RESUMO
The nucleoside hydrolase gene from Leishmania donovani was cloned and expressed in Escherichia coli as a full length 36-kDa protein (LdNH36). Following lysis and extraction, the protein was purified by anion exchange and gel filtration chromatography. The purified protein had a molecular mass of approximately 36-kDa and was confirmed to be >99% pure. Using a nucleoside hydrolase assay, the protein was found to exhibit a Km of 741 ± 246 µM. Protein integrity was confirmed by lithium dodecyl sulfate polyacrylamide gel electrophoresis (LDS-PAGE), mass spectrometry (MS), and enzymatic assay. Analysis of antibody levels from immunized mice indicated that LdNH36 alone or in a stable emulsion with the Toll-like receptor-4 ligand glucopyranosyl lipid adjuvant (GLA-SE) as immunostimulant induced high levels of antigen-specific IgG antibodies. The cellular immune response indicated a Th1 response in mice immunized with LdNH36, but only when formulated with GLA-SE. Mice immunized with the LdNH36 antigen in combination with the GLA-SE adjuvant and challenged with Leishmania mexicana showed significant reductions (>20 fold) in parasite burden, confirming the protective efficacy of this vaccine candidate.
Assuntos
Imunogenicidade da Vacina , Leishmania donovani , Vacinas contra Leishmaniose , Leishmaniose Cutânea , N-Glicosil Hidrolases , Proteínas de Protozoários , Animais , Feminino , Leishmania donovani/enzimologia , Leishmania donovani/genética , Leishmania donovani/imunologia , Vacinas contra Leishmaniose/biossíntese , Vacinas contra Leishmaniose/imunologia , Vacinas contra Leishmaniose/isolamento & purificação , Vacinas contra Leishmaniose/farmacocinética , Leishmaniose Cutânea/imunologia , Leishmaniose Cutânea/prevenção & controle , Camundongos , Camundongos Endogâmicos BALB C , N-Glicosil Hidrolases/biossíntese , N-Glicosil Hidrolases/imunologia , N-Glicosil Hidrolases/isolamento & purificação , N-Glicosil Hidrolases/farmacologia , Proteínas de Protozoários/biossíntese , Proteínas de Protozoários/imunologia , Proteínas de Protozoários/isolamento & purificação , Proteínas de Protozoários/farmacologia , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/farmacologiaRESUMO
The SARS-CoV-2 pandemic has affected more than 185 million people worldwide resulting in over 4 million deaths. To contain the pandemic, there is a continued need for safe vaccines that provide durable protection at low and scalable doses and can be deployed easily. Here, AAVCOVID-1, an adeno-associated viral (AAV), spike-gene-based vaccine candidate demonstrates potent immunogenicity in mouse and non-human primates following a single injection and confers complete protection from SARS-CoV-2 challenge in macaques. Peak neutralizing antibody titers are sustained at 1 year and complemented by functional memory T cell responses. The AAVCOVID vector has no relevant pre-existing immunity in humans and does not elicit cross-reactivity to common AAVs used in gene therapy. Vector genome persistence and expression wanes following injection. The single low-dose requirement, high-yield manufacturability, and 1-month stability for storage at room temperature may make this technology well suited to support effective immunization campaigns for emerging pathogens on a global scale.
Assuntos
Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Vacinas contra COVID-19/imunologia , COVID-19/prevenção & controle , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Vacinas contra COVID-19/administração & dosagem , Vacinas contra COVID-19/genética , Dependovirus/genética , Dependovirus/metabolismo , Feminino , Humanos , Imunogenicidade da Vacina/imunologia , Memória Imunológica/imunologia , Macaca fascicularis , Macaca mulatta , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Linfócitos T/imunologia , Transgenes/genética , Vacinação/métodos , Carga Viral/imunologiaRESUMO
The SARS-CoV-2 pandemic has affected more than 70 million people worldwide and resulted in over 1.5 million deaths. A broad deployment of effective immunization campaigns to achieve population immunity at global scale will depend on the biological and logistical attributes of the vaccine. Here, two adeno-associated viral (AAV)-based vaccine candidates demonstrate potent immunogenicity in mouse and nonhuman primates following a single injection. Peak neutralizing antibody titers remain sustained at 5 months and are complemented by functional memory T-cells responses. The AAVrh32.33 capsid of the AAVCOVID vaccine is an engineered AAV to which no relevant pre-existing immunity exists in humans. Moreover, the vaccine is stable at room temperature for at least one month and is produced at high yields using established commercial manufacturing processes in the gene therapy industry. Thus, this methodology holds as a very promising single dose, thermostable vaccine platform well-suited to address emerging pathogens on a global scale.
