RESUMO
Ebola virus causes irregular outbreaks of severe hemorrhagic fever in equatorial Africa. Case mortality remains high; there is no effective treatment and outbreaks are sporadic and unpredictable. Studies of Ebola virus vaccine platforms in non-human primates have established that the induction of protective immunity is possible and safety and human immunogenicity has been demonstrated in a previous Phase I clinical trial of a 1st generation Ebola DNA vaccine. We now report the safety and immunogenicity of a recombinant adenovirus serotype 5 (rAd5) vaccine encoding the envelope glycoprotein (GP) from the Zaire and Sudan Ebola virus species, in a randomized, placebo-controlled, double-blinded, dose escalation, Phase I human study. Thirty-one healthy adults received vaccine at 2×10(9) (n=12), or 2×10(10) (n=11) viral particles or placebo (n=8) as an intramuscular injection. Antibody responses were assessed by ELISA and neutralizing assays; and T cell responses were assessed by ELISpot and intracellular cytokine staining assays. This recombinant Ebola virus vaccine was safe and subjects developed antigen specific humoral and cellular immune responses.
Assuntos
Adenovírus Humanos/genética , Vacinas contra Ebola/imunologia , Vetores Genéticos , Doença pelo Vírus Ebola/prevenção & controle , Proteínas do Envelope Viral/imunologia , Adolescente , Adulto , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Citocinas/imunologia , Método Duplo-Cego , Vacinas contra Ebola/efeitos adversos , Vacinas contra Ebola/genética , Ebolavirus/genética , Ebolavirus/imunologia , Ensaio de Imunoadsorção Enzimática , Feminino , Doença pelo Vírus Ebola/imunologia , Humanos , Masculino , Pessoa de Meia-Idade , Testes de Neutralização , Placebos/administração & dosagem , Linfócitos T/imunologia , Vacinas Atenuadas/efeitos adversos , Vacinas Atenuadas/genética , Vacinas Atenuadas/imunologia , Proteínas do Envelope Viral/genética , Adulto JovemRESUMO
Most influenza vaccines are produced in chicken eggs but recent human influenza strains often do not grow well in this substrate. The PER.C6 cell line is an alternative platform for vaccine production. Here we demonstrate that PER.C6 cells faithfully propagate recent clinical isolates, without selecting for mutations in the HA gene. PER.C6 cells support the rescue of recombinant influenza viruses from cDNA. We used sequence data from a surveillance programme to generate a PR8-based seed virus with the HA and NA of a contemporary circulating H3N2 human strain, A/England/611/07 (E611) that did not itself grow in eggs. We engineered mutations that affected receptor-binding, G186V or L194P, into the E611 HA gene. Whilst the L194P mutation conferred efficient growth in eggs, G186V did not. The L194P mutation was also spontaneously selected during egg propagation of E611/PR8 7:1 recombinant virus. This suggests generation of a single recombinant vaccine seed might satisfy manufacturers that utilize either eggs or cells for vaccine production.
Assuntos
Vírus da Influenza A Subtipo H3N2/crescimento & desenvolvimento , Vírus da Influenza A Subtipo H3N2/imunologia , Vacinas contra Influenza/genética , Vacinas contra Influenza/imunologia , Influenza Humana/prevenção & controle , Substituição de Aminoácidos/genética , Animais , Linhagem Celular , Humanos , Vírus da Influenza A Subtipo H3N2/genética , Vírus da Influenza A Subtipo H3N2/isolamento & purificação , Influenza Humana/imunologia , Influenza Humana/virologia , Recombinação Genética , Cultura de Vírus/métodosRESUMO
Reverse genetics, the generation of influenza viruses from cDNA, presents a rapid method for creating vaccine strains. The technique necessitates the use of cultured cells. Due to technical and regulatory requirements, the choice of cell lines for production of human influenza vaccines is limited. PER.C6 cells, among the most extensively characterized and documented cells, support growth of all influenza viruses tested to date, and can be grown to high densities in large bioreactors in the absence of serum or micro carriers. Here, the suitability of these cells for the generation of influenza viruses by reverse genetics was investigated. A range of viruses reflective of vaccine strains was rescued exclusively using PER.C6 cells by various transfection methods, including an animal component-free procedure. Furthermore, a whole inactivated vaccine carrying the HA and NA segments of A/HK/156/97 (H5N1) that was both rescued from and propagated on PER.C6 cells, conferred protection in a mouse model. Thus PER.C6 cells provide an attractive platform for generation of influenza vaccine strains via reverse genetics.
