RESUMO
Antibody-based therapies are a promising treatment option for managing ebolavirus infections. Several Ebola virus (EBOV)-specific and, more recently, pan-ebolavirus antibody cocktails have been described. Here, we report the development and assessment of a Sudan virus (SUDV)-specific antibody cocktail. We produced a panel of SUDV glycoprotein (GP)-specific human chimeric monoclonal antibodies (mAbs) using both plant and mammalian expression systems and completed head-to-head in vitro and in vivo evaluations. Neutralizing activity, competitive binding groups, and epitope specificity of SUDV mAbs were defined before assessing protective efficacy of individual mAbs using a mouse model of SUDV infection. Of the mAbs tested, GP base-binding mAbs were more potent neutralizers and more protective than glycan cap- or mucin-like domain-binding mAbs. No significant difference was observed between plant and mammalian mAbs in any of our in vitro or in vivo evaluations. Based on in vitro and rodent testing, a combination of two SUDV-specific mAbs, one base binding (16F6) and one glycan cap binding (X10H2), was down-selected for assessment in a macaque model of SUDV infection. This cocktail, RIID F6-H2, provided protection from SUDV infection in rhesus macaques when administered at 50 mg/kg on days 4 and 6 postinfection. RIID F6-H2 is an effective postexposure SUDV therapy and provides a potential treatment option for managing human SUDV infection.
Assuntos
Anticorpos Antivirais/administração & dosagem , Ebolavirus/imunologia , Doença pelo Vírus Ebola/tratamento farmacológico , Animais , Anticorpos Monoclonais/administração & dosagem , Modelos Animais de Doenças , Ebolavirus/genética , Feminino , Glicoproteínas/imunologia , Doença pelo Vírus Ebola/virologia , Humanos , Imunoterapia , Macaca mulatta , Masculino , Camundongos , Proteínas Virais/imunologiaRESUMO
The potency of human and veterinary rabies vaccines is measured based on the National Institute of Health (NIH) potency test that is laborious, time-consuming, variable, and requires sacrifice of large numbers of mice. ELISA-based methods quantifying rabies glycoprotein (rGP) are being developed as potential alternatives to the NIH potency test for release of rabies vaccines. The aim of the current study was focused on the evaluation of in vitro- and in vivo-based assays in order to assess their concurrence for adequate and reliable assessment of immunogenicity and protective potency of a plant-derived recombinant rGP. The recombinant rGP of strain ERA.KK was engineered, expressed and purified from Nicotiana benthamiana plants. The recombinant rGP excluded the transmembrane and intracytoplasmic domains. It was purified by chromatography (≥90%) from the plant biomass, characterized, and mainly presented as high molecular weight forms, most likely soluble aggregates, of the rGP ectodomain. It was well-recognized and quantified by an ELISA, which utilizes two mouse monoclonal antibodies, D1-25 and 1112-1, and which should only recognize the native trimeric form of the rGP. However, in mice, the recombinant rGP did not induce the production of anti-rabies virus neutralizing antibodies and did not confer protection after intracerebral viral challenge. Similar immunogenicity was observed in guinea pigs and rabbits. Our results demonstrate that use of the ELISA method described here is not predictive of performance in vivo. These data highlight the critical need to develop in vitro potency assays that reliably define the antigen content that can induce a protective response.
Assuntos
Vacina Antirrábica , Raiva , Animais , Anticorpos Antivirais , Ensaio de Imunoadsorção Enzimática/métodos , Glicoproteínas/genética , Cobaias , Camundongos , Coelhos , Raiva/prevenção & controle , Vacina Antirrábica/química , Proteínas RecombinantesRESUMO
There are no FDA licensed vaccines or therapeutics for Venezuelan equine encephalitis virus (VEEV) which causes a debilitating acute febrile illness in humans that can progress to encephalitis. Previous studies demonstrated that murine and macaque monoclonal antibodies (mAbs) provide prophylactic and therapeutic efficacy against VEEV peripheral and aerosol challenge in mice. Additionally, humanized versions of two neutralizing mAbs specific for the E2 glycoprotein, 1A3B-7 and 1A4A-1, administered singly protected mice against aerosolized VEEV. However, no studies have demonstrated protection in nonhuman primate (NHP) models of VEEV infection. Here, we evaluated a chimeric antibody 1A3B-7 (c1A3B-7) containing mouse variable regions on a human IgG framework and a humanized antibody 1A4A-1 containing a serum half-life extension modification (Hu-1A4A-1-YTE) for their post-exposure efficacy in NHPs exposed to aerosolized VEEV. Approximately 24 hours after exposure, NHPs were administered a single bolus intravenous mAb. Control NHPs had typical biomarkers of VEEV infection including measurable viremia, fever, and lymphopenia. In contrast, c1A3B-7 treated NHPs had significant reductions in viremia and lymphopenia and on average approximately 50% reduction in fever. Although not statistically significant, Hu-1A4A-1-YTE administration did result in reductions in viremia and fever duration. Delay of treatment with c1A3B-7 to 48 hours post-exposure still provided NHPs protection from severe VEE disease through reductions in viremia and fever. These results demonstrate that post-exposure administration of c1A3B-7 protected macaques from development of severe VEE disease even when administered 48 hours following aerosol exposure and describe the first evaluations of VEEV-specific mAbs for post-exposure prophylactic use in NHPs. Viral mutations were identified in one NHP after c1A3B-7 treatment administered 24 hrs after virus exposure. This suggests that a cocktail-based therapy, or an alternative mAb against an epitope that cannot mutate without resulting in loss of viral fitness may be necessary for a highly effective therapeutic.
Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Anticorpos Neutralizantes/farmacologia , Encefalomielite Equina Venezuelana/imunologia , Vacinas Virais/farmacologia , Animais , Anticorpos Monoclonais Humanizados/imunologia , Anticorpos Neutralizantes/imunologia , Modelos Animais de Doenças , Encefalomielite Equina Venezuelana/prevenção & controle , Humanos , Macaca fascicularis , Vacinas Virais/imunologiaRESUMO
MAIN CONCLUSION : A RhoA-derived peptide fused to carrier molecules from plants showed enhanced biological activity of in vitro assays against respiratory syncytial virus compared to the RhoA peptide alone or the synthetic RhoA peptide. A RhoA-derived peptide has been reported for over a decade as a potential inhibitor of respiratory syncytial virus (RSV) infection both in vitro and in vivo and is anticipated to be a promising alternative to monoclonal antibody-based therapy against RSV infection. However, there are several challenges to furthering development of this antiviral peptide, including improvement in the peptide's bioavailability, development of an efficient delivery system and identification of a cost-effective production platform. In this study, we have engineered a RhoA peptide as a genetic fusion to two carrier molecules, either lichenase (LicKM) or the coat protein (CP) of Alfalfa mosaic virus. These constructs were introduced into Nicotiana benthamiana plants using a tobacco mosaic virus-based expression vector and targets purified. The results demonstrated that the RhoA peptide fusion proteins were efficiently expressed in N. benthamiana plants, and that two of the resulting fusion proteins, RhoA-LicKM and RhoA2-FL-d25CP, inhibited RSV growth in vitro by 50 and 80 %, respectively. These data indicate the feasibility of transient expression of this biologically active antiviral RhoA peptide in plants and the advantage of using a carrier molecule to enhance target expression and efficacy.
Assuntos
Proteínas de Plantas/farmacologia , Proteínas Recombinantes de Fusão/farmacologia , Vírus Sinciciais Respiratórios/efeitos dos fármacos , Proteína rhoA de Ligação ao GTP/farmacologia , Vetores Genéticos , Testes de Sensibilidade Microbiana , Proteínas de Plantas/química , Proteínas de Plantas/genética , Engenharia de Proteínas , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Nicotiana/genética , Nicotiana/metabolismo , Nicotiana/virologia , Vírus do Mosaico do Tabaco/genética , Proteína rhoA de Ligação ao GTP/química , Proteína rhoA de Ligação ao GTP/genéticaRESUMO
Human erythropoietin (hEPO) is one of the most in-demand biopharmaceuticals, however, its production is challenging. When produced in a plant expression system, hEPO results in extensive plant tissue damage and low expression. It is demonstrated that the modulation of the plant protein synthesis machinery enhances hEPO production. Co-expression of basic leucine zipper transcription factors with hEPO prevents plant tissue damage, boosts expression, and increases hEPO solubility. bZIP28 co-expression up-regulates genes associated with the unfolded protein response, indicating that the plant tissue damage caused by hEPO expression is due to the native protein folding machinery being overwhelmed and that this can be overcome by co-expressing bZIP28.
Assuntos
Fatores de Transcrição de Zíper de Leucina Básica , Eritropoetina , Nicotiana , Fatores de Transcrição de Zíper de Leucina Básica/genética , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Eritropoetina/genética , Eritropoetina/metabolismo , Humanos , Nicotiana/genética , Nicotiana/metabolismo , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Regulação da Expressão Gênica de Plantas , Resposta a Proteínas não Dobradas/genéticaRESUMO
Malaria kills around 409,000 people a year, mostly children under the age of five. Malaria transmission-blocking vaccines work to reduce malaria prevalence in a community and have the potential to be part of a multifaceted approach required to eliminate the parasites causing the disease. Pfs25 is a leading malaria transmission-blocking antigen and has been successfully produced in a plant expression system as both a subunit vaccine and as a virus-like particle. This study demonstrates an improved version of the virus-like particle antigen display molecule by eliminating known protease sites from the prior A85 variant. This re-engineered molecule, termed B29, displays three times the number of Pfs25 antigens per virus-like particle compared to the original Pfs25 virus-like particle. An improved purification scheme was also developed, resulting in a substantially higher yield and improved purity. The molecule was evaluated in a mouse model and found to induce improved transmission-blocking activity at lower doses and longer durations than the original molecule.