RESUMO
It is believed that an effective vaccine against leishmaniasis will require a T helper type 1 (TH 1) immune response. In this study, we investigated the adjuvanticity of the Toll-like receptor (TLR) 7/8 agonist 3M-052 in combination with the Leishmania donovani 36-kDa nucleoside hydrolase recombinant protein antigen (NH36). NH36 and 3M-052 were encapsulated in separate batches of poly(lactic-co-glycolic acid) (PLGA) microparticles (MPs). The loading efficiency for NH36 was 83% and for 3M-052 was above 95%. In vitro stimulation of bone marrow-derived dendritic cells, measured by IL-12 secretion, demonstrated that 3M-052 (free or MP-formulated) had a concentration-dependent immunostimulatory effect with an optimum concentration of 2 µg/mL. In immunogenicity studies in BALB/c mice, MP-formulated NH36 and 3M-052 elicited the highest serum titers of TH 1-associated IgG2a and IgG2b antibodies and the highest frequency of IFNγ-producing splenocytes. No dose dependency was observed among MP/NH36/3M-052 groups over a dose range of 4-60 µg 3M-052 per injection. The ability of MP-formulated NH36 and 3M-052 to elicit a TH 1-biased immune response indicates the potential for PLGA MP-formulated 3M-052 to be used as an adjuvant for leishmaniasis vaccines. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 1587-1594, 2018.
Assuntos
Antígenos de Protozoários , Compostos Heterocíclicos com 3 Anéis , Leishmania donovani/imunologia , Vacinas contra Leishmaniose , Leishmaniose Visceral , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Proteínas de Protozoários , Ácidos Esteáricos , Animais , Antígenos de Protozoários/química , Antígenos de Protozoários/farmacologia , Relação Dose-Resposta Imunológica , Compostos Heterocíclicos com 3 Anéis/química , Compostos Heterocíclicos com 3 Anéis/farmacologia , Imunogenicidade da Vacina , Vacinas contra Leishmaniose/química , Vacinas contra Leishmaniose/farmacologia , Leishmaniose Visceral/imunologia , Leishmaniose Visceral/patologia , Leishmaniose Visceral/prevenção & controle , Camundongos , Camundongos Endogâmicos BALB C , Molibdoferredoxina , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/química , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/farmacologia , Proteínas de Protozoários/química , Proteínas de Protozoários/farmacologia , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacologia , Ácidos Esteáricos/química , Ácidos Esteáricos/farmacologiaRESUMO
Chagas disease due to chronic infection with Trypanosoma cruzi is a neglected cause of heart disease, affecting approximately 6-10 million individuals in Latin America and elsewhere. T. cruzi Tc24, a calcium-binding protein in the flagellar pocket of the parasite, is a candidate antigen for an injectable therapeutic vaccine as an alternative or a complement to chemotherapy. Previously, we reported that a genetically engineered construct from which all cysteine residues had been eliminated (Tc24-C4) yields a recombinant protein with reduced aggregation and improved analytical purity in comparison to the wild-type form, without compromising antigenicity and immunogenicity. We now report that the established process for producing Escherichia coli-expressed Tc24-C4 protein is robust and reproducibly yields protein lots with consistent analytical characteristics, freeze-thaw, accelerated, and long-term stability profiles. The data indicate that, like most proteins, Tc24-C4 should be stable at -80°C, but also at 4°C and room temperature for at least 30 days, and up to 7-15 days at 37°C. Thus, the production process for recombinant Tc24-C4 is suitable for Current Good Manufacturing Practice production and clinical testing, based on process robustness, analytical characteristics, and stability profile.
