RESUMEN
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.
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Factores de Transcripción con Cremalleras de Leucina de Carácter Básico , Eritropoyetina , Nicotiana , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Eritropoyetina/genética , Eritropoyetina/metabolismo , Humanos , Nicotiana/genética , Nicotiana/metabolismo , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Regulación de la Expresión Génica de las Plantas , Respuesta de Proteína Desplegada/genéticaRESUMEN
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.
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Vacunas contra la Malaria , Malaria Falciparum , Malaria , Animales , Ratones , Plasmodium falciparum , Proteínas Protozoarias , Antígenos de Protozoos , Malaria/prevención & control , Vacunas contra la Malaria/genética , Malaria Falciparum/prevención & control , Anticuerpos AntiprotozoariosRESUMEN
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.
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Vacunas Antirrábicas , Rabia , Animales , Anticuerpos Antivirales , Ensayo de Inmunoadsorción Enzimática/métodos , Glicoproteínas/genética , Cobayas , Ratones , Conejos , Rabia/prevención & control , Vacunas Antirrábicas/química , Proteínas RecombinantesRESUMEN
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.
Asunto(s)
Anticuerpos Antivirales/administración & dosificación , Ebolavirus/inmunología , Fiebre Hemorrágica Ebola/tratamiento farmacológico , Animales , Anticuerpos Monoclonales/administración & dosificación , Modelos Animales de Enfermedad , Ebolavirus/genética , Femenino , Glicoproteínas/inmunología , Fiebre Hemorrágica Ebola/virología , Humanos , Inmunoterapia , Macaca mulatta , Masculino , Ratones , Proteínas Virales/inmunologíaRESUMEN
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.
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Anticuerpos Monoclonales Humanizados/farmacología , Anticuerpos Neutralizantes/farmacología , Encefalomielitis Equina Venezolana/inmunología , Vacunas Virales/farmacología , Animales , Anticuerpos Monoclonales Humanizados/inmunología , Anticuerpos Neutralizantes/inmunología , Modelos Animales de Enfermedad , Encefalomielitis Equina Venezolana/prevención & control , Humanos , Macaca fascicularis , Vacunas Virales/inmunologíaRESUMEN
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.
Asunto(s)
Modelos Animales de Enfermedad , Vacuna contra la Fiebre Amarilla/biosíntesis , Fiebre Amarilla/prevención & control , Animales , Ensayo de Immunospot Ligado a Enzimas/métodos , Humanos , Ratones/inmunología , Pruebas de Neutralización/métodos , Fiebre Amarilla/tratamiento farmacológico , Vacuna contra la Fiebre Amarilla/inmunología , Vacuna contra la Fiebre Amarilla/uso terapéutico , Virus de la Fiebre Amarilla/inmunologíaRESUMEN
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.
Asunto(s)
Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Glicoproteínas Hemaglutininas del Virus de la Influenza/aislamiento & purificación , Subtipo H5N1 del Virus de la Influenza A/inmunología , Vacunas contra la Influenza/inmunología , Infecciones por Orthomyxoviridae/prevención & control , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/aislamiento & purificación , Animales , Anticuerpos Antivirales/sangre , Modelos Animales de Enfermedad , Femenino , Expresión Génica , Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Inmunoglobulina G/sangre , Vacunas contra la Influenza/administración & dosificación , Vacunas contra la Influenza/genética , Ratones Endogámicos BALB C , Proteínas Recombinantes/genética , Nicotiana/genética , Nicotiana/metabolismo , Resultado del Tratamiento , Vacunas de Subunidad/administración & dosificación , Vacunas de Subunidad/genética , Vacunas de Subunidad/inmunología , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunologíaRESUMEN
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.
Asunto(s)
Proteínas de Plantas/farmacología , Proteínas Recombinantes de Fusión/farmacología , Virus Sincitiales Respiratorios/efectos de los fármacos , Proteína de Unión al GTP rhoA/farmacología , Vectores Genéticos , Pruebas de Sensibilidad Microbiana , Proteínas de Plantas/química , Proteínas de Plantas/genética , Ingeniería de Proteínas , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Nicotiana/genética , Nicotiana/metabolismo , Nicotiana/virología , Virus del Mosaico del Tabaco/genética , Proteína de Unión al GTP rhoA/química , Proteína de Unión al GTP rhoA/genéticaRESUMEN
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.
