction (70%, H1N1, 56%, H3N2) of phosphorylated eukaryotic translation initiation factor 4e (eIF4E). AdEBO suppressed CYP3A activity in vivo (44%) and in vitro (26%) 24 hours after infection. CONCLUSION: As the clinical evaluation of vaccines for SARS-CoV-2 and other global pathogens rise, studies to evaluate the impact of new vaccines and emerging pathogens on CYP3A4 and other metabolic enzymes are warranted to avoid therapeutic failures that could further compromise the public health during infectious disease emergencies.
Assuntos
Citocromo P-450 CYP3A/metabolismo , Hepatócitos/enzimologia , Hepatócitos/metabolismo , Fígado/enzimologia , Fígado/metabolismo , Preparações Farmacêuticas/metabolismo , Animais , Células Cultivadas , Fator de Iniciação 4E em Eucariotos , Humanos , Imunização/métodos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BLRESUMO
Ebolavirus is a highly infectious pathogen with a case fatality rate as high as 90%. Currently there is a lack of licensed Ebolavirus vaccines as well as pre- and post-exposure treatments. Recent increases in the frequency of natural human Ebolavirus infections and its potential use as a bioterrorism agent makes vaccine development a priority for many nations. Significant progress has been made in understanding the pathogenesis of Ebolavirus infection and several promising vaccine candidates were shown to be successful in protecting NHPs against lethal infection. These include replication-deficient adenovirus vectors, replication-competent VSV, HPIV-3 vectors and virus-like particle preparations. Recent advances in the generation of effective post-exposure immunization strategies highlight the possibility of developing a single dose vaccine that will confer full protection in humans following Ebolavirus exposure. Post-exposure protection is particularly important in outbreak and biodefense settings, as well as clinical and laboratory settings in the case of accidental exposure.
Assuntos
Vacinas contra Ebola , Ebolavirus/imunologia , Doença pelo Vírus Ebola/prevenção & controle , Adenoviridae/genética , Animais , Vias de Administração de Medicamentos , Vacinas contra Ebola/administração & dosagem , Vacinas contra Ebola/imunologia , Vacinas contra Ebola/uso terapêutico , Ebolavirus/genética , Ebolavirus/efeitos da radiação , Vetores Genéticos , Doença pelo Vírus Ebola/terapia , Humanos , Profilaxia Pós-Exposição , Vacinação , Vacinas de Subunidades Antigênicas , Vacinas de Partículas Semelhantes a VírusRESUMO
A novel, thin-film platform that preserves live viruses, bacteria, antibodies, and enzymes without refrigeration for extended periods of time is described. Studies with recombinant adenovirus in an optimized formulation that supports recovery of live virus through 16 freeze-thaw cycles revealed that production of an amorphous solid with a glass transition above room temperature and nitrogen-hydrogen bonding between virus and film components are critical determinants of stability. Administration of live influenza virus in the optimized film by the sublingual and buccal routes induced antibody-mediated immune responses as good as or better than those achieved by intramuscular injection. This work introduces the possibility of improving global access to a variety of medicines by offering a technology capable of reducing costs of production, distribution, and supply chain maintenance.
Assuntos
Adenoviridae/imunologia , Anticorpos Antivirais/biossíntese , Imunização/métodos , Vírus da Influenza A Subtipo H1N1/imunologia , Infecções por Orthomyxoviridae/prevenção & controle , Preservação Biológica/métodos , Vacinas Atenuadas/farmacologia , Adenoviridae/genética , Administração Bucal , Administração Sublingual , Animais , Anticorpos Neutralizantes/biossíntese , Células HEK293 , Humanos , Vírus da Influenza A Subtipo H1N1/patogenicidade , Injeções Intramusculares , Masculino , Membranas Artificiais , Camundongos , Camundongos Endogâmicos BALB C , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/virologia , Temperatura , Potência de Vacina , Vacinas Atenuadas/biossínteseRESUMO
In this report we test the hypothesis that long-term virus-induced alterations in CYP occur from changes initiated by the virus that may not be related to the immune response. Enzyme activity, protein expression and mRNA of CYP3A2, a correlate of human CYP3A4, and CYP2C11, responsive to inflammatory mediators, were assessed 0.25, 1, 4, and 14 days after administration of several different recombinant adenoviruses at a dose of 5.7 x 1012 virus particles (vp)/kg to male Sprague Dawley rats. Wild type adenovirus, containing all viral genes, suppressed CYP3A2 and 2C11 activity by 37% and 39%, respectively within six hours. Levels fell to 67% (CYP3A2) and 79% (CYP2C11) of control by 14 days (p Assuntos