Assuntos
Antígenos de Protozoários/química , Proteínas de Ligação ao Cálcio/química , Proteínas de Protozoários/química , Vacinas Protozoárias/química , Trypanosoma cruzi/química , Antígenos de Protozoários/imunologia , Proteínas de Ligação ao Cálcio/imunologia , Doença de Chagas/imunologia , Doença de Chagas/prevenção & controle , Congelamento , Humanos , Estabilidade Proteica , Proteínas de Protozoários/imunologia , Vacinas Protozoárias/imunologia , Proteínas Recombinantes/química , Proteínas Recombinantes/imunologia , Temperatura , Trypanosoma cruzi/imunologiaRESUMO
The pathogenic fungi Moniliophthora perniciosa causes Witches' Broom Disease (WBD) of cacao. The structure of MpPR-1i, a protein expressed by M. perniciosa when it infects cacao, are presented. This is the first reported de novo structure determined by single-wavelength anomalous dispersion phasing upon soaking with selenourea. Each monomer has flexible loop regions linking the core alpha-beta-alpha sandwich topology that comprise ~50% of the structure, making it difficult to generate an accurate homology model of the protein. MpPR-1i is monomeric in solution but is packed as a high ~70% solvent content, crystallographic heptamer. The greatest conformational flexibility between monomers is found in loops exposed to the solvent channel that connect the two longest strands. MpPR-1i lacks the conserved CAP tetrad and is incapable of binding divalent cations. MpPR-1i has the ability to bind lipids, which may have roles in its infection of cacao. These lipids likely bind in the palmitate binding cavity as observed in tablysin-15, since MpPR-1i binds palmitate with comparable affinity as tablysin-15. Further studies are required to clarify the possible roles and underlying mechanisms of neutral lipid binding, as well as their effects on the pathogenesis of M. perniciosa so as to develop new interventions for WBD.
Assuntos
Agaricales/metabolismo , Cacau/microbiologia , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Agaricales/química , Sítios de Ligação , Cristalografia por Raios X , Modelos Moleculares , Palmitatos/metabolismo , Doenças das Plantas/microbiologia , Ligação Proteica , Conformação ProteicaRESUMO
Tick-borne relapsing fever (RF) borreliosis is a neglected disease that is often misdiagnosed. RF species circulating in the United States include Borrelia turicatae, which is transmitted by argasid ticks. Environmental adaptation by RF Borrelia is poorly understood, however our previous studies indicated differential regulation of B. turicatae genes localized on the 150 kb linear megaplasmid during the tick-mammalian transmission cycle, including bta121. This gene is up-regulated by B. turicatae in the tick versus the mammal, and the encoded protein (BTA121) is predicted to be surface localized. The structure of BTA121 was solved by single-wavelength anomalous dispersion (SAD) using selenomethionine-derivative protein. The topology of BTA121 is unique with four helical domains organized into two helical bundles. Due to the sequence similarity of several genes on the megaplasmid, BTA121 can serve as a model for their tertiary structures. BTA121 has large interconnected tunnels and cavities that can accommodate ligands, notably long parallel helices, which have a large hydrophobic central pocket. Preliminary in-vitro studies suggest that BTA121 binds lipids, notably palmitate with a similar order of binding affinity as tablysin-15, a known palmitate-binding protein. The reported data will guide mechanistic studies to determine the role of BTA121 in the tick-mammalian transmission cycle of B. turicatae.
Assuntos
Proteínas de Bactérias , Infecções por Borrelia/metabolismo , Borrelia , Ácido Palmítico/química , Doenças Transmitidas por Carrapatos/metabolismo , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Borrelia/química , Borrelia/metabolismo , Cristalografia por Raios X , Humanos , Ligação Proteica , Domínios ProteicosRESUMO
From 2002 to 2003, a global pandemic of severe acute respiratory syndrome (SARS) spread to 5 continents and caused 8000 respiratory infections and 800 deaths. To ameliorate the effects of future outbreaks as well as to prepare for biodefense, a process for the production of a recombinant protein vaccine candidate is under development. Previously, we reported the 5 L scale expression and purification of a promising recombinant SARS vaccine candidate, RBD219-N1, the 218-amino acid residue receptor-binding domain (RBD) of SARS coronavirus expressed in yeast-Pichia pastoris X-33. When adjuvanted with aluminum hydroxide, this protein elicited high neutralizing antibody titers and high RBD-specific antibody titers. However, the yield of RBD219-N1 (60 mg RBD219-N1 per liter of fermentation supernatant; 60 mg/L FS) still required improvement to reach our target of >100 mg/L FS. In this study, we optimized the 10 L scale production process and increased the fermentation yield 6- to 7-fold to 400 mg/L FS with purification recovery >50%. A panel of characterization tests indicated that the process is reproducible and that the purified, tag-free RBD219-N1 protein has high purity and a well-defined structure and is therefore a suitable candidate for production under current Good Manufacturing Practice and future phase-1 clinical trials.