Assuntos
Vírus da Influenza A Subtipo H1N1/crescimento & desenvolvimento , Vírus da Influenza A Subtipo H3N2/crescimento & desenvolvimento , Virus da Influenza A Subtipo H5N1/crescimento & desenvolvimento , Vacinas contra Influenza/genética , Vírus Reordenados/crescimento & desenvolvimento , Animais , Anticorpos Antivirais/sangue , Técnicas de Cultura de Células , Linhagem Celular , Feminino , Testes de Inibição da Hemaglutinação , Humanos , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/imunologia , Vírus da Influenza A Subtipo H3N2/genética , Vírus da Influenza A Subtipo H3N2/imunologia , Virus da Influenza A Subtipo H5N1/genética , Virus da Influenza A Subtipo H5N1/imunologia , Vacinas contra Influenza/imunologia , Influenza Humana/prevenção & controle , Camundongos , Camundongos Endogâmicos BALB C , Vírus Reordenados/genética , Vírus Reordenados/imunologia , Índice de Gravidade de Doença , Análise de Sobrevida , Transfecção/métodos , Ensaio de Placa ViralRESUMO
Immediate passive immune prophylaxis as part of rabies post-exposure prophylaxis (PEP) often cannot be provided due to limited availability of human or equine rabies immunoglobulin (HRIG and ERIG, respectively). We report first clinical data from two phase I studies evaluating a monoclonal antibody cocktail CL184 against rabies. The studies included healthy adult subjects in the USA and India and involved two parts. First, subjects received a single intramuscular dose of CL184 or placebo in a double blind, randomized, dose-escalation trial. Second, open-label CL184 (20IU/kg) was co-administered with rabies vaccine. Safety was the primary objective and rabies virus neutralizing activity (RVNA) was investigated as efficacy parameter. Pain at the CL184 injection site was reported by less than 40% of subjects; no fever or local induration, redness or swelling was observed. RVNA was detectable from day 1 to day 21 after a single dose of CL184 20 or 40IU/kg. All subjects had adequate (>0.5IU/mL) RVNA levels from day 14 onwards when combined with rabies vaccine. CL184 appears promising as an alternative to RIG in PEP.
Assuntos
Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/efeitos adversos , Anticorpos Antivirais/administração & dosagem , Anticorpos Antivirais/efeitos adversos , Vírus da Raiva/imunologia , Raiva/prevenção & controle , Adolescente , Adulto , Idoso , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Linhagem Celular , Método Duplo-Cego , Feminino , Humanos , Imunização Passiva , Masculino , Pessoa de Meia-Idade , Testes de Neutralização , Raiva/imunologia , Vacina Antirrábica/administração & dosagem , Vacina Antirrábica/imunologia , Resultado do Tratamento , Adulto JovemRESUMO
Stable E1 transformed cells, like PER.C6, are able to grow at scale and to high cell densities. E1-deleted adenoviruses replicate to high titer in PER.C6 cells whereas subsequent deletion of E2A from the vector results in absence of replication in PER.C6 cells and drastically lowers the expression of adenovirus proteins in such cells. We therefore considered the use of an DeltaE1/DeltaE2 type 5 vector (Ad5) to deliver genes to PER.C6 cells growing in suspension with the aim to achieve high protein yield. To evaluate the utility of this system we constructed DeltaE1/DeltaE2 vector carrying different classes of protein, that is, the gene coding for spike protein derived from the Coronavirus causing severe acute respiratory syndrome (SARS-CoV), a gene coding for the SARS-CoV receptor or the genes coding for an antibody shown to bind and neutralize SARS-CoV (SARS-AB). The DeltaE1/DeltaE2A-vector backbones were rescued on a PER.C6 cell line engineered to constitutively over express the Ad5 E2A protein. Exposure of PER.C6 cells to low amounts (30 vp/cell) of DeltaE1/DeltaE2 vectors resulted in highly efficient (>80%) transduction of PER.C6 cells growing in suspension. The efficient cell transduction resulted in high protein yield (up to 60 picogram/cell/day) in a 4 day batch production protocol. FACS and ELISA assays demonstrated the biological activity of the transiently produced proteins. We therefore conclude that DeltaE1/DeltaE2 vectors in combination with the PER.C6 technology may provide a viable answer to the increasing demand for high quality, high yield recombinant protein.
