RESUMO
The concept of identifying appropriate scientific and regulatory principles to ensure rapid availability of pandemic influenza vaccines when needed were already developed starting in the year 2003. These principles allowed licensing of three so-called mock-up vaccines far ahead of any real presenting pandemic event. Those licenses (Marketing Authorizations) were immediately adapted to the novel H1N1 strain shortly after its identification in April 2009 ensuring that as early as September 2009 large parts of the German as well as of the EU population had access to licensed products which had undergone sufficient evaluation before first use in humans. In contrast, for pandemic vaccine concepts without a previously licensed mock-up version it generally took twice as much time to accumulate data supporting the granting of a Marketing Authorization. This article describes in detail the translation of concepts of producing, testing, and licensing of pandemic influenza vaccines into practice under real conditions.
Assuntos
Aprovação de Drogas/organização & administração , Desenho de Fármacos , Vírus da Influenza A Subtipo H1N1 , Vacinas contra Influenza/uso terapêutico , Influenza Humana/prevenção & controle , Licenciamento/legislação & jurisprudência , Pandemias/prevenção & controle , Europa (Continente)/epidemiologia , Setor de Assistência à Saúde/legislação & jurisprudência , Humanos , Vacinas contra Influenza/biossíntese , Influenza Humana/epidemiologia , Pandemias/estatística & dados numéricosRESUMO
Photobiomodulation (PBM) appears promising to treat the hallmarks of Parkinson's Disease (PD) in cellular or animal models. We measured light propagation in different areas of PD-relevant deep brain tissue during transcranial, transsphenoidal illumination (at 671 and 808 nm) of a cadaver head and modeled optical parameters of human brain tissue using Monte-Carlo simulations. Gray matter, white matter, cerebrospinal fluid, ventricles, thalamus, pons, cerebellum and skull bone were processed into a mesh of the skull (158 × 201 × 211 voxels; voxel side length: 1 mm). Optical parameters were optimized from simulated and measured fluence rate distributions. The estimated µeff for the different tissues was in all cases larger at 671 than at 808 nm, making latter a better choice for light delivery in the deep brain. Absolute values were comparable to those found in the literature or slightly smaller. The effective attenuation in the ventricles was considerably larger than literature values. Optimization yields a new set of optical parameters better reproducing the experimental data. A combination of PBM via the sphenoid sinus and oral cavity could be beneficial. A 20-fold higher efficiency of light delivery to the deep brain was achieved with ventricular instead of transcranial illumination. Our study demonstrates that it is possible to illuminate deep brain tissues transcranially, transsphenoidally and via different application routes. This opens therapeutic options for sufferers of PD or other cerebral diseases necessitating light therapy.
Assuntos
Doença de Parkinson/patologia , Doença de Parkinson/radioterapia , Fototerapia/métodos , Espectroscopia de Luz Próxima ao Infravermelho/métodos , Encéfalo/patologia , Simulação por Computador , Feminino , Cabeça , Humanos , Luz , Pessoa de Meia-Idade , Método de Monte Carlo , Óptica e Fotônica , Radiometria , CrânioRESUMO
We have investigated the requirements necessary for high-level production of the hepatitis B virus (HBV strain ayw) large surface glycoprotein preS1 with vaccinia virus (VV) recombinants. In earlier studies, only nanogram amounts of preS1 could be obtained from cells infected with an appropriate recombinant VV carrying the preS1 gene under the transcriptional control of a conventional VV promoter (p7.5). Here, we report that the use of an improved promoter system, i.e., the bacteriophage T7 polymerase/VV hybrid expression system (T7/EMC system) in combination with a G-C conversion at position 5 of the preS1 open reading frame, deleting the myristylation motif of the polypeptide, results in an at least 12-fold increase in preS1 expression compared to the wild-type preS1 expressed with the strongest homologous VV promoter system known so far. Although the T7/EMC promoter system was most effective, improved expression of the modified preS1 (preS1dMyr) is independent from the promoter system used, from the insertion locus of the modified preS1 within the VV genome and also from the cell line used for expression studies.