Assuntos
Pichia/genética , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/genética , Glicoproteína da Espícula de Coronavírus/genética , Vacinas Sintéticas/genética , Vacinas Virais/genética , Clonagem Molecular/métodos , Fermentação , Humanos , Microbiologia Industrial/métodos , Domínios Proteicos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/química , Síndrome Respiratória Aguda Grave/prevenção & controle , Síndrome Respiratória Aguda Grave/virologia , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/isolamento & purificação , Vacinas Sintéticas/química , Vacinas Sintéticas/isolamento & purificação , Vacinas Virais/química , Vacinas Virais/isolamento & purificaçãoRESUMO
A therapeutic vaccine for human Chagas disease is under development by the Sabin Vaccine Institute Product Development Partnership. The aim of the vaccine is to significantly reduce the parasite burden of Trypanosoma cruzi in humans, either as a standalone product or in combination with conventional chemotherapy. Vaccination of mice with Tc24 formulated with monophosphoryl-lipid A (MPLA) adjuvant results in a Th1 skewed immune response with elevated IgG2a and IFNγ levels and a statistically significant decrease in parasitemia following T. cruzi challenge. Tc24 was therefore selected for scale-up and further evaluation. During scale up and downstream process development, significant protein aggregation was observed due to intermolecular disulfide bond formation. To prevent protein aggregation, cysteine codons were replaced with serine codons which resulted in the production of a non-aggregated and soluble recombinant protein, Tc24-C4. No changes to the secondary structure of the modified molecule were detected by circular dichroism. Immunization of mice with wild-type Tc24 or Tc24-C4, formulated with E6020 (TLR4 agonist analog to MPLA) emulsified in a squalene-oil-in-water emulsion, resulted in IgG2a and antigen specific IFNγ production levels from splenocytes that were not significantly different, indicating that eliminating putative intermolecular disulfide bonds had no significant impact on the immunogenicity of the molecule. In addition, vaccination with either formulated wild type Tc24 or Tc24-C4 antigen also significantly increased survival and reduced cardiac parasite burden in mice. Investigations are now underway to examine the efficacy of Tc24-C4 formulated with other adjuvants to reduce parasite burden and increase survival in pre-clinical studies.
Assuntos
Doença de Chagas/prevenção & controle , Proteínas de Protozoários/imunologia , Vacinas Protozoárias/imunologia , Proteínas Recombinantes/imunologia , Trypanosoma cruzi/imunologia , Adjuvantes Imunológicos/administração & dosagem , Animais , Anticorpos Antiprotozoários/sangue , Cisteína/genética , Modelos Animais de Doenças , Feminino , Coração/parasitologia , Interferon gama/metabolismo , Leucócitos Mononucleares/imunologia , Camundongos Endogâmicos BALB C , Mutagênese , Carga Parasitária , Proteínas de Protozoários/administração & dosagem , Proteínas de Protozoários/genética , Vacinas Protozoárias/administração & dosagem , Vacinas Protozoárias/genética , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/genética , Análise de Sobrevida , Trypanosoma cruzi/genética , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologiaRESUMO
BACKGROUND: Immunity to the sand fly salivary protein SALO (Salivary Anticomplement of Lutzomyia longipalpis) protected hamsters against Leishmania infantum and L. braziliensis infection and, more recently, a vaccine combination of a genetically modified Leishmania with SALO conferred strong protection against L. donovani infection. Because of the importance of SALO as a potential component of a leishmaniasis vaccine, a plan to produce this recombinant protein for future scale manufacturing as well as knowledge of its structural characteristics are needed to move SALO forward for the clinical path. METHODOLOGY/PRINCIPAL FINDINGS: Recombinant SALO was expressed as a soluble secreted protein using Pichia pastoris, rSALO(P), with yields of 1g/L and >99% purity as assessed by SEC-MALS and SDS-PAGE. Unlike its native counterpart, rSALO(P) does not inhibit the classical pathway of complement; however, antibodies to rSALO(P) inhibit the anti-complement activity of sand fly salivary gland homogenate. Immunization with rSALO(P) produces a delayed type hypersensitivity response in C57BL/6 mice, suggesting rSALO(P) lacked anti-complement activity but retained its immunogenicity. The structure of rSALO(P) was solved by S-SAD at Cu-Kalpha to 1.94 Å and refined to Rfactor 17%. SALO is ~80% helical, has no appreciable structural similarities to any human protein, and has limited structural similarity in the C-terminus to members of insect odorant binding proteins. SALO has three predicted human CD4+ T cell epitopes on surface exposed helices. CONCLUSIONS/SIGNIFICANCE: The results indicate that SALO as expressed and purified from P. pastoris is suitable for further scale-up, manufacturing, and testing. SALO has a novel structure, is not similar to any human proteins, is immunogenic in rodents, and does not have the anti-complement activity observed in the native salivary protein which are all important attributes to move this vaccine candidate forward to the clinical path.