Assuntos
Vacinas Antimaláricas , Malária Falciparum , Malária , Animais , Camundongos , Plasmodium falciparum , Proteínas de Protozoários , Antígenos de Protozoários , Malária/prevenção & controle , Vacinas Antimaláricas/genética , Malária Falciparum/prevenção & controle , Anticorpos AntiprotozoáriosRESUMO
Application of tools of molecular biology and genomics is increasingly leading towards the development of recombinant protein-based biologics. As such, it is leading to an increased diversity of targets that have important health applications and require more flexible approaches for expression because of complex post-translational modifications. For example, Plasmodium parasites may have complex post-translationally modified proteins such as Pfs48/45 that do not carry N-linked glycans (Exp. Parasitol. 1998; 90, 165.) but contain potential N-linked glycosylation sites that can be aberrantly glycosylated during expression in mammalian and plant systems. Therefore, it is important to develop strategies for producing non-glycosylated forms of these targets to preserve biological activity and native conformation. In this study, we are describing in vivo deglycosylation of recombinant N-glycosylated proteins as a result of their transient co-expression with bacterial PNGase F (Peptide: N-glycosidase F). In addition, we show that the recognition of an in vivo deglycosylated plant-produced malaria vaccine candidate, Pfs48F1, by monoclonal antibodies I, III and V raised against various epitopes (I, III and V) of native Pfs48/45 of Plasmodium falciparum, was significantly stronger compared to that of the glycosylated form of plant-produced Pfs48F1. To our knowledge, neither in vivo enzymatic protein deglycosylation has been previously achieved in any eukaryotic system, including plants, nor has bacterial PNGase F been expressed in the plant system. Thus, here, we report for the first time the expression in plants of an active bacterial enzyme PNGase F and the production of recombinant proteins of interest in a non-glycosylated form.
Assuntos
Bactérias/enzimologia , Biotecnologia/métodos , Nicotiana/metabolismo , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/metabolismo , Proteínas Recombinantes/biossíntese , Anticorpos Monoclonais/imunologia , Antígenos de Bactérias/imunologia , Toxinas Bacterianas/imunologia , Western Blotting , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Glicosilação , Espectrometria de Massas , Mapeamento de Peptídeos , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/isolamento & purificação , Plantas Geneticamente Modificadas , Plasmodium falciparum/metabolismo , Polissacarídeos/metabolismo , Ligação Proteica , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , SolubilidadeRESUMO
In 2009, a novel H1N1 swine influenza virus was isolated from infected humans in Mexico and the United States, and rapidly spread around the world. Another virus, a highly pathogenic avian influenza virus of the H5N1 subtype, identified by the World Health Organization as a potential pandemic threat in 1997, continues to be a significant risk. While vaccination is the preferred strategy for the prevention and control of influenza infections, the traditional egg-based approach to producing influenza vaccines does not provide sufficient capacity and adequate speed to satisfy global needs to combat newly emerging strains, seasonal or potentially pandemic. Significant efforts are underway to develop and implement new cell substrates with improved efficiency for influenza vaccine development and manufacturing. In recent years, plants have been used to produce recombinant proteins including subunit vaccines and antibodies. The main advantages of using plant systems for the production of vaccine antigens against influenza are their independence from pathogenic viruses, and cost and time efficiency. Here, we describe the large-scale production of recombinant hemagglutinin proteins from A/California/04/09 (H1N1) and A/Indonesia/05/05 (H5N1) strains of influenza virus in Nicotiana benthamiana plants, and their immunogenicity (serum hemagglutination inhibition and virus neutralizing antibodies), and safety in animal models. These results support the testing of these candidate vaccines in human volunteers and also the utility of our plant expression system for large-scale recombinant influenza vaccine production.
Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Vírus da Influenza A Subtipo H1N1/imunologia , Virus da Influenza A Subtipo H5N1/imunologia , Vacinas contra Influenza/imunologia , Plantas Geneticamente Modificadas/metabolismo , Animais , Anticorpos Antivirais/sangue , Biotecnologia/métodos , Furões , Testes de Inibição da Hemaglutinação , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Humanos , Vírus da Influenza A Subtipo H1N1/genética , Virus da Influenza A Subtipo H5N1/genética , Vacinas contra Influenza/efeitos adversos , Vacinas contra Influenza/genética , Influenza Humana/prevenção & controle , Camundongos , Camundongos Endogâmicos BALB C , Plantas Geneticamente Modificadas/genética , Coelhos , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Tecnologia Farmacêutica/métodos , Nicotiana/genética , Vacinas de Subunidades Antigênicas/efeitos adversos , Vacinas de Subunidades Antigênicas/genética , Vacinas de Subunidades Antigênicas/imunologia , Vacinas Sintéticas/efeitos adversos , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologiaRESUMO
The health and economic burden of infectious diseases in general and bioterrorism in particular necessitate the development of medical countermeasures. One proven approach to reduce the disease burden and spread of pathogen is treatment with monoclonal antibodies (mAb). mAbs can prevent or reduce severity of the disease by variety of mechanisms, including neutralizing pathogen growth, limiting its spread from infected to adjacent cells, or by inhibiting biological activity of toxins, such as anthrax lethal toxin. Here, we report the production of glycosylated (pp-mAb (PA) ) and non-glycosylated (pp-mAb (PANG) ) versions of a plant-derived mAb directed against protective antigen (PA) of Bacillus anthracis in Nicotiana benthamiana plants using agroinfiltration. Both forms of the antibody were able to neutralize anthrax lethal toxin activity in vitro and protect mice against an intraperitoneal challenge with spores of B. anthracis Sterne strain. A single 180 µg intraperitoneal dose of pp-mAb (PA) or pp-mAb (PANG) provided 90% and 100% survival, respectively. When tested in non-human primates, pp-mAb (PANG) was demonstrated to be superior to pp-mAb (PA) in that it had a significantly longer terminal half-life and conferred 100% protection against a lethal dose of aerosolized anthrax spore challenge after a single 5 mg/kg intravenous dose compared to a 40% survival rate conferred by pp-mAb (PA) . This study demonstrates the potential of a plant-produced non-glycosylated antibody as a useful tool for the treatment of inhalation anthrax.
Assuntos
Antraz/terapia , Anticorpos Antibacterianos/uso terapêutico , Anticorpos Monoclonais/uso terapêutico , Antitoxinas/uso terapêutico , Toxinas Bacterianas/antagonistas & inibidores , Animais , Anticorpos Antibacterianos/genética , Anticorpos Antibacterianos/metabolismo , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/metabolismo , Antígenos de Bactérias , Antitoxinas/genética , Antitoxinas/metabolismo , Modelos Animais de Doenças , Macaca fascicularis , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Plantas Geneticamente Modificadas/genética , Doenças dos Primatas/terapia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/uso terapêutico , Doenças dos Roedores/terapia , Análise de Sobrevida , Nicotiana/genética , Resultado do TratamentoRESUMO
Malaria is a serious and sometimes fatal mosquito-borne disease caused by a protozoan parasite. Each year, it is estimated that over one million people are killed by malaria, yet the disease is preventable and treatable. Developing vaccines against the parasite is a critical component in the fight against malaria and these vaccines can target different stages of the pathogen's life cycle. We are targeting sexual stage proteins of P. falciparum which are found on the surface of the parasite reproductive cells present in the mosquito gut. Antibodies against these proteins block the progression of the parasite's life cycle in the mosquito, and thus block transmission to the next human host. Transmission blocking vaccines are essential to the malaria eradication program to ease the disease burden at the population level. We have successfully produced multiple versions of the Pfs25 antigen in a plant virus-based transient expression system and have evaluated these vaccine candidates in an animal model. The targets are expressed in plants at a high level, are soluble and most importantly, generate strong transmission blocking activity as determined by a standard membrane feeding assay. These data demonstrate the feasibility of expressing Plasmodium antigens in a plant-based system for the economic production of a transmission blocking vaccine against malaria.
Assuntos
Anticorpos Antiprotozoários/imunologia , Transmissão de Doença Infecciosa/prevenção & controle , Vacinas Antimaláricas/imunologia , Malária Falciparum/transmissão , Plasmodium falciparum/imunologia , Proteínas de Protozoários/imunologia , Animais , Culicidae/parasitologia , Culicidae/fisiologia , Comportamento Alimentar , Vacinas Antimaláricas/administração & dosagem , Camundongos , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , NicotianaRESUMO
Yellow fever (YF) is a viral disease transmitted by mosquitoes and endemic mostly in South America and Africa with 20-50% fatality. All current licensed YF vaccines, including YF-Vax® (Sanofi-Pasteur, Lyon, France) and 17DD-YFV (Bio-Manguinhos, Rio de Janeiro, Brazil), are based on live attenuated virus produced in hens' eggs and have been widely used. The YF vaccines are considered safe and highly effective. However, a recent increase in demand for YF vaccines and reports of rare cases of YF vaccine-associated fatal adverse events have provoked interest in developing a safer YF vaccine that can be easily scaled up to meet this increased global demand. To this point, we have engineered the YF virus envelope protein (YFE) and transiently expressed it in Nicotiana benthamiana as a stand-alone protein (YFE) or as fusion to the bacterial enzyme lichenase (YFE-LicKM). Immunogenicity and challenge studies in mice demonstrated that both YFE and YFE-LicKM elicited virus neutralizing (VN) antibodies and protected over 70% of mice from lethal challenge infection. Furthermore, these two YFE-based vaccine candidates induced VN antibody responses with high serum avidity in nonhuman primates and these VN antibody responses were further enhanced after challenge infection with the 17DD strain of YF virus. These results demonstrate partial protective efficacy in mice of YFE-based subunit vaccines expressed in N. benthamiana. However, their efficacy is inferior to that of the live attenuated 17DD vaccine, indicating that formulation development, such as incorporating a more suitable adjuvant, may be required for product development.