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Antígenos Helmínticos/aislamiento & purificación , Antígenos Helmínticos/metabolismo , Ácido Aspártico Endopeptidasas/aislamiento & purificación , Ácido Aspártico Endopeptidasas/metabolismo , Necator americanus/enzimología , Vacunas Sintéticas/aislamiento & purificación , Vacunas Sintéticas/metabolismo , Animales , Antígenos Helmínticos/genética , Ácido Aspártico Endopeptidasas/genética , Biotecnología/métodos , Expresión Génica , Necator americanus/genética , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismo , Tecnología Farmacéutica/métodos , Nicotiana/genética , Nicotiana/metabolismo , Vacunas Sintéticas/genéticaRESUMEN
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.
Asunto(s)
Subtipo H1N1 del Virus de la Influenza A/inmunología , Vacunas contra la Influenza/inmunología , Vacunas contra la Influenza/aislamiento & purificación , Vacunas de Partículas Similares a Virus/inmunología , Vacunas de Partículas Similares a Virus/aislamiento & purificación , Adyuvantes Inmunológicos/administración & dosificación , Hidróxido de Aluminio/administración & dosificación , Animales , Anticuerpos Antivirales/sangre , Antígenos Virales/genética , Antígenos Virales/inmunología , Antígenos Virales/metabolismo , Pruebas de Inhibición de Hemaglutinación , Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Glicoproteínas Hemaglutininas del Virus de la Influenza/metabolismo , Subtipo H1N1 del Virus de la Influenza A/genética , Vacunas contra la Influenza/administración & dosificación , Vacunas contra la Influenza/genética , Ratones Endogámicos BALB C , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismo , Nicotiana/genética , Vacunas de Partículas Similares a Virus/administración & dosificación , Vacunas de Partículas Similares a Virus/genéticaRESUMEN
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.
Asunto(s)
Anticuerpos Antiprotozoarios/sangre , Transmisión de Enfermedad Infecciosa/prevención & control , Vacunas contra la Malaria/inmunología , Malaria/prevención & control , Proteínas Protozoarias/inmunología , Animales , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Femenino , Expresión Génica , Vectores Genéticos , Glicósido Hidrolasas/genética , Glicósido Hidrolasas/metabolismo , Vacunas contra la Malaria/administración & dosificación , Vacunas contra la Malaria/genética , Ratones Endogámicos BALB C , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismo , Plasmodium falciparum/genética , Plasmodium falciparum/inmunología , Potyvirus/genética , Proteínas Protozoarias/genética , Conejos , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Factores de Tiempo , Nicotiana/genética , Nicotiana/metabolismo , Vacunas de Subunidad/administración & dosificación , Vacunas de Subunidad/genética , Vacunas de Subunidad/inmunología , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunologíaRESUMEN
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.
Asunto(s)
Anticuerpos Bloqueadores/inmunología , Vacunas contra la Malaria/inmunología , Plasmodium falciparum/inmunología , Animales , Antígenos de Protozoos/inmunología , Ratones , Proteínas Protozoarias/inmunología , Proteínas RecombinantesRESUMEN
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.
