RESUMO
In this study, we pioneered an alternative technology for manufacturing subunit influenza hemagglutinin (HA)-based vaccines. This innovative method involves harnessing the pupae of the Lepidoptera Trichoplusia ni (T. ni) as natural biofactories in combination with baculovirus vectors (using CrisBio® technology). We engineered recombinant baculoviruses encoding two versions of the HA protein (trimeric or monomeric) derived from a pandemic avian H7N1 virus A strain (A/chicken/Italy/5093/99). These were then used to infect T. ni pupae, resulting in the production of the desired recombinant antigens. The obtained HA proteins were purified using affinity chromatography, consistently yielding approximately 75 mg/L of insect extract. The vaccine antigen effectively immunized poultry, which were subsequently challenged with a virulent H7N1 avian influenza virus. Following infection, all vaccinated animals survived without displaying any clinical symptoms, while none of the mock-vaccinated control animals survived. The CrisBio®-derived antigens induced high titers of HA-specific antibodies in the vaccinated poultry, demonstrating hemagglutination inhibition activity against avian H7N1 and human H7N9 viruses. These results suggest that the CrisBio® technology platform has the potential to address major industry challenges associated with producing recombinant influenza subunit vaccines, such as enhancing production yields, scalability, and the speed of development, facilitating the global deployment of highly effective influenza vaccines.
Assuntos
Anticorpos Antivirais , Galinhas , Glicoproteínas de Hemaglutininação de Vírus da Influenza , Vacinas contra Influenza , Influenza Aviária , Pupa , Vacinas de Subunidades Antigênicas , Animais , Vacinas contra Influenza/imunologia , Vacinas contra Influenza/genética , Vacinas contra Influenza/administração & dosagem , Pupa/imunologia , Influenza Aviária/prevenção & controle , Influenza Aviária/imunologia , Vacinas de Subunidades Antigênicas/imunologia , Vacinas de Subunidades Antigênicas/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/sangue , Vírus da Influenza A Subtipo H7N1/imunologia , Vírus da Influenza A Subtipo H7N1/genética , Baculoviridae/genética , Subtipo H7N9 do Vírus da Influenza A/imunologia , Subtipo H7N9 do Vírus da Influenza A/genética , Humanos , Desenvolvimento de Vacinas , Mariposas/imunologia , Pandemias/prevenção & controleRESUMO
Prior research has established the anti-apoptotic effects in insect cell cultures of Bombyx mori (B. mori) hemolymph, as well as the heightened production yields of recombinant proteins facilitated by baculovirus vectors in insect cells cultivated in media supplemented with this hemolymph. In this study, we investigated the hemolymph of another Lepidoptera species, Trichoplusia ni (T. ni), and observed similar beneficial effects in insect cells cultivated in media supplemented with this natural substance. We observed enhancements in both production yield (approximately 1.5 times higher) and late-stage cell viabilities post-infection (30-40% higher). Storage-protein 2 from B. mori (SP2Bm) has previously been identified as one of the abundant hemolymph proteins potentially responsible for the beneficial effects observed after the use of B. mori hemolymph-supplemented cell culture media. By employing a dual baculovirus vector that co-expresses the SP2Bm protein alongside the GFP protein, we achieved a threefold increase in reporter protein production compared to a baculovirus vector expressing GFP alone. This study underscores the potential of hemolymph proteins sourced from various Lepidoptera species as biotechnological tools to augment baculovirus vector productivities, whether utilized as natural supplements in cell culture media or as hemolymph-derived recombinant proteins co-expressed by baculovirus vectors.