Assuntos
Adenoviridae/genética , Melhoramento Genético/métodos , Engenharia de Proteínas/métodos , Proteínas Recombinantes/metabolismo , Retina/metabolismo , Transfecção/métodos , Biotecnologia/métodos , Linhagem Celular , Meios de Cultura Livres de Soro , Vetores Genéticos/genética , HumanosRESUMO
Ensuring complete viral inactivation is critical for the safety of vaccines based on an inactivated virus. Detection of residual infectious virus is dependent on sensitivity of the assay, sample volume analyzed and the absence of interference with viral infection. Here we describe the development and qualification of a sensitive cell-based assay for the detection of residual infectious West Nile Virus (WNV). The results of the assay are in good agreement with the assumption that at low concentrations the number of infectious units in relatively small samples follows a Poisson distribution. The assay can detect 1 infectious unit with a confidence of 99%, provides statistical controls for interference and can easily be scaled up to test large amounts of vaccine material. Furthermore, we show equivalence in sensitivity between the cell-based assay and an in vivo assay for detection of infectious WNV. Finally, the assay has been used for successful release testing of clinical lots of inactivated WNV vaccine. Given the principle and generic setup of the method we envision broad applicability to the detection of very low concentrations of infectious virus.
Assuntos
Propiolactona/farmacologia , Vacinas de Produtos Inativados , Inativação de Vírus , Vacinas contra o Vírus do Nilo Ocidental , Vírus do Nilo Ocidental/patogenicidade , Animais , Animais Lactentes , Linhagem Celular , Chlorocebus aethiops , Modelos Animais de Doenças , Camundongos , Camundongos Endogâmicos C3H , Células Vero , Febre do Nilo Ocidental/mortalidade , Febre do Nilo Ocidental/virologia , Vírus do Nilo Ocidental/efeitos dos fármacos , Vírus do Nilo Ocidental/isolamento & purificação , Vírus do Nilo Ocidental/fisiologiaRESUMO
The presence of replication-competent adenovirus (RCA) is a safety concern for biologics based on recombinant adenoviruses and RCA testing is therefore mandatory for release of clinical material. RCA, which arises from homologous recombination between Ad5 vectors and HEK-293 cells, can be eliminated by the use of PER.C6 cells in combination with a matched vector. However, little is known on RCA formation with vectors based on adenovirus serotypes other than Ad5 and reliable RCA assays to test them are generally lacking. Here we report on the development and qualification of a sensitive RCA assay for Ad35, a promising alternative to Ad5 vectors. The assay is able to detect 1 RCA in 3x10(10) vector particles with 95% confidence, thus meeting current FDA requirements, and can discriminate between RCA and other rare CPE-causing entities, including helper dependent E1 positive particles (HDEP). Using this assay, the first batches of Ad35 vectors produced in PER.C6 cells were analysed and found to be free of RCA and HDEP. Based on the statistical model used, we anticipate that our approach to RCA assay development can be broadly applicable to other adenoviral vectors.
Assuntos
Adenoviridae/genética , Proteínas E1 de Adenovirus/genética , Bioensaio/métodos , Deleção de Genes , Replicação Viral , Adenoviridae/crescimento & desenvolvimento , Linhagem Celular , Linhagem Celular Tumoral , Vetores Genéticos/genética , Humanos , Reação em Cadeia da Polimerase , Reprodutibilidade dos TestesRESUMO
Clinical trials in malignant glioma have demonstrated excellent safety of recombinant adenovirus type 5 (Ad5) but lack of convincing efficacy. The overall low expression levels of the Coxsackie and Adenovirus receptor and the presence of high anti-Ad5-neutralizing antibody (NAb) titers in the human population are considered detrimental for consistency of clinical results. To identify an adenoviral vector better suited to infect primary glioma cells, we tested a library of fiber-chimeric Ad5-based adenoviral vectors on 12 fresh human glioma cell suspensions. Significantly improved marker gene expression was obtained with several Ad5-chimeric vectors, predominantly vectors carrying fiber molecules derived from B-group viruses (Ad11, Ad16, Ad35 and Ad50). We next tested Ad35 sero prevalence in sera derived from 90 Dutch cancer patients including 30 glioma patients and investigated the transduction efficiency of this vector in glioma cell suspensions. Our results demonstrate that the sero prevalence and the titers of NAb against Ad35 are significantly lower than against Ad5. Also, recombinant Ad35 has significantly increased ability to transfer a gene to primary glioma cells compared to Ad5. We thus conclude that Ad35 represents an interesting candidate vector for gene therapy of malignant glioma.