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Raíces de Plantas/genética , Raíces de Plantas/metabolismo , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismo , Técnicas de Cultivo de Tejidos/métodos , Vacunas/biosíntesis , Células Vegetales/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Vacunas/genéticaRESUMEN
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.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , Eritropoyetina/farmacología , Células Madre Hematopoyéticas/efectos de los fármacos , Factor I del Crecimiento Similar a la Insulina/farmacología , Interleucina-3/farmacología , Nicotiana/genética , Factor de Células Madre/farmacología , Agrobacterium tumefaciens/genética , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Clonación Molecular/métodos , Eritrocitos/citología , Eritrocitos/metabolismo , Eritropoyetina/biosíntesis , Eritropoyetina/genética , Eritropoyetina/aislamiento & purificación , Sangre Fetal/citología , Sangre Fetal/efectos de los fármacos , Sangre Fetal/metabolismo , Expresión Génica , Vectores Genéticos , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Humanos , Factor I del Crecimiento Similar a la Insulina/biosíntesis , Factor I del Crecimiento Similar a la Insulina/genética , Factor I del Crecimiento Similar a la Insulina/aislamiento & purificación , Interleucina-3/biosíntesis , Interleucina-3/genética , Interleucina-3/aislamiento & purificación , Plantas Modificadas Genéticamente , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/farmacología , Factor de Células Madre/biosíntesis , Factor de Células Madre/genética , Factor de Células Madre/aislamiento & purificación , Nicotiana/metabolismo , Nicotiana/virología , Virus del Mosaico del Tabaco/genética , TransgenesRESUMEN
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.
Asunto(s)
Glicoproteínas Hemaglutininas del Virus de la Influenza/administración & dosificación , Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Subtipo H1N1 del Virus de la Influenza A/inmunología , Vacunas contra la Influenza/inmunología , Infecciones por Orthomyxoviridae/prevención & control , Animales , Anticuerpos Antibacterianos/sangre , Modelos Animales de Enfermedad , Pruebas de Inhibición de Hemaglutinación , Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Glicoproteínas Hemaglutininas del Virus de la Influenza/aislamiento & purificación , Subtipo H1N1 del Virus de la Influenza A/genética , Vacunas contra la Influenza/administración & dosificación , Ratones Endogámicos BALB C , Infecciones por Orthomyxoviridae/inmunología , Plantas Modificadas Genéticamente/genética , Ingeniería de Proteínas , Multimerización de Proteína , Análisis de Supervivencia , Nicotiana/genética , Resultado del Tratamiento , Vacunas de Subunidad/administración & dosificación , Vacunas de Subunidad/inmunología , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/inmunologíaRESUMEN
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.
Asunto(s)
Vacunas contra el Carbunco/inmunología , Carbunco/prevención & control , Antígenos Bacterianos/administración & dosificación , Antígenos Bacterianos/inmunología , Toxinas Bacterianas/administración & dosificación , Toxinas Bacterianas/inmunología , Adyuvantes Inmunológicos/administración & dosificación , Aerosoles , Hidróxido de Aluminio/administración & dosificación , Animales , Carbunco/inmunología , Vacunas contra el Carbunco/administración & dosificación , Anticuerpos Antibacterianos/sangre , Anticuerpos Neutralizantes/sangre , Antígenos Bacterianos/genética , Antígenos Bacterianos/aislamiento & purificación , Toxinas Bacterianas/genética , Toxinas Bacterianas/aislamiento & purificación , Modelos Animales de Enfermedad , Exposición por Inhalación , Ratones Endogámicos BALB C , Plantas Modificadas Genéticamente/genética , Conejos , Análisis de Supervivencia , Nicotiana/genética , Resultado del Tratamiento , Vacunas de Subunidad/administración & dosificación , Vacunas de Subunidad/inmunología , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/inmunologíaRESUMEN
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.