Assuntos
Psychodidae/química , Proteínas Recombinantes/imunologia , Proteínas e Peptídeos Salivares/imunologia , Animais , Expressão Gênica , Camundongos Endogâmicos C57BL , Pichia/genética , Pichia/metabolismo , Conformação Proteica , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas e Peptídeos Salivares/administração & dosagem , Proteínas e Peptídeos Salivares/química , Proteínas e Peptídeos Salivares/genéticaRESUMO
The production, crystal structure, and functional characterization of the C-terminal cysteine-rich secretory protein/antigen 5/pathogenesis related-1 (CAP) domain of pathogen-related yeast protein-1 (Pry1) from Saccharomyces cerevisiae is presented. The CAP domain of Pry1 (Pry1CAP) is functional in vivo as its expression restores cholesterol export to yeast mutants lacking endogenous Pry1 and Pry2. Recombinant Pry1CAP forms dimers in solution, is sufficient for in vitro cholesterol binding, and has comparable binding properties as full-length Pry1. Two crystal structures of Pry1CAP are reported, one with Mg(2+) coordinated to the conserved CAP tetrad (His208, Glu215, Glu233 and His250) in spacegroup I41 and the other without divalent cations in spacegroup P6122. The latter structure contains four 1,4-dioxane molecules from the crystallization solution, one of which sits in the cholesterol binding site. Both structures reveal that the divalent cation and cholesterol binding sites are connected upon dimerization, providing a structural basis for the observed Mg(2+)-dependent sterol binding by Pry1.
Assuntos
Proteínas dos Microfilamentos/química , Proteínas dos Microfilamentos/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Sítios de Ligação , Cátions , Colesterol/química , Proteínas do Citoesqueleto/metabolismo , Dioxanos/química , Genoma Fúngico , Magnésio/química , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Multimerização Proteica , Proteínas Recombinantes/química , Esteróis/químicaRESUMO
Leishmania donovani is the major cause of visceral leishmaniasis (kala-azar), now recognized as the parasitic disease with the highest level of mortality second only to malaria. No human vaccine is currently available. A 36 kDa L. donovani nucleoside hydrolase (LdNH36) surface protein has been previously identified as a potential vaccine candidate antigen. Here we present data on the expression of LdNH36 in Pichia pastoris and its purification at the 20 L scale to establish suitability for future pilot scale manufacturing. To improve efficiency of process development and ensure reproducibility, 4 N-linked glycosylation sites shown to contribute to heterogeneous high-mannose glycosylation were mutated to glutamine residues. The mutant LdNH36 (LdNH36-dg2) was expressed and purified to homogeneity. Size exclusion chromatography and light scattering demonstrated that LdNH36-dg2 existed as a tetramer in solution, similar to the wild-type recombinant L. major nucleoside hydrolase. The amino acid mutations do not affect the tetrameric interface as confirmed by theoretical modeling, and the mutated amino acids are located outside the major immunogenic domain. Immunogenic properties of the LdNH36-dg2 recombinant protein were evaluated in BALB/c mice using formulations that included a synthetic CpG oligodeoxynucleotide, together with a microparticle delivery platform (poly(lactic-co-glycolic acid)). Mice exhibited high levels of IgG1, IgG2a, and IgG2b antibodies that were reactive to both LdNH36-dg2 and LdNH36 wild-type. While the point mutations did affect the hydrolase activity of the enzyme, the IgG antibodies elicited by LdNH36-dg2 were shown to inhibit the hydrolase activity of the wild-type LdNH36. The results indicate that LdNH36-dg2 as expressed in and purified from P. pastoris is suitable for further scale-up, manufacturing, and testing in support of future first-in-humans phase 1 clinical trials.