Assuntos
Adenoviridae/genética , Neoplasias Encefálicas/terapia , Terapia Genética , Vetores Genéticos , Glioma/terapia , Sequência de Bases , Neoplasias Encefálicas/imunologia , Primers do DNA , Glioma/imunologia , Humanos , Transdução GenéticaRESUMO
Adenoviral vectors based on adenovirus type 35 (rAd35) have the advantage of low natural vector immunity and induce strong, insert-specific T- and B-cell responses, making them prime-candidate vaccine carriers. However, severe vector-genome instability of E1-deleted rAd35 vectors was observed, hampering universal use. The instability of E1-deleted rAd35 vector proved to be caused by low pIX expression induced by removal of the pIX promoter, which was located in the E1B region of B-group viruses. Reinsertion of a minimal pIX promoter resulted in stable vectors able to harbour large DNA inserts (> 5 kb). In addition, it is shown that replacement of the E4-Orf6 region of Ad35 by the E4-Orf6 region of Ad5 resulted in successful propagation of an E1-deleted rAd35 vector on existing E1-complementing cell lines, such as PER.C6 cells. The ability to produce these carriers on PER.C6 contributes significantly to the scale of manufacturing of rAd35-based vaccines. Next, a stable rAd35 vaccine was generated carrying Mycobacterium tuberculosis antigens Ag85A, Ag85B and TB10.4. The antigens were fused directly, resulting in expression of a single polyprotein. This vaccine induced dose-dependent CD4+ and CD8+ T-cell responses against multiple antigens in mice. It is concluded that the described improvements to the rAd35 vector contribute significantly to the further development of rAd35 carriers for mass-vaccination programmes for diseases such as tuberculosis, AIDS and malaria.
Assuntos
Adenoviridae/genética , Adenoviridae/isolamento & purificação , Vetores Genéticos , Vacinas Sintéticas , Adenoviridae/fisiologia , Proteínas E4 de Adenovirus/genética , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Proteínas do Capsídeo/biossíntese , Proteínas do Capsídeo/genética , Linhagem Celular , Teste de Complementação Genética , Humanos , Imunidade Celular , Interferon gama/biossíntese , Camundongos , Modelos Animais , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/imunologia , Regiões Promotoras Genéticas , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Recombinação Genética , Vacinas contra a Tuberculose/genética , Vacinas contra a Tuberculose/imunologia , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Replicação ViralRESUMO
A major obstacle to successful oral vaccination is the lack of antigen delivery systems that are both safe and highly efficient. Conventional replication-incompetent adenoviral vectors, derived from human adenoviruses of subgroup C, are poorly efficient in delivering genetic material to differentiated intestinal epithelia. To date, 51 human adenovirus serotypes have been identified and shown to recognize different cellular receptors with different tissue distributions. This natural diversity was exploited in the present study to identify suitable adenoviral vectors for efficient gene delivery to the human intestinal epithelium. In particular, we compared the capacities of a library of adenovirus type 5-based vectors pseudotyped with fibers of several human serotypes for transduction, binding, and translocation toward the basolateral pole in human and murine tissue culture models of differentiated intestinal epithelia. In addition, antibody-based inhibition was used to gain insight into the molecular interactions needed for efficient attachment. We found that vectors differing merely in their fiber proteins displayed vastly different capacities for gene transfer to differentiated human intestinal epithelium. Notably, vectors bearing fibers derived from subgroup B and subgroup D serotypes transduced the apical pole of human epithelium with considerably greater efficiency than a subgroup C vector. Such efficiency was correlated with the capacity to use CD46 or sialic acid-containing glycoconjugates as opposed to CAR as attachment receptors. These results suggest that substantial gains could be made in gene transfer to digestive epithelium by exploiting the tropism of existing serotypes of human adenoviruses.