Assuntos
Regulação Viral da Expressão Gênica , Vetores Genéticos , Antígenos de Superfície da Hepatite B/genética , Vírus da Hepatite B/genética , Ácidos Mirísticos/metabolismo , Precursores de Proteínas/genética , Vaccinia virus/genética , Adaptação Fisiológica , Animais , Linhagem Celular , Chlorocebus aethiops , Conversão Gênica , Humanos , Ácido Mirístico , Fases de Leitura Aberta , Proteínas Recombinantes de Fusão/genética , Células VeroRESUMO
Factor X/Xa plays a pivotal role in the coagulation cascade and exhibits a therapeutic potential for the treatment of factor X-deficient as well as FVIII and FIX inhibitor patients. This report describes the establishment of Chinese hamster ovary cell clones expressing recombinant human factor X up to 120 microg/mL x day and 78 microg/10(6) cells x day, that is to 100-fold higher levels than reported previously. Although propeptide removal and single chain precursor to light and heavy chain processing as well as vitamin K-dependent gamma-carboxylation became impaired at these expression levels, up to 25% of the recombinant human factor X produced was active. This represents the highest functional activity ever reported for a vitamin K-dependent protein at such an expression level. Expression of recombinant human factor X in Chinese hamster ovary cells lacking the endoprotease Furin revealed that propeptide removal still occurred, whereas single chain precursor to light/heavy chain processing was abolished. This suggests that a protease different from Furin mediates propeptide removal, a unique finding compared with the other vitamin K-dependent coagulation factors. In contrast, exposure of incompletely processed rFX molecules to soluble recombinant Furin in vitro mediated both of these cleavage reactions despite the absence of a typical argP4-xP3-lys/argP2-argP1 Furin cleavage site in the propeptide, indicating relaxed specificity in vitro. Concomitantly with the degree of processing, the functional activity of recombinant human factor X increased. Interestingly, Furin was shown to even perform correct N-terminal proteolytic trimming of FX molecules truncated amino-terminal to the P3 residue in vitro. Depending on the absence or presence of warfarin in the culture media, as well as on the processing state, four distinct recombinant human factor X light chain isoforms were observed and their structure characterized. One of these light chain forms correlated with the functional activity. Finally, the distribution of the individual light chain isoforms suggests that gamma-carboxylation may be a prerequisite for propeptide removal.
Assuntos
Fator X/biossíntese , Fator X/efeitos dos fármacos , Subtilisinas/farmacologia , Animais , Células CHO/enzimologia , Células CHO/metabolismo , Clonagem Molecular , Cricetinae , Eletroforese em Gel de Poliacrilamida , Furina , Humanos , Peso Molecular , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Estrutura Quaternária de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/metabolismo , Análise de Sequência de Proteína , Relação Estrutura-Atividade , Subtilisinas/metabolismoRESUMO
Achieving stability is a prerequisite for allowing bone to grow into the porous surface of non-cemented acetabular cups. The purpose of this study is to estimate the effects of interfacial characteristics on relative cyclical micromotion between cup and bone during gait in the immediate postoperative phase. The technique used is finite element analysis. Six models with different interfacial characteristics are created in order to study the effects of fixation technique. These include representation of a 1 mm press-fit, 2 mm press-fits (with and without an initial polar gap) and exact-fit conditions (with and without additional screw fixation). Although direct validation of the model has not been performed, the calculated micromotions under a static load of 1112 N are compared with appropriate experimental data. Generally, the model tends to underestimate micromotion and this underestimate is significant in the case of relative surface-normal micromotion in polar regions for models with low- and no-interference. The most likely cause of this significant underestimate is a failure of the model to accurately represent penetration of rough contacting surfaces under compression. Other types of micromotion, although low, are within standard deviations reported by Kwong et al. (1994 Journal of Arthroplasty 9, 163-170). Quasi-static joint contact and muscle forces, representative of the stance phase of gait are then applied and maximum micromotions are found to occur consistently prior to toe off: this being the point of maximum force. With regard to the press-fit simulations, good cup-bone contact in the superior region of the interface is required for stability and the greatest micromotions occur in the models with the larger interference and larger polar gaps. In contrast to the press-fit models, muscle activity in exact-fit models influences the calculations. Specifically, the early activity of m.semimembranosus modelled causes opening of the peripheral seal. Taken together it is found that polar gaps reduce the stability of the model and lack of pre-compresssion in the periphery allows this region of the interface to be opened up.
