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The growing requirements regarding the safety of using polymers and their composites are related to the emergence of more effective, sustainable, and hazardous-limited fire retardants (FRs). Significant amounts of FRs are usually required to effectively affect a polymer's burning behavior, while the knowledge of their recycling potential is still insufficient. At the same time, concerns are related not only to the reduced effectiveness of flame retardancy but also, above all, to the potential deterioration of mechanical properties caused by the degradation of temperature-affected additives under processing conditions. This study describes the impact of the four-time reprocessing of bio-based polyamide 11 (PA11) modified with an intumescent flame-retardant (IFR) system composed of ammonium polyphosphate (APP), melamine cyanurate (MC), and pentaerythritol (PER) and its composites containing additional short basalt fibers (BFs). Composites manufactured via twin-screw extrusion were subjected to four reprocessing cycles using injection molding. A comprehensive analysis of their structural, mechanical, and fire behavior changes in each cycle was conducted. The obtained results confirmed the safety of using the proposed fire-retarded polyamide and its composites while reprocessing under the recommended process parameters without the risk of significant changes in the structure. The partial increase in flammability of reprocessed PA-based materials caused mainly by polymer degradation has been described.
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After its emergence in late 2019 SARS-CoV-2 was declared a pandemic by the World Health Organization on 11 March 2020 and has claimed more than 2.8 million lives. There has been a massive global effort to develop vaccines against SARS-CoV-2 and the rapid and low cost production of large quantities of vaccine is urgently needed to ensure adequate supply to both developed and developing countries. Virus-like particles (VLPs) are composed of viral antigens that self-assemble into structures that mimic the structure of native viruses but lack the viral genome. Thus they are not only a safer alternative to attenuated or inactivated vaccines but are also able to induce potent cellular and humoral immune responses and can be manufactured recombinantly in expression systems that do not require viral replication. VLPs have successfully been produced in bacteria, yeast, insect and mammalian cell cultures, each production platform with its own advantages and limitations. Plants offer a number of advantages in one production platform, including proper eukaryotic protein modification and assembly, increased safety, low cost, high scalability as well as rapid production speed, a critical factor needed to control outbreaks of potential pandemics. Plant-based VLP-based viral vaccines currently in clinical trials include, amongst others, Hepatitis B virus, Influenza virus and SARS-CoV-2 vaccines. Here we discuss the importance of plants as a next generation expression system for the fast, scalable and low cost production of VLP-based vaccines.
Assuntos
Vacinas contra COVID-19/biossíntese , Plantas Geneticamente Modificadas/metabolismo , SARS-CoV-2/imunologia , Vacinas de Partículas Semelhantes a Vírus/biossíntese , Antígenos Virais/genética , Antígenos Virais/metabolismo , Vacinas contra COVID-19/economia , Vacinas contra COVID-19/genética , Expressão Gênica , Plantas Geneticamente Modificadas/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Vacinas de Partículas Semelhantes a Vírus/economia , Vacinas de Partículas Semelhantes a Vírus/genética , Vacinas Virais/biossíntese , Vacinas Virais/genéticaRESUMO
BACKGROUND: African horse sickness (AHS) is a severe arthropod-borne viral disease of equids, with a mortality rate of up to 95% in susceptible naïve horses. Due to safety concerns with the current live, attenuated AHS vaccine, alternate safe and effective vaccination strategies such as virus-like particles (VLPs) are being investigated. Transient plant-based expression systems are a rapid and highly scalable means of producing such African horse sickness virus (AHSV) VLPs for vaccine purposes. RESULTS: In this study, we demonstrated that transient co-expression of the four AHSV capsid proteins in agroinfiltrated Nicotiana benthamiana dXT/FT plants not only allowed for the assembly of homogenous AHSV-1 VLPs but also single, double and triple chimeric VLPs, where one capsid protein originated from one AHS serotype and at least one other capsid protein originated from another AHS serotype. Following optimisation of a large scale VLP purification procedure, the safety and immunogenicity of the plant-produced, triple chimeric AHSV-6 VLPs was confirmed in horses, the target species. CONCLUSIONS: We have successfully shown assembly of single and double chimeric AHSV-7 VLPs, as well as triple chimeric AHSV-6 VLPs, in Nicotiana benthamiana dXT/FT plants. Plant produced chimeric AHSV-6 VLPs were found to be safe for administration into 6 month old foals as well as capable of eliciting a weak neutralizing humoral immune response in these target animals against homologous AHSV virus.