Assuntos
Adenovírus Humanos/patogenicidade , Proteínas do Capsídeo/metabolismo , Vetores Genéticos , Mucosa Intestinal/virologia , Transdução Genética , Adenovírus Humanos/classificação , Adenovírus Humanos/genética , Animais , Células CACO-2 , Proteínas do Capsídeo/genética , Linhagem Celular , Polaridade Celular , Humanos , Proteína Cofatora de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Ácido N-Acetilneuramínico/metabolismo , Receptores Virais/metabolismo , SorotipagemRESUMO
To study the molecular epidemiology of HIV-1 in Belarus, the genetic sequences of HIV-1 variants were obtained from 50 infected persons, which represented the main stages, risk groups, and geographic areas of the epidemic. The env and gag sequences were studied for HIV-1 variants from 31 persons, the env sequences were for HIV-1 variants from 18 persons, and the gag sequence was for HIV-1 variant from 1 person. Phylogenetic analysis indicated that the sequences of HIV-1 variants from 46 persons were homogenic and evolutionally closely related to IDU-A strains specific for other epidemics in the former Soviet Union are dominating in the epidemic in Belarus. Circulation of epidemiologically unrelated subtype B viruses was also established.
Assuntos
Infecções por HIV/epidemiologia , HIV-1/genética , Feminino , Produtos do Gene gag/genética , Genes Virais/genética , HIV-1/classificação , Humanos , Masculino , Epidemiologia Molecular , Filogenia , República de Belarus/epidemiologia , Proteínas do Envelope Viral/genéticaRESUMO
Given the promise of recombinant adenovirus type 5 (rAd5) as a malaria vaccine carrier in preclinical models, we evaluated the potency of rAd35 coding for Plasmodium yoelii circumsporozoite protein (rAd35PyCS). We chose rAd35 since a survey with serum samples from African subjects demonstrated that human Ad35 has a much lower seroprevalence of 20% and a much lower geometric mean neutralizing antibody titer (GMT) of 48 compared to Ad5 (seroprevalence, 85%; GMT, 1,261) in countries with a high malaria incidence. We also demonstrated that immunization with rAd35PyCS induced a dose-dependent and potent, CS-specific CD8(+) cellular and humoral immune response and conferred significant inhibition (92 to 94%) of liver infection upon high-dose sporozoite challenge. Furthermore, we showed that in mice carrying neutralizing antibody activity against Ad5, mimicking a human situation, CS-specific T- and B-cell responses were significantly dampened after rAd5PyCS vaccination, resulting in loss of inhibition of liver infection upon sporozoite challenge. In contrast, rAd35 vaccine was as potent in naive mice as in Ad5-preimmunized mice. Finally, we showed that heterologous rAd35-rAd5 prime-boost regimens were more potent than rAd35-rAd35 because of induction of anti-Ad35 antibodies after rAd35 priming. The latter data provide a further rationale for developing rAd prime-boost regimens but indicate that priming and boosting Ad vectors must be immunologically distinct and also should be distinct from Ad5. Collectively, the data presented warrant further development of rAd35-based vaccines against human malaria.
Assuntos
Adenovírus Humanos/imunologia , Vacinas Antimaláricas/imunologia , Malária/imunologia , Plasmodium yoelii/imunologia , Adenovírus Humanos/genética , Animais , Anticorpos Antiprotozoários/biossíntese , Anticorpos Antiprotozoários/sangue , Feminino , Vetores Genéticos/imunologia , Humanos , Imunização Secundária , Fígado/imunologia , Fígado/parasitologia , Hepatopatias Parasitárias/imunologia , Malária/prevenção & controle , Vacinas Antimaláricas/administração & dosagem , Vacinas Antimaláricas/genética , Camundongos , Camundongos Endogâmicos BALB C , Plasmodium yoelii/genética , Proteínas de Protozoários/administração & dosagem , Proteínas de Protozoários/genética , Proteínas de Protozoários/imunologia , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologiaRESUMO
BACKGROUND: Peripartum antiretroviral regimens have been shown to prevent mother-to-child transmission of HIV (MTCT) in randomized clinical trials; however, direct comparison of published results is impossible given methodological and population differences. OBJECTIVE: To directly compare the efficacy of different antiretroviral regimens in reducing the risk of 6-week MTCT rate in African breastfeeding populations. METHODS: Pooled analysis including all mother-infant pairs from any relevant trial: West African ZDV-placebo trials, Petra ZDV+3TC [two regimens A (pre/intra/post-partum) and B (intra/post-partum), placebo from Uganda and Tanzania], SAINT (NVP and Petra arm B), HIVNET012 (NVP, ultra short ZDV pp) and the Vitamin A trial (as placebo arm in South Africa). Peripartum HIV infection was any positive RNA or DNA polymerase chain reaction test < day 60. The MTCT risk was estimated at 6 weeks for each treatment arm and compared with placebo or single-dose NVP using logistic regression adjusting for maternal CD4 cell count, breastfeeding and birthweight. RESULTS: Overall, 4125 singleton live-births were included; 3629 (88%) were assessed for HIV status at 6 weeks of age. In comparison with placebo, zidovudine + lamivudine (ZDV+3TC) arm A [adjusted odds ratio (AOR), 0.23; P < 0.0001], ZDV+3TC arm B (AOR, 0.49; P < 0.001), antenatal ZDV short (AOR, 0.55; P = 0.006) and nevirapine (NVP) (AOR, 0.60; P = 0.0007) significantly reduced MTCT. In comparison with NVP, only the longest regimen of ZDV+3TC (AOR, 0.39, P < 0.0005) was significantly more effective. CONCLUSION: These results are in line with current World Health Organisation guidelines suggesting equivalence of choice between single-dose NVP and short-course ZDV, and confirm the greater efficacy of ZDV+3TC than with any single antiretroviral drug.
Assuntos
Fármacos Anti-HIV/administração & dosagem , Infecções por HIV/transmissão , Transmissão Vertical de Doenças Infecciosas/prevenção & controle , Lamivudina/administração & dosagem , Zidovudina/administração & dosagem , Adulto , Aleitamento Materno/efeitos adversos , Combinação de Medicamentos , Feminino , Infecções por HIV/tratamento farmacológico , Humanos , Lactente , Recém-Nascido , Masculino , Assistência Perinatal , Ensaios Clínicos Controlados Aleatórios como Assunto , Análise de Regressão , Fatores de Risco , Resultado do TratamentoRESUMO
This paper describes a new automatic hinge system for leg orthoses, which provides knee stability in stance, and allows knee-flexion during swing. Indications for the hinge system are a paresis or paralysis of the quadriceps muscles. Instrumented gait analysis was performed in three patients, fitted with this new hinge system in a knee orthosis. The orthosis proves to satisfy the patients allowing them to walk without a knee-lock system.
Assuntos
Membros Artificiais , Aparelhos Ortopédicos , Adulto , Desenho de Equipamento , Marcha , Humanos , Articulação do Joelho/fisiopatologia , Perna (Membro) , Masculino , Pessoa de Meia-Idade , Neoplasias Musculares/cirurgia , Distrofia Muscular de Duchenne/reabilitação , Paresia/reabilitação , Amplitude de Movimento ArticularRESUMO
Gene transfer with recombinant adenoviruses (rAds) is a powerful means of inducing an immune response against a transgene product. However, little is known about the mechanisms that underlie the induction of the immune response after intramuscular inoculation of adenovirus and, in particular, the relative role of the different cell types transduced. Several studies have suggested that CD8+ cytotoxic T lymphocyte responses elicited after inoculation of adenoviruses (Ads) are induced both by direct transduction of antigen presenting cells (APCs) and by cross-priming. In the present study, a library of fibre-chimeric rAds was screened in order to identify rAds with distinct capacities to express transgene product in murine cell types naturally found in muscle, i.e. myoblasts, endothelial cells (both representing non-APCs) and dendritic cells (representing APCs). Four selected pseudotypes, differing in their ability to infect muscular cells were used to immunize C57BL/6 mice. The relationship between the capacity to transduce non-APC or APC in vitro and the ability to induce humoral and cellular responses against the beta-galactosidase antigen after intramuscular inoculation were studied. Results indicate that CD8+ T cell responses against the beta-galactosidase antigen were similar after inoculation of the four viruses, thus revealing no direct relationship with their ability to transduce myoblasts, endothelial cells or dendritic cells in vitro.