Assuntos
Acetábulo/fisiopatologia , Prótese de Quadril , Osseointegração/fisiologia , Simulação por Computador , Análise de Elementos Finitos , Marcha , Humanos , Modelos Biológicos , Período Pós-OperatórioRESUMO
Interfacial gaps and relative micromotions during activities are widely believed to restrict the boney in-growth process of non-cemented acetabular cups. Using finite element modeling of the cup-bone system, relative micromotions and interfacial gaps are calculated for walking slow, normal and fast and for climbing upstairs, downstairs and standing up from a chair. A 2mm press-fit is simulated and interfacial conditions in the immediate postoperative period (i.e. prior to boney in-growth) calculated between paired nodes covering the whole of the interface. In regions of 'safe' micromotions and 'allowable' gaps, boney in-growth is simulated by specifying zero relative displacement between nodal pairs. The modified model is then resubjected to the loads associated with climbing upstairs, which was shown to be the worst activity. Interfacial conditions are recalculated for subsequent iterations. The procedure is repeated until no further in-growth is predicted. The final pattern of in-growth calculated with the model compares reasonably well with histological evidence from explanted canine cups (Cha et al., 1998. Transactions of the Orthopaedic Research Society, 23, p. 373). Bridging between adjacent regions of in-growth is observed. Notably, in-growth occurs at most of the periphery but not in the polar region. The lack of polar in-growth is caused by the interfacial gap assumed to exist after cup implantation. It is suggested that increasing/decreasing hip-joint loads would have little effect on this lack of polar in-growth. However, excessive micromotions as a result of high hip-joint loads cause a lack of in-growth in the anterior region of the periphery in the model. Although such results were not found in the canine study, if relevant to the general human population, the avoidance of harsh weight-bearing activities may encourage complete peripheral in-growth but is speculated to do little for polar in-growth.
Assuntos
Prótese de Quadril , Modelos Biológicos , Osseointegração/fisiologia , Acetábulo/fisiologia , Acetábulo/cirurgia , Atividades Cotidianas , Animais , Fenômenos Biomecânicos , Cães , Humanos , Movimento/fisiologiaRESUMO
The formation of gaps in the polar region of acetabular cups is seen as a drawback of press-fit fixation of non-cemented acetabular cups. Recent findings indicate a link between long-term polar gaps and the gaps present directly after implantation. In this study the process of press-fitting is simulated with a linear-elastic two-dimensional axisymmetric finite-element model. The aim of this paper is to investigate the possible importance of friction and interference on the formation of these gaps. A range of cup-bone friction coefficients (mu = 0.1-0.5) is assigned to the cup-bone interface in order to represent the unknown amount of friction occurring during press-fitting. The cup is modeled with a radius of 27 mm, whereas the radius of the cavity is varied between 26.50 and 26.75 mm, thus, creating 0.50 and 0.25 mm radial interference fits. The difference in cavity radius represents the discrepancy between the radius of the last-reamer-used and radius of the cavity it creates. The subchondral plate is considered as being completely removed during reaming. The effects of impact blows via the surgeon's mallet during surgery are modeled as a series of four load pulses, in which peak force is gradually increased from 0.5 to 4.0 kN. The effects of load removal as well as those of load application are investigated. On load application, the cup penetrates into the cavity, and on load removal, the cup rebounds. Depending on the friction, interference and load applied, the position of the cup after the load pulse is somewhere between its position at peak force and its position at the beginning of the pulse. Although the simplifications and conditions involved in the creation of the model necessitate caution when interpreting the results for all clinical cases, it is found that the seating of hemispherical cups in trabecular bone could be more satisfactory for intermediate values of friction (mu = 0.2-0.3) and smaller interference fits (0.25 mm).
