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
The main objective of this study was to characterize the N-linked glycosylation profiles of recombinant hemagglutinin (HA) proteins expressed in either insect or plant hosts, and to develop a mass spectrometry based workflow that can be used in quality control to assess batch-to-batch reproducibility for recombinant HA glycosylation. HA is a surface glycoprotein of the influenza virus that plays a key role in viral infectivity and pathogenesis. Characterization of the glycans for plant recombinant HA from the viral strain A/California/04/09 (H1N1) has not yet been reported. In this study, N-linked glycosylation patterns of the recombinant HAs from both insect and plant hosts were characterized by precursor ion scan-driven data-dependent analysis followed by high-resolution MS/MS analysis of the deglycosylated tryptic peptides. Five glycosylation sites (N11, N23, N276, N287, and N481) were identified containing high mannose type glycans in plant-expressed HAs, and complex type glycoforms for the insect-expressed HA. More than 95% site occupancy was observed for all glycosylation sites except N11, which was 60% occupied. Multiple-reaction monitoring based quantitation analysis was developed for each glycopeptide isoform and the quantitative results indicate that the Man(8) GlcNAc(2) is the dominant glycan for all sites in plant-expressed HAs. The relative abundance of the glycoforms at each specific glycosylation site and the relative quantitation for each glycoform among three HAs were determined. Few differences in the glycosylation profiles were detected between the two batches of plant HAs studied, but there were significant differences between the glycosylation patterns in the HAs generated in plant and insect expression hosts.
Assuntos
Baculoviridae/química , Cromatografia Líquida/métodos , Glicoproteínas de Hemaglutininação de Vírus da Influenza/análise , Vírus da Influenza A Subtipo H1N1/química , Nicotiana/química , Espectrometria de Massas em Tandem/métodos , Sequência de Aminoácidos , Animais , Baculoviridae/genética , Glicosilação , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Humanos , Dados de Sequência Molecular , Peptídeos/análise , Polissacarídeos/análise , Controle de Qualidade , Proteínas Recombinantes/análise , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Spodoptera/virologia , Nicotiana/genética , Tripsina/químicaRESUMO
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
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
The affinity-matured human antibody repertoire may be ideal as a source for antibody therapeutics against infectious diseases and bioterror agents. Hybridoma methods for cloning these antibodies have many potential advantages, including convenience, high-yield antibody expression, and the ability to capture the antibodies in their native configurations. However, they have been hindered by hybridoma instability and limited accessibility of antigen-specific, class-switched human B-cells. Here, we describe an efficient, three-step method that uses human peripheral blood B-cells to produce stable hybridoma populations that are highly-enriched for affinity-matured human IgG antibodies. Peripheral blood mononuclear cells (PBMCs) are (a) selected for expression of CD27, a marker of post-germinal center B-cells, (b) cultured in vitro to promote B-cell proliferation and class-switching, and (c) fused to a genetically modified myeloma cell line. Using this strategy, we cloned 5 IgG antibodies that bind botulinum neurotoxins (BoNT), the causes of the food-borne paralytic illness, botulism, and Category A Select Bioterror agents. Two of these antibodies bind BoNT with low picomolar affinities. One (30B) is the first high-affinity human antibody to bind serotype B BoNT, and another (6A) is able to neutralize a lethal dose of serotype A BoNT in vivo in pre- and post-exposure models. This optimized hybridoma method will broadly enable access to the native human antibody repertoire.