Assuntos
Baculoviridae , Hemolinfa , Proteínas de Insetos , Proteínas Recombinantes , Animais , Hemolinfa/metabolismo , Proteínas Recombinantes/genética , Baculoviridae/genética , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Lepidópteros/virologia , Vetores Genéticos/genética , Linhagem Celular , Expressão Gênica , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Bombyx/genética , Bombyx/virologia , Bombyx/metabolismo , Meios de Cultura/química , Mariposas/virologia , Sobrevivência CelularRESUMO
The VP60 capsid protein from rabbit haemorrhagic disease virus (RHDV), the causative agent of one of the most economically important disease in rabbits worldwide, forms virus-like particles (VLPs) when expressed using heterologous protein expression systems such as recombinant baculovirus, yeasts, plants or mammalian cell cultures. To prevent RHDV dissemination, it would be beneficial to develop a bivalent vaccine including both RHDV GI.1- and RHDV GI.2-derived VLPs to achieve robust immunisation against both serotypes. In the present work, we developed a strategy of production of a dual-serving RHDV vaccine co-expressing the VP60 proteins from the two RHDV predominant serotypes using CrisBio technology, which uses Tricholusia ni insect pupae as natural bioreactors, which are programmed by recombinant baculovirus vectors. Co-infecting the insect pupae with two baculovirus vectors expressing the RHDV GI.1- and RHDV GI.2-derived VP60 proteins, we obtained chimeric VLPs incorporating both proteins as determined by using serotype-specific monoclonal antibodies. The resulting VLPs showed the typical size and shape of this calicivirus as determined by electron microscopy. Rabbits immunised with the chimeric VLPs were fully protected against a lethal challenge infection with the two RHDV serotypes. This study demonstrates that it is possible to generate a dual cost-effective vaccine against this virus using a single production and purification process, greatly simplifying vaccine manufacturing.
RESUMO
Bovine viral diarrhoea virus (BVDV) is a major cause of economic loss in the cattle industry, worldwide. Infection results in reduced productive performance, growth retardation, reduced milk production and increased susceptibility to other diseases leading to early culling of animals. There are two primary methods used to control the spread of BVDV: the elimination of persistently infected (PI) animals and vaccination. Currently, modified live or inactivated vaccines are used in BVDV vaccination programmes, but there are safety risks or insufficient protection, respectively, with these vaccines. Here, we report the development and efficacy of the first targeted subunit vaccine against BVDV. The core of the vaccine is the fusion of the BVDV structural protein, E2, to a single-chain antibody, APCH, together termed, APCH-E2. The APCH antibody targets the E2 antigen to the major histocompatibility type II molecule (MHC-II) present on antigen-presenting cells. Industrial production of the vaccine is carried out using the baculovirus expression vector system (BEVS) using single-use manufacturing technologies. This new subunit vaccine induces strong BVDV-specific neutralizing antibodies in guinea pigs and cattle. Importantly, in cattle with low levels of natural BVDV-specific neutralizing antibodies, the vaccine induced strong neutralizing antibody levels to above the protective threshold, as determined by a competition ELISA. The APCH-E2 vaccine induced a rapid and sustained neutralizing antibody response compared with a conventional vaccine in cattle.
Assuntos
Doença das Mucosas por Vírus da Diarreia Viral Bovina , Doenças dos Bovinos , Vírus da Diarreia Viral Bovina , Vacinas Virais , Animais , Anticorpos Antivirais , Doença das Mucosas por Vírus da Diarreia Viral Bovina/prevenção & controle , Bovinos , Cobaias , Vacinas de Subunidades AntigênicasRESUMO
The immunization of poultry where H5 and H7 influenza viruses (IVs) are endemic is one of the strategies to prevent unexpected zoonoses. Our group has been focused on conserved HA-epitopes as potential vaccine candidates to obtain multivalent immune responses against distinct IV subtypes. In this study, two conserved epitopes (NG-34 and CS-17) fused to flagellin were produced in a Baculovirus platform based on Trichoplusia ni larvae as living biofactories. Soluble extracts obtained from larvae expressing "flagellin-NG34/CS17 antigen" were used to immunize chickens and the efficacy of the vaccine was evaluated against a heterologous H7N1 HPAIV challenge in chickens. The flagellin-NG34/CS17 vaccine protected the vaccinated chickens and blocked viral shedding orally and cloacally. Furthermore, no apparent clinical signs were monitored in 10/12 vaccinated individuals. The mechanism of protection conferred is under investigation.