Asunto(s)
Bacterias/enzimología , Biotecnología/métodos , Nicotiana/metabolismo , Péptido-N4-(N-acetil-beta-glucosaminil) Asparagina Amidasa/metabolismo , Proteínas Recombinantes/biosíntesis , Anticuerpos Monoclonales/inmunología , Antígenos Bacterianos/inmunología , Toxinas Bacterianas/inmunología , Western Blotting , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Glicosilación , Espectrometría de Masas , Mapeo Peptídico , Péptido-N4-(N-acetil-beta-glucosaminil) Asparagina Amidasa/aislamiento & purificación , Plantas Modificadas Genéticamente , Plasmodium falciparum/metabolismo , Polisacáridos/metabolismo , Unión Proteica , Proteínas Protozoarias/química , Proteínas Protozoarias/metabolismo , SolubilidadRESUMEN
BACKGROUND: Influenza virus is a globally important respiratory pathogen that causes a high degree of annual morbidity and mortality. Significant antigenic drift results in emergence of new, potentially pandemic, virus variants. The best prophylactic option for controlling emerging virus strains is to manufacture and administer pandemic vaccines in sufficient quantities and to do so in a timely manner without impacting the regular seasonal influenza vaccine capacity. Current, egg-based, influenza vaccine production is well established and provides an effective product, but has limited capacity and speed. OBJECTIVES: To satisfy the additional global demand for emerging influenza vaccines, high-performance cost-effective technologies need to be developed. Plants have a potential as an economic and efficient large-scale production platform for vaccine antigens. METHODS: In this study, a plant virus-based transient expression system was used to produce hemagglutinin (HA) proteins from the three vaccine strains used during the 2008-2009 influenza season, A/Brisbane/59/07 (H1N1), A/Brisbane/10/07 (H3N2), and B/Florida/4/06, as well as from the recently emerged novel H1N1 influenza A virus, A/California/04/09. RESULTS: The recombinant plant-based HA proteins were engineered and produced in Nicotiana benthamiana plants within 2 months of obtaining the genetic sequences specific to each virus strain. These antigens expressed at the rate of 400-1300 mg/kg of fresh leaf tissue, with >70% solubility. Immunization of mice with these HA antigens induced serum anti-HA IgG and hemagglutination inhibition antibody responses at the levels considered protective against these virus infections. CONCLUSIONS: These results demonstrate the feasibility of our transient plant expression system for the rapid production of influenza vaccine antigens.
Asunto(s)
Antígenos Virales/genética , Expresión Génica , Vacunas contra la Influenza/genética , Gripe Humana/inmunología , Nicotiana/genética , Animales , Anticuerpos Antivirales/inmunología , Antígenos Virales/inmunología , Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Humanos , Subtipo H1N1 del Virus de la Influenza A/genética , Subtipo H1N1 del Virus de la Influenza A/inmunología , Subtipo H3N2 del Virus de la Influenza A/genética , Subtipo H3N2 del Virus de la Influenza A/inmunología , Vacunas contra la Influenza/inmunología , Gripe Humana/prevención & control , Gripe Humana/virología , Ratones , Ratones Endogámicos BALB C , Orthomyxoviridae/genética , Orthomyxoviridae/inmunología , Nicotiana/metabolismoRESUMEN
Plasmodium falciparum is transmitted to a new host after completing its sexual cycle within a mosquito. Developing vaccines against the parasite sexual stages is a critical component in the fight against malaria. We are targeting multiple proteins of P. falciparum which are found only on the surfaces of the sexual forms of the parasite and where antibodies against these proteins have been shown to block the progression of the parasite's life cycle in the mosquito and thus block transmission to the next human host. We have successfully produced a region of the Pfs230 antigen in our plant-based transient-expression system and evaluated this vaccine candidate in an animal model. This plant-produced protein, 230CMB, is expressed at approximately 800 mg/kg in fresh whole leaf tissue and is 100% soluble. Administration of 230CMB with >90% purity induces strong immune responses in rabbits with high titers of transmission-blocking antibodies, resulting in a greater than 99% reduction in oocyst counts in the presence of complement, as determined by a standard membrane feeding assay. Our data provide a clear perspective on the clinical development of a Pfs230-based transmission-blocking malaria vaccine.
Asunto(s)
Antígenos de Protozoos/inmunología , Proteínas del Sistema Complemento/inmunología , Vacunas contra la Malaria/inmunología , Malaria Falciparum/prevención & control , Malaria Falciparum/transmisión , Plantas Modificadas Genéticamente/genética , Proteínas Protozoarias/inmunología , Animales , Anopheles/parasitología , Antígenos de Protozoos/biosíntesis , Antígenos de Protozoos/genética , Humanos , Vacunas contra la Malaria/genética , Plantas , Proteínas Protozoarias/biosíntesis , Proteínas Protozoarias/genética , Conejos , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunologíaRESUMEN
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.