Assuntos
Vírus da Doença Equina Africana/imunologia , Doença Equina Africana/prevenção & controle , Proteínas do Capsídeo/imunologia , Nicotiana/metabolismo , Vacinas Virais , Animais , Anticorpos Neutralizantes/imunologia , Proteínas do Capsídeo/metabolismo , Regulação da Expressão Gênica de Plantas , Cavalos , Plantas Geneticamente Modificadas , Proteínas Recombinantes de Fusão , Proteínas Recombinantes , Vacinas Atenuadas , Vacinas de Partículas Semelhantes a VírusRESUMO
African horse sickness (AHS) is caused by multiple serotypes of the dsRNA AHSV and is a major scourge of domestic equids in Africa. While there are well established commercial live attenuated vaccines produced in South Africa, risks associated with these have encouraged attempts to develop new and safer recombinant vaccines. Previously, we reported on the immunogenicity of a plant-produced AHS serotype 5 virus-like particle (VLP) vaccine, which stimulated high titres of AHS serotype 5-specific neutralizing antibodies in guinea pigs. Here, we report a similar response to the vaccine in horses. This is the first report demonstrating the safety and immunogenicity of plant-produced AHS VLPs in horses.
Assuntos
Vírus da Doença Equina Africana , Doença Equina Africana/prevenção & controle , Anticorpos Antivirais/imunologia , Nicotiana/metabolismo , Vacinas Virais/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Cavalos , Vacinas Atenuadas/imunologiaRESUMO
The bulk of the major core protein VP7 in African horse sickness virus (AHSV) self-assembles into flat, hexagonal crystalline particles in a process appearing unrelated to viral replication. Why this unique characteristic of AHSV VP7 is genetically conserved, and whether VP7 aggregation and particle formation have an effect on cellular biology or the viral life cycle, is unknown. Here we investigated how different small peptide and enhanced green fluorescent protein (eGFP) insertions into the VP7 top domain affected VP7 localization, aggregation, and particle formation. This was done using a dual laser scanning confocal and transmission electron microscopy approach in conjunction with analyses of the solubility, aggregation, and fluorescence profiles of the proteins. VP7 top domain modifications did not prevent trimerization, or intracellular trafficking, to one or two discrete sites in the cell. However, modifications that resulted in a misfolded and insoluble VP7-eGFP component blocked trafficking, and precluded protein accumulation at a single cellular site, perhaps by interfering with normal trimer-trimer interactions. Furthermore, the modifications disrupted the stable layering of the trimers into characteristic AHSV VP7 crystalline particles. It was concluded that VP7 trafficking is driven by a balance between VP7 solubility, trimer forming ability, and trimer-trimer interactions.
Assuntos
Vírus da Doença Equina Africana/metabolismo , Microscopia Confocal/métodos , Microscopia Eletrônica de Transmissão/métodos , Proteínas do Core Viral/fisiologia , Proteínas do Core Viral/ultraestrutura , Vírus da Doença Equina Africana/genética , Animais , Baculoviridae/genética , Regulação Viral da Expressão Gênica , Vetores Genéticos , Proteínas de Fluorescência Verde , Estágios do Ciclo de Vida , Transporte Proteico , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/ultraestrutura , Células Sf9 , Proteínas do Core Viral/química , Proteínas do Core Viral/genética , Proteínas Virais de Fusão/fisiologia , Proteínas Virais de Fusão/ultraestrutura , Replicação ViralRESUMO
A safe, highly immunogenic multivalent vaccine to protect against all nine serotypes of African horse sickness virus (AHSV), will revolutionise the AHS vaccine industry in endemic countries and beyond. Plant-produced AHS virus-like particles (VLPs) and soluble viral protein 2 (VP2) vaccine candidates were developed that have the potential to protect against all nine serotypes but can equally well be formulated as mono- and bi-valent formulations for localised outbreaks of specific serotypes. In the first interferon α/ß receptor knock-out (IFNAR-/-) mice trial conducted, a nine-serotype (nonavalent) vaccine administered as two pentavalent (5 µg per serotype) vaccines (VLP/VP2 combination or exclusively VP2), were directly compared to the commercially available AHS live attenuated vaccine. In a follow up trial, mice were vaccinated with an adjuvanted nine-serotype multivalent VP2 vaccine in a prime boost strategy and resulted in the desired neutralising antibody titres of 1:320, previously demonstrated to confer protective immunity in IFNAR-/- mice. In addition, the plant-produced VP2 vaccine performed favourably when compared to the commercial vaccine. Here we provide compelling data for a nonavalent VP2-based vaccine candidate, with the VP2 from each serotype being antigenically distinguishable based on LC-MS/MS and ELISA data. This is the first preclinical trial demonstrating the ability of an adjuvanted nonavalent cocktail of soluble, plant-expressed AHS VP2 proteins administered in a prime-boost strategy eliciting high antibody titres against all 9 AHSV serotypes. Furthermore, elevated T helper cells 2 (Th2) and Th1, indicative of humoral and cell-mediated memory T cell immune responses, respectively, were detected in mouse serum collected 14 days after the multivalent prime-boost vaccination. Both Th2 and Th1 may play a role to confer protective immunity. These preclinical immunogenicity studies paved the way to test the safety and protective efficacy of the plant-produced nonavalent VP2 vaccine candidate in the target animals, horses.