Assuntos
Flagelina/administração & dosagem , Granulovirus , Glicoproteínas de Hemaglutininação de Vírus da Influenza/administração & dosagem , Vírus da Influenza A Subtipo H7N1 , Influenza Aviária/prevenção & controle , Administração Intranasal , Sequência de Aminoácidos , Animais , Galinhas , Cães , Flagelina/imunologia , Granulovirus/imunologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Imunização/métodos , Vírus da Influenza A Subtipo H7N1/fisiologia , Vacinas contra Influenza/administração & dosagem , Vacinas contra Influenza/imunologia , Influenza Aviária/imunologia , Larva/imunologia , Células Madin Darby de Rim Canino , Zoonoses/imunologia , Zoonoses/prevenção & controleAssuntos
Varizes , Humanos , Ablação por Radiofrequência , Procedimentos Cirúrgicos Vasculares , VeiasRESUMO
The baculovirus vector expression system (BEVS) combines cultured insect cells and genetically modified Autographa californica nuclear polyhedrosis virus (AcMNPV)-derived baculovirus vectors. This expression system has been widely used for the expression of hundred of proteins for more than 30 years, existing commercial products manufactured at large scale by this methodology, mainly subunit vaccines. At an industrial scale, insect cells, as any other cultured cells, require artificial media and a strict control of environmental sterile conditions in the complex and expensive bioreactors. Here we describe an efficient alternative to produce recombinant biologics using the versatile and productive baculovirus vectors. It consists in natural biocapsules (pupae from Trichoplusia ni (Hübner) Lepidoptera), containing millions of insect cells in perfect physiological conditions, ready to be programmed by a genetically modified AcMNPV-derived baculovirus vector to produce large quantities of any recombinant protein. This technology, denominated CrisBio, has been tested to produce dozens of proteins, reaching productivities on the range of milligrams per infected pupa, that can be translated into dozens of vaccine doses, for example. The biologics production by CrisBio was industrialized with the design of both insect rearing and pupae storage single-use plastic devices, compatible with machines specifically designed for the automation of pupae manipulation and inoculation. These devices and machines reduce manual operations, increase batches consistency and facilitate the scaled production of any recombinant protein. As a mode of examples, the productivity in CrisBio technology platform of two virus-like particle (VLP) vaccine antigens is described in this work.
Assuntos
Mariposas , Nucleopoliedrovírus , Animais , Baculoviridae/genética , Nucleopoliedrovírus/genética , Pupa , Proteínas Recombinantes/genética , Vacinas de Subunidades Antigênicas/genéticaRESUMO
BACKGROUND: Identifying risk factors for complications or death associated with influenza remains crucial to target preventive interventions. Scores like the Charlson comorbidity index (CCI) may be of help. The aims of this study were to assess the effect of vaccination and comorbidities on severe influenza disease and influenza-related death among hospitalized patients during the season 2016/17; and to evaluate the validity of the CCI to predict death among these patients. METHODS: Data from adult patients (≥18 years old) with influenza infection admitted to La Paz University Hospital (LPUH) were recorded during the 2016/17 epidemic. The effect of influenza vaccine to prevent severe influenza or death was evaluated using multivariate logistic regression models. The area under the curve of the CCI and the age-adjusted CCI were compared to assess the predictive effect on mortality. RESULTS: A total of 342 adult patients with influenza infection were admitted, of which 83 developed severe influenza and 25 died during hospitalization. There were no differences between patients who survived and those who died concerning the CCI, but the age-adjusted CCI was higher in fatal cases (p-value=0.005). Influenza vaccine had no statistically significant effect on the risk of mortality (p-value=0.162) while age (OR: 1.12, p-value<0.001) and dementia (OR: 3.05, p-value=0.016) proved to be independent predictors for mortality. The seasonal vaccine was found to be protective for severe infection (OR: 0.54, p-value=0.019). The age-adjusted CCI was a better predictor of mortality than the crude CCI. CONCLUSIONS: Age and dementia are significant independent risk factors for mortality associated with influenza among hospitalized patients. The age-adjusted CCI seems to be a better predictor of mortality than the crude CCI. Influenza vaccine has shown to be effective in preventing severe influenza in the season 2016/17 among hospitalized patients and should be promoted in population at risk.
Assuntos
Influenza Humana/mortalidade , Influenza Humana/prevenção & controle , Vacinação/estatística & dados numéricos , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Comorbidade , Epidemias/estatística & dados numéricos , Feminino , Hospitalização/estatística & dados numéricos , Humanos , Vacinas contra Influenza/uso terapêutico , Modelos Logísticos , Masculino , Pessoa de Meia-Idade , Prognóstico , Medição de Risco , Fatores de Risco , Índice de Gravidade de Doença , Espanha/epidemiologia , Centros de Atenção TerciáriaRESUMO
The family Asfarviridae includes the single species African swine fever virus, isolates of which have linear dsDNA genomes of 170-194 kbp. Virions have an internal core, an internal lipid membrane, an icosahedral capsid and an outer lipid envelope. Infection of domestic pigs and wild boar results in an acute haemorrhagic fever with transmission by contact or ingestion, or by ticks of the genus Ornithodoros. Indigenous pigs act as reservoirs in Africa, where infection is endemic, and from where introductions occur periodically to Europe. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of the Asfarviridae, which is available at www.ictv.global/report/asfarviridae.