Assuntos
Acetábulo/fisiologia , Prótese de Quadril , Modelos Biológicos , Fricção , Humanos , Pelve/fisiologia , Desenho de Prótese , Ajuste de Prótese , Estresse Mecânico , Suporte de Carga/fisiologiaRESUMO
The surface glycoprotein (S) of the murine hepatitis coronavirus MHV normally undergoes proteolytic cleavage during transport to the cell surface. To determine whether the cleavage of the MHV-JHM S glycoprotein is required to activate its ability to fuse cellular membranes, the protease recognition sequence in a cDNA copy of the S gene was altered from Arg-Arg-Ala-Arg-Arg into Ser-Val-Ser-Gly-Gly by site directed mutagenesis. The mutated and wild type S genes were expressed by means of recombinant vaccinia viruses and it could be shown that the mutated S protein was not cleaved when it was expressed in mouse DBT cells, in contrast to the wild type S protein. Nevertheless, the non-cleaved S protein induced extensive syncytium formation in mouse DBT cells. These results clearly indicate that the non-cleaved form of the MHV S protein is able to mediate cell membrane fusion. Thus, proteolytic cleavage is not an absolute requirement for its fusion function.
Assuntos
Efeito Citopatogênico Viral , Glicoproteínas de Membrana/metabolismo , Vírus da Hepatite Murina/fisiologia , Processamento de Proteína Pós-Traducional , Proteínas do Envelope Viral/metabolismo , Sequência de Aminoácidos , Animais , Astrocitoma , Fusão Celular , Endopeptidases/metabolismo , Células HeLa/microbiologia , Humanos , Glicoproteínas de Membrana/genética , Camundongos , Dados de Sequência Molecular , Vírus da Hepatite Murina/genética , Glicoproteína da Espícula de Coronavírus , Células Tumorais Cultivadas/microbiologia , Proteínas do Envelope Viral/genéticaAssuntos
Coronaviridae/fisiologia , Hemaglutininas Virais/metabolismo , Vaccinia virus/genética , Proteínas Virais de Fusão , Proteínas Virais/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Clonagem Molecular , Coronaviridae/genética , Esterases/metabolismo , Genes Virais , Hemaglutininas Virais/genética , Cinética , Substâncias Macromoleculares , Fusão de Membrana , Dados de Sequência Molecular , Proteínas Recombinantes/metabolismo , Recombinação Genética , Homologia de Sequência do Ácido Nucleico , Transfecção , Proteínas Virais/genéticaRESUMO
The principle of live attenuated influenza vaccines has been known for many decades. However, the pharmaceutical and clinical development according to current regulations, of modern live influenza vaccines based on cold adapted influenza viruses (CAIV) started only recently and these vaccines will most probably become an alternative within the next couple of years to licensed inactivated influenza vaccines that have been used routinely since the early 1940s. In contrast to contemporary trivalent inactivated influenza vaccines, which are administered intramuscularly, trivalent CAIV-based vaccines will be administered intranasally as a spray. Quality, safety and efficacy aspects related to CAIV vaccines as well as possible risks linked to the widespread use of these vaccines will be discussed in the following overview and compared to established influenza vaccines. Moreover, issues of practicality of CAIV vaccines focusing on the necessity of an annual update of influenza vaccines are addressed.
Assuntos
Vacinas contra Influenza/efeitos adversos , Vacinas contra Influenza/farmacologia , Animais , Galinhas/virologia , Temperatura Baixa , Contaminação de Medicamentos , Hipersensibilidade a Drogas , Humanos , Vacinas contra Influenza/genética , Vacinas contra Influenza/normas , Influenza Humana/epidemiologia , Influenza Humana/prevenção & controle , Medição de Risco , Segurança , Superinfecção , Fatores de Tempo , Vacinas Atenuadas/efeitos adversos , Vacinas Atenuadas/genética , Vacinas Atenuadas/farmacologia , Vacinas Atenuadas/normasRESUMO
We have expressed the murine coronavirus haemagglutinin-esterase protein in a vaccinia virus/T7 RNA polymerase system. The levels of expression observed are significantly higher than those found in virus-infected cells. The expressed protein has both receptor-destroying (esterase) and receptor-binding (haemadsorption) activities. The use of this system will greatly facilitate analysis of the structure-function relationships of this protein.