Assuntos
Adenoviridae/imunologia , Linfócitos T CD8-Positivos/imunologia , Vetores Genéticos/imunologia , Adenoviridae/genética , Animais , Células CHO , Cricetinae , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Células Endoteliais/imunologia , Células Endoteliais/metabolismo , Feminino , Imunização/métodos , Interferon gama/biossíntese , Contagem de Linfócitos , Camundongos , Camundongos Endogâmicos C57BL , Mioblastos/imunologia , Mioblastos/metabolismo , Transdução Genética , Tropismo , Proteínas Virais/genética , Proteínas Virais/metabolismo , beta-Galactosidase/imunologiaRESUMO
We had previously collected and analyzed, by phylogenetic methods, all genetic data available now for the Parvoviridae and Astroviridae families, which made it possible to define the evolutionary relations between the viruses as well as to depict a variety of events in the evolutionary history of the two families. The offered case study is dedicated to investigating the stabilizing and splitting selection types in the evolution of the discussed viral families. We analyzed the number of synonymous and non-synonymous nucleotide substitutions in the coding genomes' regions of the viruses. Finally, the stabilizing selection was shown to be a key factor in the evolution of parvoviruses and astroviruses.
Assuntos
Astroviridae/genética , Evolução Biológica , Parvovirus/genética , Animais , Astroviridae/classificação , Parvovirus/classificaçãoRESUMO
BACKGROUND: Most toxicities associated with nucleoside analogue reverse transcriptase inhibitors (NRTIs) are thought to result from mitochondrial toxicity. These toxicities include peripheral neuropathy, pancreatitis, lactic acidosis, and peripheral lipoatrophy. Unfortunately, there are no validated laboratory markers for clinically assessing, let alone predicting, the onset of mitochondrial toxicity associated with NRTI therapy. OBJECTIVES: To provide preliminary evidence of the potential clinical utility of an assay which has been developed for quantifying mitochondrial DNA (mtDNA) in clinical samples from HIV-infected patients. METHODS: A single-tube duplex real-time DNA-nucleic acid sequence-based amplification (NASBA) assay (Mitox, Primagen, Amsterdam, the Netherlands) was used to quantify mtDNA in cryopreserved peripheral blood mononuclear cells (PBMC) obtained from HIV-1-infected patients during their prior participation in a randomized placebo-controlled trial comparing zidovudine (ZDV) monotherapy with combinations of ZDV plus either dideoxycytidine (ddC) or didanosine (ddI) (the Delta trial). Patients were antiretroviral naïve prior to entering the trial. Samples obtained during the initial 48 weeks of treatment were tested. RESULTS: A significant decline of mtDNA, both in an intent-to-treat and in an as-treated analysis, was observed in patients treated with ZDV+ddC and ZDV+ddI, but not with ZDV alone, consistent with the results expected from the degree of mtDNA depletion described for each of these drugs in vitro. CONCLUSIONS: This single-tube duplex real-time DNA-NASBA assay was shown to measure mtDNA accurately in PBMC. Treatment with a combination of two NRTIs was associated with greater reductions in mtDNA than obtained for ZDV monotherapy. The relevance of these results in predicting treatment toxicity requires further evaluation.
Assuntos
Fármacos Anti-HIV/efeitos adversos , DNA Mitocondrial/efeitos dos fármacos , Infecções por HIV/tratamento farmacológico , Doenças Mitocondriais/induzido quimicamente , Inibidores da Transcriptase Reversa/efeitos adversos , Análise de Variância , Fármacos Anti-HIV/administração & dosagem , DNA Mitocondrial/isolamento & purificação , Didanosina/administração & dosagem , Didanosina/efeitos adversos , Combinação de Medicamentos , Humanos , Inibidores da Transcriptase Reversa/administração & dosagem , Replicação de Sequência Autossustentável/normas , Sensibilidade e Especificidade , Zalcitabina/administração & dosagem , Zalcitabina/efeitos adversos , Zidovudina/administração & dosagem , Zidovudina/efeitos adversosRESUMO
Reliable assays for accurate titration of influenza virus in infectious samples are pivotal to both influenza research and vaccine development. A titration assay adopted commonly for this purpose is the plaque assay on Madin-Darby canine kidney (MDCK) cells, despite it being time and labour consuming. A novel assay is described for titration of influenza viruses based on the detection of intracellular viral nucleoprotein (NP) by fluorescence-activated cell sorting (FACS). By using a panel of viruses of different type, subtype and origin, it is demonstrated that there is a mathematical correlation between titres measured by immunotitration and by classical plaque assay on MDCK cells. Moreover, the availability of NP antibodies specific for type A or type B influenza virus ensures the specificity of the assay. Based on speed, accuracy and specificity, it is concluded that the FACS-based immunotitration of influenza virus represents a valid and efficient alternative to the classical plaque assay.