Assuntos
Asfarviridae/classificação , Asfarviridae/genética , África , Febre Suína Africana , Vírus da Febre Suína Africana , Animais , Doenças Endêmicas , Europa (Continente) , Genoma Viral , Sus scrofa/virologia , Suínos/virologia , VírionRESUMO
Trichoplusia ni insect larvae infected with vectors derived from the Autographa californica multiple nucleopolyhedrovirus (AcMNPV), are an excellent alternative to insect cells cultured in conventional bioreactors to produce recombinant proteins because productivity and cost-efficiency reasons. However, there is still a lot of work to do to reduce the manual procedures commonly required in this production platform that limit its scalability. To increase the scalability of this platform technology, a current bottleneck to be circumvented in the future is the need of injection for the inoculation of larvae with polyhedrin negative baculovirus vectors (Polh-) because of the lack of oral infectivity of these viruses, which are commonly used for production in insect cell cultures. In this work we have developed a straightforward alternative to obtain orally infective vectors derived from AcMNPV and expressing recombinant proteins that can be administered to the insect larvae (Trichoplusia ni) by feeding, formulated in the insect diet. The approach developed was based on the use of a recombinant polyhedrin protein expressed by a recombinant vector (Polh+), able to co-occlude any recombinant Polh- baculovirus vector expressing a recombinant protein. A second alternative was developed by the generation of a dual vector co-expressing the recombinant polyhedrin protein and the foreign gene of interest to obtain the occluded viruses. Additionally, by the incorporation of a reporter gene into the helper Polh+ vector, it was possible the follow-up visualization of the co-occluded viruses infection in insect larvae and will help to homogenize infection conditions. By using these methodologies, the production of recombinant proteins in per os infected larvae, without manual infection procedures, was very similar in yield to that obtained by manual injection of recombinant Polh- AcMNPV-based vectors expressing the same proteins. However, further analyses will be required for a detailed comparison of production yields reached by injection vs oral infections for different recombinant proteins. In conclusion, these results open the possibility of future industrial scaling-up production of recombinant proteins in insect larvae by reducing manual operations.
Assuntos
Vetores Genéticos , Biologia Molecular/métodos , Mariposas/genética , Mariposas/virologia , Nucleopoliedrovírus/genética , Proteínas Recombinantes/biossíntese , Animais , Larva/genética , Larva/metabolismo , Larva/virologia , Mariposas/metabolismo , Proteínas de Matriz de Corpos de Inclusão , Proteínas Recombinantes/isolamento & purificação , Células Sf9 , Proteínas Estruturais Virais/genéticaRESUMO
Current challenges in global immunization indicate the demand for new delivery strategies, which could be applied to the development of new vaccines against emerging diseases, as well as to improve safety and efficacy of currently existing vaccine formulations. Here, we report a novel antigen nanocarrier consisting of an oily core and a protamine shell, further stabilized with pegylated surfactants. These nanocarriers, named protamine nanocapsules, were rationally designed to promote the intracellular delivery of antigens to immunocompetent cells and to trigger an efficient and long-lasting immune response. Protamine nanocapsules have nanometric size, positive zeta potential and high association capacity for H1N1 influenza hemagglutinin, a protein that was used here as a model antigen. The new formulation shows an attractive stability profile both, as an aqueous suspension or a freeze-dried powder formulation. In vitro studies showed that protamine nanocapsules were efficiently internalized by macrophages without eliciting significant toxicity. In vivo studies indicate that antigen-loaded nanocapsules trigger immune responses comparable to those achieved with alum, even when using significantly lower antigen doses, thus indicating their adjuvant properties. These promising in vivo data, alongside with their versatility for the loading of different antigens and oily immunomodulators and their excellent stability profile, make these nanocapsules a promising platform for the delivery of antigens. CHEMICAL COMPOUNDS: Protamine sulphate (PubChem SID: 7849283), Sodium Cholate (PubChem CID: 23668194), Miglyol (PubChem CID: 53471835), α tocopherol (PubChem CID: 14985), Tween® 20(PubChem CID: 443314), Tween® 80(PubChem CID: 5281955), TPGS (PubChem CID: 71406).