Assuntos
Hemaglutininas Virais/genética , Vírus da Hepatite Murina/genética , Vaccinia virus/genética , Proteínas Virais de Fusão , Proteínas Virais/genética , Sequência de Bases , Linhagem Celular , Clonagem Molecular , Esterases/genética , Esterases/isolamento & purificação , Esterases/metabolismo , Glicoproteínas/genética , Células HeLa/fisiologia , Hemadsorção , Hemaglutininas Virais/isolamento & purificação , Hemaglutininas Virais/metabolismo , Humanos , Cinética , Dados de Sequência Molecular , Peso Molecular , Vírus da Hepatite Murina/imunologia , Vírus da Hepatite Murina/metabolismo , Sondas de Oligonucleotídeos , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Virais/isolamento & purificação , Proteínas Virais/metabolismoRESUMO
Coronavirus MHV-JHM infections of rats provide useful models to study the pathogenesis of virus-induced central nervous system disease. To analyze the role of the immune response against defined MHV-JHM antigens, we tested the protective efficacy of vaccinia virus (VV) recombinants expressing either the nucleocapsid (N) or the spike (S) protein. A strong protection was mediated in animals by immunization with recombinant VV encoding a wild-type S protein (VV-Swildtype), whereas VV recombinant expressing a mutant S354CR protein (VV-S354CR) had no protective effect. Recombinant VV encoding N protein (VV-N) induces a humoral and a CD4+ T cell response, but did not prevent acute disease regardless of the immunization protocol. In these experiments, challenge with an otherwise lethal dose of MHV-JHM was performed prior to the induction of virus-neutralizing antibodies and studies with the anti-CD8+ monoclonal antibody. MRC OX8 showed that elimination of the CD8+ subset of T cells abrogates the protective effect. This result indicates that CD8+ T cells primed by recombinant VV expressing wild-type S protein are a primary mechanism of immunological defense against MHV-JHM infection in rats.
Assuntos
Infecções por Coronaviridae/imunologia , Encefalomielite/imunologia , Vírus da Hepatite Murina/imunologia , Linfócitos T/imunologia , Vacinas Sintéticas/imunologia , Vacinas Virais/imunologia , Animais , Anticorpos Antivirais/biossíntese , Antígenos CD8/análise , Imunização , Ratos , Ratos Endogâmicos Lew , Vaccinia virus/imunologiaRESUMO
The murine coronavirus surface glycoprotein gene was expressed as a fusion protein in bacteria, and the expressed protein was used to generate S protein-specific monoclonal antibodies (MAbs). Three of the MAbs, 11F, 30B, and 10G, were able to neutralize virus infectivity, and two of them, 11F and 10G, were able to block virus-induced, cell-to-cell fusion. The binding sites of the 11F, 30B, and 10G MAbs were determined by Western immunoblotting and epitope mapping. The 11F and 30B MAbs bound to sites located, respectively, between amino acids 33 to 40 and 395 to 406 in the amino-terminal (S1) subunit of the S protein, and the 10G MAb bound to a site located between amino acids 1123 and 1137 in the carboxy-terminal (S2) subunit. These data define more precisely the interactions between the S1 and S2 subunits of the murine coronavirus S protein and provide further insights into its structure and function.