Assuntos
Modelos Animais de Doenças , Vacina contra Febre Amarela/biossíntese , Febre Amarela/prevenção & controle , Animais , ELISPOT/métodos , Humanos , Camundongos/imunologia , Testes de Neutralização/métodos , Febre Amarela/tratamento farmacológico , Vacina contra Febre Amarela/imunologia , Vacina contra Febre Amarela/uso terapêutico , Vírus da Febre Amarela/imunologiaRESUMO
Highly pathogenic avian influenza (HPAI) H5N1 is an ongoing global health concern due to its severe sporadic outbreaks in Asia, Africa and Europe, which poses a potential pandemic threat. The development of safe and cost-effective vaccine candidates for HPAI is considered the best strategy for managing the disease and addressing the pandemic preparedness. The most potential vaccine candidate is the antigenic determinant of influenza A virus, hemagglutinin (HA). The present research was aimed at developing optimized expression in Nicotiana benthamiana and protein purification process for HA from the Malaysian isolate of H5N1 as a vaccine antigen for HPAI H5N1. Expression of HA from the Malaysian isolate of HPAI in N. benthamiana was confirmed, and more soluble protein was expressed as truncated HA, the HA1 domain over the entire ectodomain of HA. Two different purification processes were evaluated for efficiency in terms of purity and yield. Due to the reduced yield, protein degradation and length of the 3-column purification process, the 2-column method was chosen for target purification. Purified HA1 was found immunogenic in mice inducing H5 HA-specific IgG and a hemagglutination inhibition antibody. This paper offers an alternative production system of a vaccine candidate against a locally circulating HPAI, which has a regional significance.
Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/isolamento & purificação , Virus da Influenza A Subtipo H5N1/imunologia , Vacinas contra Influenza/imunologia , Infecções por Orthomyxoviridae/prevenção & controle , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Animais , Anticorpos Antivirais/sangue , Modelos Animais de Doenças , Feminino , Expressão Gênica , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Imunoglobulina G/sangue , Vacinas contra Influenza/administração & dosagem , Vacinas contra Influenza/genética , Camundongos Endogâmicos BALB C , Proteínas Recombinantes/genética , Nicotiana/genética , Nicotiana/metabolismo , Resultado do Tratamento , Vacinas de Subunidades Antigênicas/administração & dosagem , Vacinas de Subunidades Antigênicas/genética , Vacinas de Subunidades Antigênicas/imunologia , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologiaRESUMO
Over 400 million people living in the world's poorest developing nations are infected with hookworms, mostly of the genus Necator americanus. A bivalent human hookworm vaccine composed of the Necator americanus Glutathione S-Transferase-1 (Na-GST-1) and the Necator americanus Aspartic Protease-1 (Na-APR-1 (M74)) is currently under development by the Sabin Vaccine Institute Product Development Partnership (Sabin PDP). Both monovalent vaccines are currently in Phase 1 trials. Both Na-GST-1 and Na-APR-1 antigens are expressed as recombinant proteins. While Na-GST-1 was found to express with high yields in Pichia pastoris, the level of expression of Na-APR-1 in this host was too low to be suitable for a manufacturing process. When the tobacco plant Nicotiana benthamiana was evaluated as an expression system, acceptable levels of solubility, yield, and stability were attained. Observed expression levels of Na-APR-1 (M74) using this system are â¼300 mg/kg. Here we describe the achievements and obstacles encountered during process development as well as characterization and stability of the purified Na-APR-1 (M74) protein and formulated vaccine. The expression, purification and analysis of purified Na-APR-1 (M74) protein obtained from representative 5 kg reproducibility runs performed to qualify the Na-APR-1 (M74) production process is also presented. This process has been successfully transferred to a pilot plant and a 50 kg scale manufacturing campaign under current Good Manufacturing Practice (cGMP) has been performed. The 50 kg run has provided a sufficient amount of protein to support the ongoing hookworm vaccine development program of the Sabin PDP.