Assuntos
Antígenos Virais/administração & dosagem , Portadores de Fármacos/administração & dosagem , Glicoproteínas de Hemaglutininação de Vírus da Influenza/administração & dosagem , Nanocápsulas/administração & dosagem , Protaminas/administração & dosagem , Animais , Antígenos Virais/química , Sobrevivência Celular/efeitos dos fármacos , Portadores de Fármacos/química , Feminino , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Imunização , Imunoglobulina G/sangue , Camundongos , Camundongos Endogâmicos BALB C , Nanocápsulas/química , Protaminas/química , Células RAW 264.7RESUMO
The baculovirus expression vector system (BEVS) has been widely used to produce a large number of recombinant proteins, and is becoming one of the most powerful, robust, and cost-effective systems for the production of eukaryotic proteins. Nevertheless, as in any other protein expression system, it is important to improve the production capabilities of this vector. The orf46 viral gene was identified among the most highly abundant sequences in the transcriptome of Spodoptera exigua larvae infected with its native baculovirus, the S. exigua multiple nucleopolyhedrovirus (SeMNPV). Different sequences upstream of the orf46 gene were cloned, and their promoter activities were tested by the expression of the GFP reporter gene using the Autographa californica nucleopolyhedrovirus (AcMNPV) vector system in different insect cell lines (Sf21, Se301, and Hi5) and in larvae from S. exigua and Trichoplusia ni. The strongest promoter activity was defined by a 120 nt sequence upstream of the ATG start codon for the orf46 gene. On average, GFP expression under this new promoter was more than two fold higher than the expression obtained with the standard polyhedrin (polh) promoter. Additionally, the orf46 promoter was also tested in combination with the polh promoter, revealing an additive effect over the polh promoter activity. In conclusion, this new characterized promoter represents an excellent alternative to the most commonly used baculovirus promoters for the efficient expression of recombinant proteins using the BEVS.
RESUMO
Vaccines based on virus-like particles (VLPs) have proven effective in humans and animals. In this regard, the baculovirus expression vector system (BEVS) is one of the technologies of choice to generate such highly immunogenic vaccines. The extended use of these vaccines for human and animal populations is constrained because of high production costs, therefore a significant improvement in productivity is crucial to ensure their commercial viability. Here we describe the use of the previously described baculovirus expression cassette, called TB, to model the production of two VLP-forming vaccine antigens in insect cells. Capsid proteins from porcine circovirus type 2 (PCV2 Cap) and from the calicivirus that causes rabbit hemorrhagic disease (RHDV VP60) were expressed in insect cells using baculoviruses genetically engineered with the TB expression cassette. Productivity was compared to that obtained using standard counterpart vectors expressing the same proteins under the control of the polyhedrin promoter. Our results demonstrate that the use of the TB expression cassette increased the production yields of these vaccine antigens by around 300% with respect to the standard vectors. The recombinant proteins produced by TB-modified vectors were fully functional, forming VLPs identical in size and shape to those generated by the standard baculoviruses, as determined by electron microscopy analysis. The use of the TB expression cassette implies a simple modification of the baculovirus vectors that significantly improves the cost efficiency of VLP-based vaccine production, thereby facilitating the commercial viability and broad application of these vaccines for human and animal health.