Assuntos
Coronaviridae/genética , Proteínas do Envelope Viral/genética , Animais , Anticorpos Monoclonais , Sequência de Bases , Sítios de Ligação de Anticorpos , Fusão Celular , Clonagem Molecular , DNA Viral/genética , DNA Viral/isolamento & purificação , Ensaio de Imunoadsorção Enzimática , Feminino , Genes Virais , Células L/metabolismo , Camundongos , Camundongos Endogâmicos BALB C/imunologia , Dados de Sequência Molecular , Sondas de Oligonucleotídeos , Proteínas Recombinantes de Fusão/imunologia , Mapeamento por Restrição , Proteínas do Envelope Viral/imunologia , Proteínas Estruturais Virais/genéticaRESUMO
Selection of an optimal promoter is necessary for efficient expression of foreign genes with vaccinia virus. Since a variety of powerful (homologous) vaccinia virus promoters and foreign (heterologous) promoter systems have been described for use in vaccinia, we have addressed the question of whether a general rule exists that allows the prediction of the optimal promoter/gene combination. We have compared the expression properties of four secreted proteins, the human blood clotting factor IX (FIX), the human blood glycoprotein Protein S (ProtS), the human von Willebrand factor (vWF), and the Hepatitis B virus (HBV) middle surface glycoprotein preS2, with proteins that were reported not to be secreted, the HBV large surface glycoprotein preS1 and the murine leukemia virus (MuLV) BM-5 Eco gag protein. In addition, we have included in our study an internal control protein, the vaccinia virus p11 protein, to monitor possible side effects of the promoter system used. Genes encoding the foreign proteins were placed either under control of a synthetic vaccinia virus early/late promoter (selP) or under control of the bacteriophage T7 promoter (T7/emc system). The secreted proteins were more efficiently expressed when fused to the homologous promoter. Direct comparison of the two promoters indicated that the expression level ranged between 1.4 (ProtS) and 3.9 (FIX)-fold higher with the selP than with the T7 promoter. In contrast, the cell-associated HBV preS1 was more efficiently expressed under the T7 promoter and the MuLV BM-5 Eco gag polypeptide was expressed equally well from both promoters. These data indicate that a careful prediction of optimal promoter/foreign gene combinations for the vaccinia virus expression system is possible. The choice of the optimal promoter/expression system is based on a simple classification scheme, discriminating secreted and nonsecreted proteins.
Assuntos
Proteínas Recombinantes de Fusão/biossíntese , Vaccinia virus/genética , Animais , Bacteriófago T7 , Sequência de Bases , Linhagem Celular , Citoplasma , Fator IX/biossíntese , Fator IX/genética , Expressão Gênica , Produtos do Gene gag/biossíntese , Produtos do Gene gag/genética , Glicoproteínas/biossíntese , Glicoproteínas/genética , Haplorrinos , Vírus da Hepatite B/química , Humanos , Vírus da Leucemia Murina/química , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Proteína S/biossíntese , Proteína S/genética , Proteínas Recombinantes de Fusão/classificação , Proteínas Recombinantes de Fusão/genética , Transcrição Gênica , Proteínas do Envelope Viral/biossíntese , Proteínas do Envelope Viral/genética , Fator de von Willebrand/biossíntese , Fator de von Willebrand/genéticaRESUMO
Vaccinia virus is one of the most widely applied expression systems for use in eukaryotes in molecular biology. Expression of heterologous genes in the vaccinia virus system, however, requires integration of the foreign DNA into the vaccinia virus genome by means of homologous recombination or by direct molecular cloning. In both cases, plasmid vector constructs are required that contain the gene of interest and, usually, a marker gene, both of which are controlled by suitable promoter sequences. In order to simplify the construction of recombinants and to eliminate the need for a marker gene we have developed a modified vaccinia virus genome that allows the direct targeted insertion of DNA fragments downstream of a strong vaccinia virus promoter without any further cloning steps. The gene of interest is amplified by PCR using oligonucleotide primers that provide an SfiI site at the 5' end and an RsrII site at the 3' end of the PCR product. Following digestion with these restriction enzymes, the PCR product is operationally linked to a synthetic early/late promoter within the viral genomic DNA via the unique SfiI/RsrII sites of the modified vaccinia virus genome. Using this approach, intermediate plasmid constructs and bacterial hosts are not required and time consuming screening steps can be omitted, because of 90% of the virus progeny carry the foreign DNA.