Assuntos
Citometria de Fluxo/métodos , Vírus da Influenza A/isolamento & purificação , Vírus da Influenza B/isolamento & purificação , Nucleoproteínas/metabolismo , Proteínas do Core Viral/metabolismo , Animais , Linhagem Celular , Cães , Humanos , Vírus da Influenza A/metabolismo , Vírus da Influenza B/metabolismo , Proteínas do Nucleocapsídeo , Fatores de Tempo , Ensaio de Placa ViralRESUMO
BACKGROUND: Human herpesvirus-8 (HHV-8) is linked to the pathogenesis of Kaposi's sarcoma (KS), and the HHV-8 DNA load in peripheral blood mononuclear cells (PBMC) is associated with the clinical stage of KS. To examine the expression of HHV-8 in PBMC, four HHV-8 mRNA specific NASBA assays were developed METHODS: We have developed four quantitative nucleic acid sequence-based amplification assays (NASBA-QT) specifically to detect mRNA coding for ORF 73 (latency-associated nuclear antigen, LANA), vGCR (a membrane receptor), vBcl-2 (a viral inhibitor of apoptosis) and vIL-6 (a viral growth factor). The NASBA technique amplifies nucleic acids without thermocycling and mRNA can be amplified in a dsDNA background. A molecular beacon is used during amplification to enable real-time detection of the product. The assays were tested on PBMC samples of two AIDS-KS patients from the Amsterdam Cohort. RESULTS: For all four assays, the limit of detection (LOD) of 50 molecules and the limit of quantification (LOQ) of 100 molecules were determined using in vitro transcribed RNA. The linear dynamic range was 50 to 10(7) molecules of HHV-8 mRNA. We found HHV-8 mRNA expression in 9 out of the 10 tested samples. CONCLUSION: These real-time NASBA assays with beacon detection provide tools for further study of HHV-8 expression in patient material.
Assuntos
Proteínas de Transporte/genética , Herpesvirus Humano 8/genética , RNA Mensageiro/genética , RNA Viral/genética , Replicação de Sequência Autossustentável/métodos , Proteínas Virais/genética , Latência Viral/genética , Antígenos Virais , Fatores de Transcrição de Zíper de Leucina Básica , Genes Virais/genética , Herpesvirus Humano 8/isolamento & purificação , Herpesvirus Humano 8/fisiologia , Humanos , Interleucina-6/genética , Leucócitos Mononucleares/virologia , Proteínas Nucleares/genética , Proteínas Oncogênicas , RNA Mensageiro/metabolismo , Proteínas Repressoras , Sarcoma de Kaposi/virologia , Sensibilidade e Especificidade , Proteínas Estruturais Virais/genéticaRESUMO
Site-specific proteases, which catalyze cleavage of peptide bonds in specific amino acid sequences of target proteins, play important roles in various biological events of many living organisms. In humans, disruption in regulation of these site-specific proteases can lead to pathological consequences. Here, we report a simple in vitro assay for enzymatic activities of site-specific proteases. This assay system employs a protein substrate molecule that is comprised of (i) His-tag binding module, (ii) cleavage sites, and (iii) green fluorescent protein (GFP) detection module. In this study, prostate-specific antigen (PSA) and Thrombin-specific cleavage sites were introduced into the substrate molecules. The overexpressed GFP substrate protein was purified with the aid of Ni++-charged magnetic beads. On cleavage by either PSA or Thrombin, GFP was released from the bound magnetic beads, enabling a direct measurement of the cleaved product by fluorescence. Detection sensitivity, as well as the kinetics of reaction of PSA cleavage with the GFP substrate, was similar or better than commercially available PSA fluorogenic peptide substrate. This bead-attached GFP substrate was also used for an inhibition assay using a competitive inhibitor of Thrombin. In conclusion, this assay offers a simple fluorescent method for monitoring the activity of the site-specific proteases. Furthermore, this system provides flexible means of incorporating varying sizes of flanking sequences adjacent to the cleavage site, which can be essential for studying the regulatory macromolecular interactions between proteases and their substrates.