Assuntos
Baculoviridae/genética , Baculoviridae/imunologia , Vetores Genéticos/biossíntese , Vacinas de Partículas Semelhantes a Vírus/biossíntese , Animais , Linhagem Celular , Análise Custo-Benefício , Vetores Genéticos/imunologia , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Regiões Promotoras Genéticas , Coelhos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Spodoptera/citologia , Suínos , Vacinas de Partículas Semelhantes a Vírus/genéticaRESUMO
Bovine viral diarrhea virus (BVDV) is an important cause of economic losses worldwide. E2 is an immunodominant protein and a promising candidate to develop subunit vaccines. To improve its immunogenicity, a truncated E2 (tE2) was fused to a single chain antibody named APCH, which targets to antigen-presenting cells. APCH-tE2 and tE2 proteins were expressed in the baculovirus system and their immunogenicity was firstly compared in guinea pigs. APCH-tE2 vaccine was the best one to evoke a humoral response, and for this reason, it was selected for a cattle vaccination experiment. All the bovines immunized with 1.5Ag of APCH-tE2 developed high levels of neutralizing antibodies against BVDV up to a year post-immunization, demonstrating its significant potential as a subunit vaccine. This novel vaccine is undergoing scale-up and was transferred to the private sector. Nowadays, it is being evaluated for registration as the first Argentinean subunit vaccine for cattle
El virus de la diarrea viral bovina (BVDV) es causante de importantes pérdidas económicas a nivel mundial. La proteína E2 es la inmunodominante del virus y es la candidata para desarrollar vacunas de subunidad. Para mejorar su inmunogenicidad, una versión truncada de la E2 (tE2) se fusionó a un anticuerpo de cadena simple (APCH), que se dirige a las células presentadoras de antígeno. Se expresaron las proteínas APCH-tE2 y tE2 en el sistema de baculovirus y su inmunogenicidad fue evaluada y comparada en cobayos; la proteína APCH-tE2 fue la que indujo la mejor respuesta humoral. Por dicha razón se la evaluó en bovinos utilizando 1,5µg de antígeno. Los animales presentaron altos títulos de anticuerpos neutralizantes contra BVDV hasta un año posinmunización. Esta nueva vacuna está en proceso de escalado y se transfirió al sector privado. Actualmente se está evaluando para su registro como la primera vacuna argentina de subunidad para bovinos
Assuntos
Animais , Bovinos , Cobaias , Vírus da Diarreia Viral Bovina/imunologia , Vacinas de Subunidades Antigênicas/biossíntese , Células Apresentadoras de Antígenos/efeitos dos fármacos , Baculoviridae/imunologia , Imunização/veterinária , Proteínas E2 de Adenovirus/imunologia , Vírus da Diarreia Viral Bovina/efeitos dos fármacos , Anticorpos Neutralizantes/análiseRESUMO
Bovine viral diarrhea virus (BVDV) is an important cause of economic losses worldwide. E2 is an immunodominant protein and a promising candidate to develop subunit vaccines. To improve its immunogenicity, a truncated E2 (tE2) was fused to a single chain antibody named APCH, which targets to antigen-presenting cells. APCH-tE2 and tE2 proteins were expressed in the baculovirus system and their immunogenicity was firstly compared in guinea pigs. APCH-tE2 vaccine was the best one to evoke a humoral response, and for this reason, it was selected for a cattle vaccination experiment. All the bovines immunized with 1.5 µg of APCH-tE2 developed high levels of neutralizing antibodies against BVDV up to a year post-immunization, demonstrating its significant potential as a subunit vaccine. This novel vaccine is undergoing scale-up and was transferred to the private sector. Nowadays, it is being evaluated for registration as the first Argentinean subunit vaccine for cattle.
Assuntos
Células Apresentadoras de Antígenos/imunologia , Doenças dos Bovinos/prevenção & controle , Doenças dos Bovinos/virologia , Diarreia/veterinária , Glicoproteínas/imunologia , Anticorpos de Cadeia Única/imunologia , Vacinas de Subunidades Antigênicas , Animais , Bovinos , Diarreia/prevenção & controle , Diarreia/virologia , CobaiasRESUMO
Growth factors (GFs) are naturally signalling proteins, which bind to specific receptors on the cell surface. Numerous families of GFs have already been identified and remarkable progresses have been made in understanding the pathways that these proteins use to activate/regulate the complex signalling network involved in cell proliferation or wound healing processes. The bottleneck for a wider clinical and commercial application of these factors relay on their scalable cost-efficient production as bioactive molecules. The present work describes the capacity of Trichoplusia ni insect larvae used as living bioreactors in combination with the baculovirus vector expression system to produce three fully functional human GFs, the human epidermal growth factor (huEGF), the human fibroblast growth factor 2 (huFGF2) and the human keratinocyte growth factor 1 (huKGF1). The expression levels obtained per g of insect biomass were of 9.1, 2.6 and 3mg for huEGF, huFGF2 and huKGF1, respectively. Attempts to increase the productivity of the insect/baculovirus system we have used different modifications to optimize their production. Additionally, recombinant proteins were expressed fused to different tags to facilitate their purification. Interestingly, the expression of huKGF1 was significantly improved when expressed fused to the fragment crystallizable region (Fc) of the human antibody IgG. The insect-derived recombinant GFs were finally characterized in terms of biological activity in keratinocytes and fibroblasts. The present work opens the possibility of a cost-efficient and scalable production of these highly valuable molecules in a system that favours its wide use in therapeutic or cosmetic applications.