Assuntos
Anticorpos Antibacterianos/biossíntese , Toxinas Botulínicas Tipo A/imunologia , Toxinas Botulínicas/imunologia , Hibridomas/imunologia , Imunoglobulina G/biossíntese , Animais , Anticorpos Antibacterianos/genética , Anticorpos Antibacterianos/imunologia , Especificidade de Anticorpos , Linfócitos B/imunologia , Toxinas Botulínicas/genética , Toxinas Botulínicas Tipo A/genética , Ensaio de Imunoadsorção Enzimática , Humanos , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Cinética , Modelos Lineares , Camundongos , Testes de Neutralização , RNA Bacteriano/química , RNA Bacteriano/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ressonância de Plasmônio de Superfície , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/imunologiaRESUMO
Malaria continues to be one of the world's most devastating infectious tropical diseases, and alternative strategies to prevent infection and disease spread are urgently needed. These strategies include the development of effective vaccines, such as malaria transmission blocking vaccines (TBV) directed against proteins found on the sexual stages of Plasmodium falciparum parasites present in the mosquito midgut. The Pfs25 protein, which is expressed on the surface of gametes, zygotes and ookinetes, has been a primary target for TBV development. One such vaccine strategy based on Pfs25 is a plant-produced malaria vaccine candidate engineered as a chimeric non-enveloped virus-like particle (VLP) comprising Pfs25 fused to the Alfalfa mosaic virus coat protein. This Pfs25 VLP-FhCMB vaccine candidate has been engineered and manufactured in Nicotiana benthamiana plants at pilot plant scale under current Good Manufacturing Practice guidelines. The safety, reactogenicity and immunogenicity of Pfs25 VLP-FhCMB was assessed in healthy adult volunteers. This Phase 1, dose escalation, first-in-human study was designed primarily to evaluate the safety of the purified plant-derived Pfs25 VLP combined with Alhydrogel® adjuvant. At the doses tested in this Phase 1 study, the vaccine was generally shown to be safe in healthy volunteers, with no incidence of vaccine-related serious adverse events and no evidence of any dose-limiting or dose-related toxicity, demonstrating that the plant-derived Pfs25 VLP-FhCMB vaccine had an acceptable safety and tolerability profile. In addition, although the vaccine did induce Pfs25-specific IgG in vaccinated patients in a dose dependent manner, the transmission reducing activity of the antibodies generated were weak, suggesting the need for an alternative vaccine adjuvant formulation. This study was registered at www.ClinicalTrials.gov under reference identifier NCT02013687.
Assuntos
Imunogenicidade da Vacina , Vacinas Antimaláricas/imunologia , Proteínas de Protozoários/imunologia , Vacinas Sintéticas/imunologia , Vacinas de Partículas Semelhantes a Vírus/imunologia , Adjuvantes Imunológicos/administração & dosagem , Adolescente , Adulto , Vírus do Mosaico da Alfafa , Anticorpos Antiprotozoários/sangue , Antígenos de Protozoários/imunologia , Feminino , Voluntários Saudáveis , Humanos , Vacinas Antimaláricas/efeitos adversos , Malária Falciparum/prevenção & controle , Masculino , Pessoa de Meia-Idade , Plasmodium falciparum , Nicotiana/metabolismo , Vacinas Sintéticas/efeitos adversos , Adulto JovemRESUMO
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
Bovine spongiform encephalopathy (BSE) is a fatal neurodegenerative prion disease affecting cattle that is transmissible to humans, manifesting as a variant of Creutzfeldt-Jakob disease (vCJD) likely following the consumption of meat contaminated with BSE prions. High-affinity antibodies are a prerequisite for the development of simple, highly sensitive and non-invasive diagnostic tests that are able to detect even small amounts of the disease-associated PrP conformer (PrP(Sc)). We describe here the affinity maturation of a single-chain Fv antibody fragment with a binding affinity of 1 pM to a peptide derived from the unstructured region of bovine PrP (BoPrP (90-105)). This is the tightest peptide-binding antibody reported to date and may find useful application in diagnostics, especially when PrP(Sc) is pretreated by denaturation and/or proteolysis for peptide-like presentation. Several rounds of directed evolution and off-rate selection with ribosome display were performed using an antibody library generated from a single PrP binder with error-prone PCR and DNA-shuffling. As the correct determinations of affinities in this range are not straightforward, competition biosensor techniques and KinExA methods were both applied and compared. Structural interpretation of the affinity improvement was performed based on the crystal structure of the original prion binder in complex with the BoPrP (95-104) peptide by modeling the corresponding mutations.