Assuntos
Clonagem Molecular/métodos , DNA Recombinante , Expressão Gênica , Vaccinia virus/genética , Sequência de Bases , Desoxirribonucleases de Sítio Específico do Tipo II , Fator IX/genética , Vetores Genéticos/genética , Genoma Viral , Vírus Auxiliares , Humanos , Dados de Sequência Molecular , Fases de Leitura Aberta/genética , Reação em Cadeia da Polimerase/métodos , Regiões Promotoras Genéticas/genética , Proteínas Recombinantes de Fusão/biossínteseRESUMO
We have described recently the construction of a defective vaccinia virus (VV) lacking the essential D4R open reading frame and have shown furthermore the selection of a complementing cell line providing the essential D4R gene product. The D4R gene belongs to the group of early transcribed vaccinia genes preventing a virus defective in D4R from entering into the intermediate and late phase of replication under noncomplementing conditions. Here we show that this property, which is unique among the group of so called nonreplicating poxviruses, is helpful for the production of (secretable) recombinant human proteins. Recombinant VV based on a D4R-defective parental strain expressing cDNAs coding for the human blood coagulation factors VII and XI produced significantly more recombinant protein than the corresponding recombinants based on wild-type VV. Moreover, the complementing cell line RK-D4R-44.20 was a more effective production cell system for both vD4 and wild-type VV recombinants compared to wild-type RK-13 cells. Surprisingly, recombinant human factor VII was more efficiently produced with the defective vaccinia recombinant even under noncomplementing conditions, suggesting that persistence of the early phase of vaccinia replication in combination with a delayed host shutoff is advantageous for the overproduction of certain recombinant proteins using the VV expression system.
Assuntos
DNA Glicosilases , Vírus Defeituosos/genética , Fator VII/biossíntese , Fator XI/biossíntese , Vetores Genéticos/genética , Proteínas Recombinantes de Fusão/biossíntese , Vaccinia virus/genética , Animais , Linhagem Celular , Chlorocebus aethiops , Clonagem Molecular , DNA Complementar/genética , Vírus Defeituosos/fisiologia , Fator VII/genética , Fator VII/isolamento & purificação , Fator XI/genética , Fator XI/isolamento & purificação , Teste de Complementação Genética , Humanos , N-Glicosil Hidrolases/deficiência , N-Glicosil Hidrolases/genética , Fases de Leitura Aberta , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Segurança , Uracila-DNA Glicosidase , Vaccinia virus/fisiologia , Proteínas Virais/genética , Replicação ViralRESUMO
Vaccinia viruses defective in the essential gene coding for the enzyme uracil DNA glycosylase (UDG) do not undergo DNA replication and do not express late genes in wild-type cells. A UDG-deficient vaccinia virus vector carrying the tick-borne encephalitis (TBE) virus prM/E gene, termed vD4-prME, was constructed, and its potential as a vaccine vector was evaluated. High-level expression of the prM/E antigens could be demonstrated in infected complementing cells, and moderate levels were found under noncomplementing conditions. The vD4-prME vector was used to vaccinate mice; animals receiving single vaccination doses as low as 10(4) PFU were fully protected against challenge with high doses of virulent TBE virus. Single vaccination doses of 10(3) PFU were sufficient to induce significant neutralizing antibody titers. With the corresponding replicating virus, doses at least 10-fold higher were needed to achieve protection. The data indicate that late gene expression of the vaccine vector is not required for successful vaccination; early vaccinia virus gene expression induces a potent protective immune response. The new vaccinia virus-based defective vectors are therefore promising live vaccines for prophylaxis and cancer immunotherapy.
Assuntos
Vírus Defeituosos/genética , Vírus da Encefalite Transmitidos por Carrapatos/imunologia , Vetores Genéticos , Vacinas Sintéticas/imunologia , Vaccinia virus/genética , Vacinas Virais/imunologia , Animais , Replicação do DNA , Camundongos , Vacinação , Replicação ViralRESUMO
A cDNA clone encoding a cellular protein that interacts with murine leukemia virus (MuLV) Gag proteins was isolated from a T-cell lymphoma library. The sequence of the clone is identical to the C terminus of a cellular protein, KIF4, a microtubule-associated motor protein that belongs to the kinesin superfamily. KIF4-MuLV Gag associations have been detected in vitro and in vivo in mammalian cells. We suggest that KIF4 could be involved in Gag polyprotein translocation from the cytoplasm to the cell membrane.