Assuntos
Antígenos de Protozoários/imunologia , Expressão Gênica , Leishmania donovani/imunologia , Proteínas Mutantes/imunologia , N-Glicosil Hidrolases/imunologia , Proteínas Recombinantes/imunologia , Animais , Anticorpos Antiprotozoários/sangue , Antígenos de Protozoários/genética , Cromatografia em Gel , Difusão Dinâmica da Luz , Feminino , Imunoglobulina G/sangue , Leishmania donovani/genética , Camundongos Endogâmicos BALB C , Modelos Moleculares , Peso Molecular , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/isolamento & purificação , N-Glicosil Hidrolases/genética , Pichia/genética , Pichia/metabolismo , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificaçãoRESUMO
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 Tc24 calcium binding protein from the flagellar pocket of Trypanosoma cruzi is under evaluation as a candidate vaccine antigen against Chagas disease. Previously, a DNA vaccine encoding Tc24 was shown to be an effective vaccine (both as a preventive and therapeutic intervention) in mice and dogs, as evidenced by reductions in T. cruzi parasitemia and cardiac amastigotes, as well as reduced cardiac inflammation and increased host survival. Here we developed a suitable platform for the large scale production of recombinant Tc24 (rTc24) and show that when rTc24 is combined with a monophosphoryl-lipid A (MPLA) adjuvant, the formulated vaccine induces a Th1-biased immune response in mice, comprised of elevated IgG2a antibody levels and interferon-gamma levels from splenocytes, compared to controls. These immune responses also resulted in statistically significant decreased T. cruzi parasitemia and cardiac amastigotes, as well as increased survival following T. cruzi challenge infections, compared to controls. Partial protective efficacy was shown regardless of whether the antigen was expressed in Escherichia coli or in yeast (Pichia pastoris). While mouse vaccinations will require further modifications in order to optimize protective efficacy, such studies provide a basis for further evaluations of vaccines comprised of rTc24, together with alternative adjuvants and additional recombinant antigens.
Assuntos
Antígenos de Protozoários/imunologia , Doença de Chagas/prevenção & controle , Vacinas Protozoárias/imunologia , Trypanosoma cruzi/imunologia , Adjuvantes Imunológicos/administração & dosagem , Animais , Anticorpos Antiprotozoários/sangue , Antígenos de Protozoários/genética , Doença de Chagas/imunologia , Clonagem Molecular , Modelos Animais de Doenças , Escherichia coli/genética , Feminino , Expressão Gênica , Interferon gama/metabolismo , Leucócitos Mononucleares/imunologia , Lipídeo A/administração & dosagem , Camundongos Endogâmicos BALB C , Carga Parasitária , Parasitemia/prevenção & controle , Pichia/genética , Vacinas Protozoárias/administração & dosagem , Vacinas Protozoárias/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Baço/imunologia , Análise de Sobrevida , Células Th1/imunologia , Trypanosoma cruzi/genética , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologiaRESUMO
Infection by the human hookworm Necator americanus is a leading cause of anemia and disability in the developing countries of Africa, Asia, and the Americas. In order to prevent childhood hookworm disease in resource poor settings, a recombinant vaccine is under development by the Sabin Vaccine Institute and Texas Children's Hospital Center for Vaccine Development, a Product Development Partnership (PDP). Previously, we reported on the expression and purification of a highly promising hookworm vaccine candidate, Na-GST-1, an N. americanus glutathione s-transferase expressed in Pichia pastoris (yeast), which led to production of 1.5 g of 95% pure recombinant protein at a 20L scale. (1) (,) (2) (,) (3) This yield and purity of Na-GST-1 was sufficient for early pilot manufacturing and initial phase 1 clinical testing. However, based on the number of doses which would be required to allow mass vaccination and a potential goal to deliver a vaccine as inexpensively as possible, a higher yield of expression of the recombinant antigen at the lowest possible cost is highly desirable. Here we report on modifications to the fermentation (upstream process) of the antigen expressed in P. pastoris, and to the purification (downstream process) of the recombinant protein that allowed for a 2-3-fold improvement in the final yield of Na-GST-1 purified protein. The major improvements included upstream process changes such as the addition of a sorbitol pulse and co-feed during methanol induction as well as an extension of the induction stage to approximately 96 hours; downstream process changes included modifying the UFDF to flat sheet with a 10 kDa Molecular Weight cut-off (MWCO), adjusting the capacity of an ion-exchange chromatography step utilizing a gradient elution as opposed to the original step elution, and altering the hydrophobic interaction chromatography conditions. The full process, as well as the purity and stability profiles of the target Na-GST-1, and its formulation on Alhydrogel(®), is described.