Assuntos
Vírus da Doença Equina Africana , Doença Equina Africana , Vacinas Virais , Animais , Camundongos , Cavalos , Vírus da Doença Equina Africana/genética , Doença Equina Africana/prevenção & controle , Vacinas Combinadas , Cromatografia Líquida , Proteínas do Capsídeo , Espectrometria de Massas em Tandem , Anticorpos AntiviraisRESUMO
Brucellosis is an important bacterial disease of livestock and the most common zoonotic disease. The current vaccines are effective but unsafe, as they result in animal abortions and are pathogenic to humans. Virus-like particles are being investigated as molecular scaffolds for foreign antigen presentation to the immune system. Here, we sought to develop a new-generation vaccine by presenting selected Brucella melitensis T cell epitopes on the surface of Orbivirus core-like particles (CLPs) and transiently expressing these chimeric particles in Nicotiana benthamiana plants. We successfully demonstrated the assembly of five chimeric CLPs in N. benthamiana plants, with each CLP presenting a different T cell epitope. The safety and protective efficacy of three of the highest-yielding CLPs was investigated in a mouse model of brucellosis. All three plant-expressed chimeric CLPs were safe when inoculated into BALB/c mice at specific antigen doses. However, only one chimeric CLP induced protection against the virulent Brucella strain challenge equivalent to the protection induced by the commercial Rev1 vaccine. Here, we have successfully shown the assembly, safety and protective efficacy of plant-expressed chimeric CLPs presenting B. melitensis T cell epitopes. This is the first step in the development of a safe and efficacious subunit vaccine against brucellosis.
RESUMO
Next generation vaccines have the capability to contribute to and revolutionise the veterinary vaccine industry. African horse sickness (AHS) is caused by an arbovirus infection and is characterised by respiratory distress and/or cardiovascular failure and is lethal to horses. Mandatory annual vaccination in endemic areas curtails disease occurrence and severity. However, development of a next generation AHSV vaccine, which is both safe and efficacious, has been an objective globally for years. In this study, both AHSV serotype 5 chimaeric virus-like particles (VLPs) and soluble viral protein 2 (VP2) were successfully produced in Nicotiana benthamiana ΔXT/FT plants, partially purified and validated by gel electrophoresis, transmission electron microscopy and liquid chromatography-mass spectrometry (LC-MS/MS) based peptide sequencing before vaccine formulation. IFNAR-/- mice vaccinated with the adjuvanted VLPs or VP2 antigens in a 10 µg prime-boost regime resulted in high titres of antibodies confirmed by both serum neutralising tests (SNTs) and enzyme-linked immunosorbent assays (ELISA). Although previous studies reported high titres of antibodies in horses when vaccinated with plant-produced AHS homogenous VLPs, this is the first study demonstrating the protective efficacy of both AHSV serotype 5 chimaeric VLPs and soluble AHSV-5 VP2 as vaccine candidates. Complementary to this, coating ELISA plates with the soluble VP2 has the potential to underpin serotype-specific serological assays.