Assuntos
Antígenos de Helmintos/isolamento & purificação , Antígenos de Helmintos/metabolismo , Ácido Aspártico Endopeptidases/isolamento & purificação , Ácido Aspártico Endopeptidases/metabolismo , Necator americanus/enzimologia , Vacinas Sintéticas/isolamento & purificação , Vacinas Sintéticas/metabolismo , Animais , Antígenos de Helmintos/genética , Ácido Aspártico Endopeptidases/genética , Biotecnologia/métodos , Expressão Gênica , Necator americanus/genética , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Tecnologia Farmacêutica/métodos , Nicotiana/genética , Nicotiana/metabolismo , Vacinas Sintéticas/genéticaRESUMO
The H1N1 influenza pandemic of 2009 stimulated interest in developing safe and effective subunit influenza vaccines using rapid and cost-effective recombinant technologies that can avoid dependence on hens' eggs supply and live viruses for production. Among alternative approaches to subunit vaccine development, virus-like particles (VLPs) represent an attractive strategy due to their safety and immunogenicity. Previously, we have produced a recombinant monomeric hemagglutinin (HA) protein derived from the A/California/04/09 (H1N1) strain of influenza virus in a plant-based transient expression system and demonstrated immunogenicity and safety of this monomeric HA in animal models and human volunteers. In an effort to produce higher potency influenza vaccine in plants, we have designed and generated enveloped VLPs using the ectodomain of HA from the A/California/04/09 strain and heterologous sequences. The resulting H1 HA VLPs (HAC-VLPs) elicited robust hemagglutination inhibition antibody responses in mice at doses lower than 1 µg in the presence or absence of Alhydrogel adjuvant. These results suggest enhanced immunogenicity of recombinant HA in the form of an enveloped VLP over soluble antigen.
Assuntos
Vírus da Influenza A Subtipo H1N1/imunologia , Vacinas contra Influenza/imunologia , Vacinas contra Influenza/isolamento & purificação , Vacinas de Partículas Semelhantes a Vírus/imunologia , Vacinas de Partículas Semelhantes a Vírus/isolamento & purificação , Adjuvantes Imunológicos/administração & dosagem , Hidróxido de Alumínio/administração & dosagem , Animais , Anticorpos Antivirais/sangue , Antígenos Virais/genética , Antígenos Virais/imunologia , Antígenos Virais/metabolismo , Testes de Inibição da Hemaglutinação , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Vírus da Influenza A Subtipo H1N1/genética , Vacinas contra Influenza/administração & dosagem , Vacinas contra Influenza/genética , Camundongos Endogâmicos BALB C , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Nicotiana/genética , Vacinas de Partículas Semelhantes a Vírus/administração & dosagem , Vacinas de Partículas Semelhantes a Vírus/genéticaRESUMO
Malaria transmission blocking vaccines (TBV) directed against proteins expressed on sexual stages of Plasmodium falciparum in the mosquito midgut are considered an effective means to reduce malaria transmission. Antibodies induced by TBV block sporogonic development in the mosquito, and thus transmission to the next human host. The Pfs25 protein, expressed on the surface of gametes, zygotes and ookinetes, is one of the primary targets for TBV development. Using a plant virus-based transient expression system, we have successfully produced Pfs25 fused to a modified lichenase (LicKM) carrier in Nicotiana benthamiana, purified and characterized the protein (Pfs25-FhCMB), and evaluated this vaccine candidate in animal models for the induction of transmission blocking antibodies. Soluble Pfs25-FhCMB was expressed in plants at a high level, and induced transmission blocking antibodies that persisted for up to 6 months post immunization in mice and rabbits. These data demonstrate the potential of the new malaria vaccine candidate and also support feasibility of expressing Plasmodium antigens in a plant-based system.