Assuntos
Evolução Molecular Direcionada , Fragmentos de Imunoglobulinas/química , Região Variável de Imunoglobulina/química , Príons/imunologia , Sequência de Aminoácidos , Substituição de Aminoácidos/genética , Encéfalo/imunologia , Encéfalo/metabolismo , Química Encefálica , Cristalografia por Raios X , Humanos , Fragmentos de Imunoglobulinas/genética , Fragmentos de Imunoglobulinas/metabolismo , Região Variável de Imunoglobulina/genética , Região Variável de Imunoglobulina/metabolismo , Dados de Sequência Molecular , Ligação ProteicaRESUMO
Second generation anthrax vaccines focus on the use of recombinant protective antigen (rPA) to elicit a strong, toxin neutralizing antibody responses in immunized subjects. The main difference between the rPA vaccines compared to the current licensed vaccine, anthrax vaccine absorbed (AVA), is the rPA vaccines are highly purified preparations of only rPA. These second generation rPA vaccines strive to elicit strong immune responses with substantially fewer doses than AVA while provoking less side effects. Many of the rPA candidates have shown to be effective in pre-clinical studies, but most of the second generation molecules have stability issues which reduce their efficacy over time. These stability issues are evident even under refrigerated conditions and thus emphasis has been directed to stabilizing the rPA molecule and determining an optimized final formulation. Stabilization of vaccines for long-term storage is a major challenge in the product development life cycle. The effort required to identify suitable formulations can be slow and expensive. The ideal storage for stockpiled vaccines would allow the candidate to withstand years of storage at ambient temperatures. The Fraunhofer Center for Molecular Biotechnology is developing a plant-produced rPA vaccine candidate that shows instability when stored under refrigerated conditions in a solution, as is typical for rPA vaccines. Increased stability of our plant-produced rPA vaccine candidate was achieved in a spray dried powder formulation that could eliminate the need for conventional cold chain allowing greater confidence to stockpile vaccine for civilian and military biodefense.
Assuntos
Vacinas contra Antraz/sangue , Plantas/química , Vacinas Sintéticas/química , Animais , Antraz/imunologia , Vacinas contra Antraz/imunologia , Anticorpos Antibacterianos , Anticorpos Neutralizantes/imunologia , Antígenos de Bactérias/imunologia , Bacillus anthracis/imunologia , Toxinas Bacterianas/imunologia , Química Farmacêutica/métodos , Estabilidade de Medicamentos , Armazenamento de Medicamentos/métodos , Imunização/métodos , Camundongos , Camundongos Endogâmicos BALB C , Pós/química , Vacinas Sintéticas/imunologiaRESUMO
In this study, hydrophilic and hydrolytically degradable poly (ethylene glycol) (PEG) hydrogels were formed via Michael-type addition and employed for sustained delivery of a monoclonal antibody against the protective antigen of anthrax. Taking advantage of the PEG-induced precipitation of the antibody, burst release from the matrix was avoided. These hydrogels were able to release active antibodies in a controlled manner from 14 days to as long as 56 days in vitro by varying the polymer architectures and molecular weights of the precursors. Analysis of the secondary and tertiary structure and the in vitro activity of the released antibody showed that the encapsulation and release did not affect the protein conformation or functionality. The results suggest the promise for developing PEG-based carriers for sustained release of therapeutic antibodies against toxins in various applications.