Infecções por Adenoviridae/enzimologia
, Adenoviridae/genética
, Hidrocarboneto de Aril Hidroxilases/metabolismo
, Vetores Genéticos/genética
, Fígado/enzimologia
, Proteínas de Membrana/metabolismo
, Esteroide 16-alfa-Hidroxilase/metabolismo
, Adenoviridae/metabolismo
, Infecções por Adenoviridae/genética
, Infecções por Adenoviridae/virologia
, Alanina Transaminase/sangue
, Animais
, Hidrocarboneto de Aril Hidroxilases/genética
, Linhagem Celular
, Células Cultivadas
, Citocromo P-450 CYP3A
, Família 2 do Citocromo P450
, Expressão Gênica
, Vetores Genéticos/metabolismo
, Hepatócitos/enzimologia
, Hepatócitos/virologia
, Humanos
, Fígado/virologia
, Masculino
, Proteínas de Membrana/genética
, Ratos
, Ratos Sprague-Dawley
, Esteroide 16-alfa-Hidroxilase/genética
RESUMO
Inactivated viruses are important tools for vaccine development and gene transfer. 8-Methoxypsoralen (8-MOP) and long-wavelength ultraviolet irradiation (LWUVI) inactivates many viruses. Toxicity limits its use in animals and humans. Toxicological and photosensitizing properties of riboflavin make it suitable for virus inactivation in preparations for biological use. Viruses expressing beta-galactosidase were mixed with either 8-MOP (1.5mM) or riboflavin (50 microM) and exposed to LWUVI (365 nm) for 2 h. Virus activity was determined by limiting dilution. The half-life of the adenovirus preparation treated with 8-MOP was 8.28 ns(-1) and 36.5 ns(-1) after treatment with riboflavin. Despite the difference in half-life, both preparations were completely inactivated within 45 min. In contrast, the half-lives for adeno-associated virus (AAV) preparations were similar (63 ns(-1) 8-MOP vs. 67 ns(-1) riboflavin). Each AAV preparation was fully inactivated within 90 min. The half-life of lentivirus was 193.4 ns(-1) after treatment with 8-MOP and 208 ns(-1) after exposure to riboflavin. Virus treated with riboflavin was inactivated within 20 min. Virus exposed to 8-MOP was inactivated in 90 min. DNA and RNA viruses can be inactivated by riboflavin and LWUVI and used in physiological systems sensitive to other photochemicals.
Assuntos
Adenoviridae/efeitos dos fármacos , Antivirais/farmacologia , Dependovirus/efeitos dos fármacos , Lentivirus/efeitos dos fármacos , Fármacos Fotossensibilizantes/farmacologia , Riboflavina/farmacologia , Inativação de Vírus , Adenoviridae/efeitos da radiação , Adenoviridae/ultraestrutura , Animais , Dependovirus/efeitos da radiação , Dependovirus/ultraestrutura , Genes Reporter , Humanos , Lentivirus/efeitos da radiação , Lentivirus/ultraestrutura , Fígado/virologia , Metoxaleno/farmacologia , Microscopia Eletrônica de Transmissão , Ratos , beta-Galactosidase/biossíntese , beta-Galactosidase/genéticaRESUMO
Recombinant adenovirus (Ad) significantly alters hepatic cytochrome P450 (CYP). Because changes in renal function can alter hepatic CYP, the effect of Ad on renal CYPs 4A1, 4A2, 4F1, and 2E1 was evaluated. Male Sprague-Dawley rats were given one of six intravenous doses (5.7x10(6)-5.7x10(12) viral particles/kg [VP/kg]) of Ad expressing beta-galactosidase or saline. CYP protein, activity, gene expression, and serum creatinine (SCr) were evaluated 0.25, 1, 4, and 14 days later. Doses of 5.7x10(11) and 5.7x10(12) VP/kg increased CYP4A protein within 24 hr by 35 and 48%, respectively (p<0.05). A similar trend was observed on day 4. CYP4A1 mRNA doubled 6 hr after doses of 5.7x10(10)-10(12) VP/kg (p<0.01). Similar effects were observed 1 day after each dose tested. CYP4A2 gene expression was 20% above control 1 day after treatment with 5.7x10(10)-10(12) VP/kg and remained high through day 14. CYP4F1 expression was unaffected by all doses (p=0.08). CYP2E1 activity and gene expression were significantly suppressed 24 hr after administration of all doses and began to normalize by day 14 (p<0.01). SCr was significantly reduced (approximately 50%) throughout the study for doses at and below 5.7x10(11) VP/kg. SCr was increased by a factor of 3 by 5.7x10(12) VP/kg and glomerular filtration was significantly reduced (p<0.01). This suggests that changes in renal CYP and corresponding arachidonic acid metabolites may play a role in the documented toxicity associated with the systemic administration of recombinant Ad.