Assuntos
Anticorpos Antiprotozoários/sangue , Transmissão de Doença Infecciosa/prevenção & controle , Vacinas Antimaláricas/imunologia , Malária/prevenção & controle , Proteínas de Protozoários/imunologia , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Feminino , Expressão Gênica , Vetores Genéticos , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Vacinas Antimaláricas/administração & dosagem , Vacinas Antimaláricas/genética , Camundongos Endogâmicos BALB C , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Plasmodium falciparum/genética , Plasmodium falciparum/imunologia , Potyvirus/genética , Proteínas de Protozoários/genética , Coelhos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Fatores de Tempo , Nicotiana/genética , Nicotiana/metabolismo , Vacinas de Subunidades Antigênicas/administração & dosagem , Vacinas de Subunidades Antigênicas/genética , Vacinas de Subunidades Antigênicas/imunologia , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologiaRESUMO
The potential use of Bacillus anthracis as a bioterrorism weapon threatens the security of populations globally, requiring the immediate availability of safe, efficient and easily delivered anthrax vaccine for mass vaccination. Extensive research efforts have been directed toward the development of recombinant subunit vaccines based on protective antigen (PA), the principal virulence factor of B. anthracis. Among the emerging technologies for the production of these vaccine antigens is our launch vector-based plant transient expression system. Using this system, we have successfully engineered, expressed, purified and characterized full-length PA (pp-PA83) in Nicotiana benthamiana plants using agroinfiltration. This plant-produced antigen elicited high toxin neutralizing antibody titers in mice and rabbits after two vaccine administrations with Alhydrogel. In addition, immunization with this vaccine candidate protected 100% of rabbits from a lethal aerosolized B. anthracis challenge. The vaccine effects were dose-dependent and required the presence of Alhydrogel adjuvant. In addition, the vaccine antigen formulated with Alhydrogel was stable and retained immunogenicity after two-week storage at 4°C, the conditions intended for clinical use. These results support the testing of this vaccine candidate in human volunteers and the utility of our plant expression system for the production of a recombinant anthrax vaccine.
Assuntos
Vacinas contra Antraz/imunologia , Antraz/prevenção & controle , Antígenos de Bactérias/administração & dosagem , Antígenos de Bactérias/imunologia , Toxinas Bacterianas/administração & dosagem , Toxinas Bacterianas/imunologia , Adjuvantes Imunológicos/administração & dosagem , Aerossóis , Hidróxido de Alumínio/administração & dosagem , Animais , Antraz/imunologia , Vacinas contra Antraz/administração & dosagem , Anticorpos Antibacterianos/sangue , Anticorpos Neutralizantes/sangue , Antígenos de Bactérias/genética , Antígenos de Bactérias/isolamento & purificação , Toxinas Bacterianas/genética , Toxinas Bacterianas/isolamento & purificação , Modelos Animais de Doenças , Exposição por Inalação , Camundongos Endogâmicos BALB C , Plantas Geneticamente Modificadas/genética , Coelhos , Análise de Sobrevida , Nicotiana/genética , Resultado do Tratamento , Vacinas de Subunidades Antigênicas/administração & dosagem , Vacinas de Subunidades Antigênicas/imunologia , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/imunologiaRESUMO
The increased worldwide awareness of seasonal and pandemic influenza, including pandemic H1N1 virus, has stimulated interest in the development of economic platforms for rapid, large-scale production of safe and effective subunit vaccines. In recent years, plants have demonstrated their utility as such a platform and have been used to produce vaccine antigens against various infectious diseases. Previously, we have produced in our transient plant expression system a recombinant monomeric hemagglutinin (HA) protein (HAC1) derived from A/California/04/09 (H1N1) strain of influenza virus and demonstrated its immunogenicity and safety in animal models and human volunteers. In the current study, to mimic the authentic HA structure presented on the virus surface and to improve stability and immunogenicity of the HA antigen, we generated trimeric HA by introducing a trimerization motif from a heterologous protein into the HA sequence. Here, we describe the engineering, production in Nicotiana benthamiana plants, and characterization of the highly purified recombinant trimeric HA protein (tHA-BC) from A/California/04/09 (H1N1) strain of influenza virus. The results demonstrate the induction of serum hemagglutination inhibition antibodies by tHA-BC and its protective efficacy in mice against a lethal viral challenge. In addition, the immunogenic and protective doses of tHA-BC were much lower compared with monomeric HAC1. Further investigation into the optimum vaccine dose and/or regimen as well as the stability of trimerized HA is necessary to determine whether trimeric HA is a more potent vaccine antigen than monomeric HA.
Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/administração & dosagem , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Vírus da Influenza A Subtipo H1N1/imunologia , Vacinas contra Influenza/imunologia , Infecções por Orthomyxoviridae/prevenção & controle , Animais , Anticorpos Antibacterianos/sangue , Modelos Animais de Doenças , Testes de Inibição da Hemaglutinação , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/isolamento & purificação , Vírus da Influenza A Subtipo H1N1/genética , Vacinas contra Influenza/administração & dosagem , Camundongos Endogâmicos BALB C , Infecções por Orthomyxoviridae/imunologia , Plantas Geneticamente Modificadas/genética , Engenharia de Proteínas , Multimerização Proteica , Análise de Sobrevida , Nicotiana/genética , Resultado do Tratamento , Vacinas de Subunidades Antigênicas/administração & dosagem , Vacinas de Subunidades Antigênicas/imunologia , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/imunologiaRESUMO
Clinically available red blood cells (RBCs) for transfusions are at high demand, but in vitro generation of RBCs from hematopoietic stem cells requires significant quantities of growth factors. Here, we describe the production of four human growth factors: erythropoietin (EPO), stem cell factor (SCF), interleukin 3 (IL-3), and insulin-like growth factor-1 (IGF-1), either as non-fused proteins or as fusions with a carrier molecule (lichenase), in plants, using a Tobacco mosaic virus vector-based transient expression system. All growth factors were purified and their identity was confirmed by western blotting and peptide mapping. The potency of these plant-produced cytokines was assessed using TF1 cell (responsive to EPO, IL-3 and SCF) or MCF-7 cell (responsive to IGF-1) proliferation assays. The biological activity estimated here for the cytokines produced in plants was slightly lower or within the range cited in commercial sources and published literature. By comparing EC50 values of plant-produced cytokines with standards, we have demonstrated that all four plant-produced growth factors stimulated the expansion of umbilical cord blood-derived CD34+ cells and their differentiation toward erythropoietic precursors with the same potency as commercially available growth factors. To the best of our knowledge, this is the first report on the generation of all key bioactive cytokines required for the erythroid development in a cost-effective manner using a plant-based expression system.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Eritropoetina/farmacologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Fator de Crescimento Insulin-Like I/farmacologia , Interleucina-3/farmacologia , Nicotiana/genética , Fator de Células-Tronco/farmacologia , Agrobacterium tumefaciens/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Clonagem Molecular/métodos , Eritrócitos/citologia , Eritrócitos/metabolismo , Eritropoetina/biossíntese , Eritropoetina/genética , Eritropoetina/isolamento & purificação , Sangue Fetal/citologia , Sangue Fetal/efeitos dos fármacos , Sangue Fetal/metabolismo , Expressão Gênica , Vetores Genéticos , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , Fator de Crescimento Insulin-Like I/biossíntese , Fator de Crescimento Insulin-Like I/genética , Fator de Crescimento Insulin-Like I/isolamento & purificação , Interleucina-3/biossíntese , Interleucina-3/genética , Interleucina-3/isolamento & purificação , Plantas Geneticamente Modificadas , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/farmacologia , Fator de Células-Tronco/biossíntese , Fator de Células-Tronco/genética , Fator de Células-Tronco/isolamento & purificação , Nicotiana/metabolismo , Nicotiana/virologia , Vírus do Mosaico do Tabaco/genética , TransgenesRESUMO
Prevention of infectious diseases by vaccination is often limited because of the lack of safe, effective, and accessible vaccines. Traditional vaccines are expensive and require special conditions for storage, distribution, and administration. Plants have potential for large-scale production of a variety of inexpensive and highly effective recombinant proteins for biomedical and pharmaceutical applications, including subunit vaccines. There are several approaches for the production of vaccine antigens in plants, including transient expression systems based on Agrobacterium delivery of binary vectors or plant viral vectors, stable transgenic plants, and plant cell or tissue cultures. Axenic plant cultures maintained under defined physical and chemical conditions appear to be an attractive production platform when target proteins need to be synthesized in a fully controlled environment. Hairy root cultures meet the criteria for such a system. Hairy root cultures, generated from edible plants and producing target antigens, provide a potential approach for the development of vaccines for oral delivery. With this approach, there are no protein extraction and purification costs and the active biomolecule is protected by the plant cell wall during passage through the upper gastrointestinal tract. This allows for gradual release of antigen at mucosal surfaces in the gut. Lyophilized hairy root cultures expressing vaccine antigens can be stored at ambient temperature for extended periods of time, which should facilitate storage and distribution, ultimately allowing for large populations to be vaccinated.
Assuntos
Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Técnicas de Cultura de Tecidos/métodos , Vacinas/biossíntese , Células Vegetais/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Vacinas/genéticaRESUMO
Malaria transmission blocking vaccines (TBVs) are considered an effective means to control and eventually eliminate malaria. The Pfs25 protein, expressed predominantly on the surface of the sexual and sporogonic stages of Plasmodium falciparum including gametes, zygotes and ookinetes, is one of the primary targets for TBV. It has been demonstrated that plants are an effective, highly scalable system for the production of recombinant proteins, including virus-like particles (VLPs). We engineered VLPs (Pfs25-CP VLP) comprising Pfs25 fused to the Alfalfa mosaic virus coat protein (CP) and produced these non-enveloped hybrid VLPs in Nicotiana benthamiana plants using a Tobacco mosaic virus-based 'launch' vector. Purified Pfs25-CP VLPs were highly consistent in size (19.3±2.4 nm in diameter) with an estimated 20-30% incorporation of Pfs25 onto the VLP surface. Immunization of mice with one or two doses of Pfs25-CP VLPs plus Alhydrogel® induced serum antibodies with complete transmission blocking activity through the 6 month study period. These results support the evaluation of Pfs25-CP VLP as a potential TBV candidate and the feasibility of the 'launch' vector technology for the production of VLP-based recombinant vaccines against infectious diseases.