Assuntos
Vírus da Doença Equina Africana , Doença Equina Africana , Vacinas Virais , Animais , Anticorpos Neutralizantes , Anticorpos Antivirais , Proteínas do Capsídeo , Cromatografia Líquida , Cavalos , Camundongos , Sorogrupo , Espectrometria de Massas em Tandem , Proteínas ViraisRESUMO
Virus-like particles (VLPs) are a class of structures formed by the self-assembly of viral capsid protein subunits and contain no infective viral genetic material. The Hepatitis B core (HBc) antigen is capable of assembling into VLPs that can elicit strong immune responses and has been licensed as a commercial vaccine against Hepatitis B. The HBc VLPs have also been employed as a platform for the presentation of foreign epitopes to the immune system and have been used to develop vaccines against, for example, influenza A and Foot-and-mouth disease. Plant expression systems are rapid, scalable and safe, and are capable of providing correct post-translational modifications and reducing upstream production costs. The production of HBc-based virus-like particles in plants would thus greatly increase the efficiency of vaccine production. This review investigates the application of plant-based HBc VLP as a platform for vaccine production.
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Bluetongue (BT) is a hemorrhagic non-contagious, biting midge-transmitted disease of wild and domestic ruminants that is caused by bluetongue virus (BTV). Annual vaccination plays a pivotal role in BT disease control in endemic regions. Due to safety concerns of the current BTV multivalent live attenuated vaccine (LAV), a safe efficacious new generation subunit vaccine such as a plant-produced BT virus-like particle (VLP) vaccine is imperative. Previously, homogenous BTV serotype 8 (BTV-8) VLPs were successfully produced in Nicotiana benthamiana plants and provided protective immunity in sheep. In this study, combinations of BTV capsid proteins from more than one serotype were expressed and assembled to form chimaeric BTV-3 and BTV-4 VLPs in N. benthamiana plants. The assembled homogenous BTV-8, as well as chimaeric BTV-3 and chimaeric BTV-4 VLP serotypes, were confirmed by SDS-PAGE, Transmission Electron microscopy (TEM) and protein confirmation using liquid chromatography-mass spectrometry (LC-MS/MS) based peptide sequencing. As VP2 is the major determinant eliciting protective immunity, the percentage coverage and number of unique VP2 peptides detected in assembled chimaeric BT VLPs were used as a guide to assemble the most appropriate chimaeric combinations. Both plant-produced chimaeric BTV-3 and BTV-4 VLPs were able to induce long-lasting serotype-specific neutralizing antibodies equivalent to the monovalent LAV controls. Antibody levels remained high to the end of the trial. Combinations of homogenous and chimaeric BT VLPs have great potential as a safe, effective multivalent vaccine with the ability to distinguish between vaccinated and infected individuals (DIVA) due to the absence of non-structural proteins.
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Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Vírus Bluetongue/imunologia , Bluetongue/prevenção & controle , Ovinos/imunologia , Vacinação/veterinária , Animais , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/imunologia , Nicotiana/virologia , Vacinas Atenuadas/imunologia , Vacinas de Partículas Semelhantes a Vírus/imunologia , Vacinas Virais/imunologiaRESUMO
We have investigated the use of soluble chimeric trimers of the major capsid protein VP7 of African horse sickness virus (AHSV) as a vaccine delivery system by targeting some of the natural hydrophilic loops on the VP7 top domain for the insertion of foreign peptides. Key to this trimer display strategy is the solubility of AHSV VP7 and how the solubility of this hydrophobic protein can be manipulated by inserting peptides into the top domain. To investigate, we generated different cloning vectors by inserting multiple cloning sites at three different positions in the VP7 gene. These modifications inserted six amino acids at the cloning sites and in some cases this converted VP7 to a largely soluble protein without affecting the ability of the modified proteins to form trimers. The vectors were used to generate a number of soluble VP7 fusion proteins including a fusion with a 36 amino acid insert that overlaps important immunological domains on protein VP1 of foot and mouth disease virus (FMDV) as well as a 110 amino acid peptide derived from AHSV VP2. Soluble trimers of these fusion proteins were able to elicit a good insert-specific immune response in guinea pigs. l-Arginine was found to reverse protein aggregation and was employed as an effective strategy to isolate relatively pure soluble chimeric VP7 trimers. Another factor that increased VP7 solubility in both wild-type VP7 and one of the VP7 vector proteins was the substitution of the leucine residue in position 345 of the VP7 C-terminus with a hydrophilic arginine residue.