Assuntos
Antígenos de Helmintos/isolamento & purificação , Glutationa Transferase/isolamento & purificação , Infecções por Uncinaria/prevenção & controle , Necator americanus/enzimologia , Tecnologia Farmacêutica/métodos , Vacinas Sintéticas/isolamento & purificação , Animais , Antígenos de Helmintos/genética , Biotecnologia/métodos , Química Farmacêutica , Cromatografia Líquida/métodos , Meios de Cultura/química , Estabilidade de Medicamentos , Glutationa Transferase/genética , Infecções por Uncinaria/imunologia , Humanos , Necator americanus/imunologia , Pichia/genética , Pichia/crescimento & desenvolvimento , Pichia/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Ativação Transcricional , Vacinas Sintéticas/genéticaRESUMO
Development of vaccines for preventing a future pandemic of severe acute respiratory syndrome (SARS) caused by SARS coronavirus (SARS-CoV) and for biodefense preparedness is urgently needed. Our previous studies have shown that a candidate SARS vaccine antigen consisting of the receptor-binding domain (RBD) of SARS-CoV spike protein can induce potent neutralizing antibody responses and protection against SARS-CoV challenge in vaccinated animals. To optimize expression conditions for scale-up production of the RBD vaccine candidate, we hypothesized that this could be potentially achieved by removing glycosylation sites in the RBD protein. In this study, we constructed two RBD protein variants: 1) RBD193-WT (193-aa, residues 318-510) and its deglycosylated forms (RBD193-N1, RBD193-N2, RBD193-N3); 2) RBD219-WT (219-aa, residues 318-536) and its deglycosylated forms (RBD219-N1, RBD219-N2, and RBD219-N3). All constructs were expressed as recombinant proteins in yeast. The purified recombinant proteins of these constructs were compared for their antigenicity, functionality and immunogenicity in mice using alum as the adjuvant. We found that RBD219-N1 exhibited high expression yield, and maintained its antigenicity and functionality. More importantly, RBD219-N1 induced significantly stronger RBD-specific antibody responses and a higher level of neutralizing antibodies in immunized mice than RBD193-WT, RBD193-N1, RBD193-N3, or RBD219-WT. These results suggest that RBD219-N1 could be selected as an optimal SARS vaccine candidate for further development.
Assuntos
Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Vacinas Virais/imunologia , Adjuvantes Imunológicos/administração & dosagem , Compostos de Alúmen/administração & dosagem , Animais , Feminino , Expressão Gênica , Glicosilação , Camundongos Endogâmicos BALB C , Pichia/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Glicoproteína da Espícula de Coronavírus/genética , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Vacinas Virais/administração & dosagem , Vacinas Virais/genéticaRESUMO
CONTEXT: Posttransplant diabetes mellitus (PTDM) is a major complication after solid organ transplantation. The use of corticosteroids and calcineurin inhibitors, especially tacrolimus, are significant risk factors. However, it is not clear what genetic factors modify the risk. Evidence suggests vitamin D deficiency, perturbed glucose homeostasis, and increased inflammation all play roles in the development of diabetes. OBJECTIVE: To investigate whether common vitamin D receptor (VDR), cytokine, and peroxisome proliferator-activated receptor γ (PPARγ) polymorphisms are correlated with the development of PTDM. DESIGN: DNA was isolated from the peripheral blood of 51 kidney transplant recipients with PTDM and 72 patients without diabetes pretransplant or posttransplant at the time of follow-up. The genotypes for 5 polymorphisms, 1 each in VDR, PPARγ, INFγ, TGFß1, and TNF, were determined using direct sequencing. Age, sex, number of acute rejection episodes, follow-up length, ethnicity, body mass index, and the frequency of alleles and genotypes for each polymorphism were compared between the 2 groups. RESULTS: Body mass index was the only factor that was statistically different between the 2 groups (P â=â .001). The frequency of different alleles and genotypes for each of the 5 polymorphisms did not differ between the 2 groups. CONCLUSIONS: These results indicate that increased body mass index is a significant risk factor for the development of PTDM. However, none of the genetic polymorphisms studied confer predisposition to PTDM with the current sample size.