Assuntos
Fator de Crescimento Epidérmico/biossíntese , Fator 2 de Crescimento de Fibroblastos/biossíntese , Fator 7 de Crescimento de Fibroblastos/biossíntese , Mariposas/genética , Animais , Reatores Biológicos , Fator de Crescimento Epidérmico/genética , Fator 2 de Crescimento de Fibroblastos/genética , Fator 7 de Crescimento de Fibroblastos/genética , Expressão Gênica , Humanos , Larva/genética , Larva/metabolismo , Mariposas/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genéticaRESUMO
Here we describe the development of a baculovirus vector expression cassette containing rearranged baculovirus-derived genetic regulatory elements. This newly designed expression cassette conferred significant production improvements to the baculovirus expression vector system (BEVS), including prolonged cell integrity after infection, improved protein integrity, and around 4-fold increase in recombinant protein production yields in insect cells with respect to a standard baculovirus vector. The expression cassette consisted of a cDNA encoding for the baculovirus transactivation factors IE1 and IE0, expressed under the control of the polyhedrin promoter, and a homologous repeated transcription enhancer sequence operatively cis-linked to the p10 promoter or to chimeric promoters containing p10. The prolonged cell integrity observed in cells infected by baculoviruses harbouring the novel expression cassette reduced the characteristic proteolysis and aberrant forms frequently found in baculovirus-derived recombinant proteins. The new expression cassette developed here has the potential to significantly improve the productivity of the BEVS.
Assuntos
Baculoviridae/genética , Vetores Genéticos , Regiões Promotoras Genéticas , Proteínas Recombinantes/genética , Animais , Linhagem Celular , Expressão Gênica , Insetos/genéticaRESUMO
AIM: To develop a new core-shell type (nanocapsules) adjuvant system composed of squalene and polyglucosamine (PG) and to evaluate its immunostimulant capacity. RESULTS: The defined PG nanocapsules exhibited the capacity to efficiently associate the selected antigens (recombinant hepatitis B surface antigen and hemagglutinin of influenza virus) onto their polymeric surface (70-75%), and the immunostimulant imiquimod within the oily core. The resulting nanovaccines, with a particle size of 200-250 nm and a positive zeta-potential (â¼+60 mV), were able to significantly potentiate and modulate the immune response to the selected antigens upon intramuscular administration to mice. Their efficacy as novel adjuvants was attributed to their enhanced cell internalization and effective intracellular imiquimod/antigen delivery, together with their prolonged residence time at the injection site. CONCLUSION: The nanocapsules described herein have the capacity to enhance, prolong and modulate the immune response of subunit antigens and, therefore, they could be proposed as a platform for the codelivery of different antigens and immunostimulators.
Assuntos
Adjuvantes Imunológicos/administração & dosagem , Nanocápsulas , Animais , Anticorpos/sangue , Ensaio de Imunoadsorção Enzimática , Feminino , Camundongos , Camundongos Endogâmicos BALB CRESUMO
West Nile virus (WNV) is maintained in nature in an enzootic transmission cycle between birds and mosquitoes, although it occasionally infects other vertebrates, including humans, in which it may result fatal. To date, no licensed vaccines against WNV infection are available for birds, but its availability would certainly benefit certain populations, as birds grown for restocking, hunting activities, or alimentary purposes, and those confined to wildlife reservations and recreation installations. We have tested the protective capability of WNV envelope recombinant (rE) protein in red-legged partridges (Alectoris rufa). Birds (n=28) were intramuscularly immunized three times at 2-weeks interval with rE and a control group (n=29) was sham-immunized. Except for 5 sham-immunized birds that were not infected and housed as contact controls, partridges were subcutaneously challenged with WNV. Oropharyngeal and cloacal swabs and feather pulps were collected at several days after infection and blood samples were taken during vaccination and after infection. All rE-vaccinated partridges elicited anti-WNV antibodies before challenge and survived to the infection, while 33.3% of the sham-immunized birds succumbed, as did 25% of the contact animals. Most (84%) unvaccinated birds showed viremia 3 d.p.i., but virus was only detected in 14% of the rE vaccinated birds. WNV-RNA was detected in feathers and swabs from sham-immunized partridges from 3 to 7 d.p.i., mainly in birds that succumbed to the infection, but not in rE vaccinated birds. Thus, rE vaccination fully protected partridges against WND and reduced the risk of virus spread.