Assuntos
Anticorpos Neutralizantes/farmacologia , Antígenos de Bactérias/imunologia , Toxinas Bacterianas/imunologia , Hidrogéis/química , Polietilenoglicóis/química , Antígenos de Bactérias/química , Toxinas Bacterianas/química , Cromatografia em Gel , Dicroísmo Circular , Preparações de Ação Retardada , Eletroforese em Gel de Poliacrilamida , Glicosilação , Hidrólise , Polietilenoglicóis/síntese química , Conformação ProteicaRESUMO
Bacillus anthracis has long been considered a potential biological warfare agent, and therefore, there is a need for a safe, low-cost and highly efficient anthrax vaccine with demonstrated long-term stability for mass vaccination in case of an emergency. Many efforts have been made towards developing an anthrax vaccine based on recombinant protective antigen (rPA) of B. anthracis, a key component of the anthrax toxin, produced using different expression systems. Plants represent a promising recombinant protein production platform due to their relatively low cost, rapid scalability and favorable safety profile. Previous studies have shown that full-length rPA produced in Nicotiana benthamiana (pp-PA83) is immunogenic and can provide full protection against lethal spore challenge; however, further improvement in the potency and stability of the vaccine candidate is necessary. PA of B. anthracis is not a glycoprotein in its native host; however, this protein contains potential N-linked glycosylation sites, which can be aberrantly glycosylated during expression in eukaryotic systems including plants. This glycosylation could affect the availability of certain key epitopes either due to masking or misfolding of the protein. Therefore, a non-glycosylated form of pp-PA83 was engineered and produced in N. benthamiana using an in vivo deglycosylation approach based on co-expression of peptide-N-glycosidase F (PNGase F) from Flavobacterium meningosepticum. For comparison, versions of pp-PA83 containing point mutations in six potential N-glycosylation sites were also engineered and expressed in N. benthamiana. The in vivo deglycosylated pp-PA83 (pp-dPA83) was shown to have in vitro activity, in contrast to glycosylated pp-PA83, and to induce significantly higher levels of toxin-neutralizing antibody responses in mice compared with glycosylated pp-PA83, in vitro deglycosylated pp-PA83 or the mutated versions of pp-PA83. These results suggest that pp-dPA83 may offer advantages in terms of dose sparing and enhanced immunogenicity as a promising candidate for a safe, effective and low-cost subunit vaccine against anthrax.
Assuntos
Vacinas contra Antraz/genética , Antígenos de Bactérias/genética , Bacillus anthracis/genética , Toxinas Bacterianas/genética , Flavobacterium/enzimologia , Manosil-Glicoproteína Endo-beta-N-Acetilglucosaminidase/metabolismo , Nicotiana/genética , Animais , Antraz/imunologia , Antraz/prevenção & controle , Vacinas contra Antraz/imunologia , Vacinas contra Antraz/metabolismo , Antígenos de Bactérias/imunologia , Antígenos de Bactérias/metabolismo , Toxinas Bacterianas/imunologia , Toxinas Bacterianas/metabolismo , Clonagem Molecular , Flavobacterium/genética , Glicosilação , Imunidade , Manosil-Glicoproteína Endo-beta-N-Acetilglucosaminidase/genética , Camundongos Endogâmicos BALB C , Plantas Geneticamente Modificadas/genética , Engenharia de Proteínas , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismoRESUMO
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 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
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.
Assuntos
Anticorpos Bloqueadores/imunologia , Vacinas Antimaláricas/imunologia , Plasmodium falciparum/imunologia , Animais , Antígenos de Protozoários/imunologia , Camundongos , Proteínas de Protozoários/imunologia , Proteínas RecombinantesRESUMO
Recently, we have reported [1,2] on a subunit influenza vaccine candidate based on the recombinant hemagglutinin protein from the A/Indonesia/05/2005 (H5N1) strain of influenza virus, produced it using 'launch vector'-based transient expression technology in Nicotiana benthamiana, and demonstrated its immunogenicity in pre-clinical studies. Here, we present the results of a first-in-human, Phase 1 randomized, double-blind, placebo-controlled study designed to investigate safety, reactogenicity and immunogenicity of three escalating dose levels of this vaccine, HAI-05, (15, 45 and 90 µg) adjuvanted with Alhydrogel® (0.75 mg aluminum per dose) and the 90 µg dose level without Alhydrogel®. Vaccine was administered intramuscularly in two injections three weeks apart to healthy adults of 18-49 years of age. At all dose levels the vaccine was generally safe and well tolerated, with no reported serious adverse events or dose-limiting toxicities. Mild local and systemic reactions were observed in all vaccine dose groups and the placebo group and their occurrence was not dose related. The incidence rates were higher in the groups receiving vaccine with Alhydrogel®. The immune response elicited by the HAI-05 vaccine was variable with respect to both hemagglutination-inhibition and virus microneutralization antibody titers, with the highest responses observed in the 90 µg unadjuvanted group.