RESUMEN
Multivalent live-attenuated or inactivated vaccines are often used to control the bovine viral diarrhea disease (BVD). Still, they retain inherent disadvantages and do not provide the expected protection. This study developed a new vaccine prototype, including the external segment of the E2 viral protein from five different subgenotypes selected after a massive screening. The E2 proteins of every subgenotype (1aE2, 1bE2, 1cE2, 1dE2, and 1eE2) were produced in mammalian cells and purified by IMAC. An equimolar mixture of E2 proteins formulated in an oil-in-water adjuvant made up the vaccine candidate, inducing a high humoral response at 50, 100, and 150 µg doses in sheep. A similar immune response was observed in bovines at 50 µg. The cellular response showed a significant increase in the transcript levels of relevant Th1 cytokines, while those corresponding to the Th2 cytokine IL-4 and the negative control were similar. High levels of neutralizing antibodies against the subgenotype BVDV1a demonstrated the effectiveness of our vaccine candidate, similar to that observed in the sera of animals vaccinated with the commercial vaccine. These results suggest that our vaccine prototype could become an effective recombinant vaccine against the BVD.
Asunto(s)
Anticuerpos Antivirales , Diarrea Mucosa Bovina Viral , Vacunas de Subunidad , Vacunas Sintéticas , Vacunas Virales , Animales , Bovinos , Vacunas Virales/inmunología , Vacunas de Subunidad/inmunología , Anticuerpos Antivirales/inmunología , Anticuerpos Antivirales/sangre , Vacunas Sintéticas/inmunología , Diarrea Mucosa Bovina Viral/prevención & control , Diarrea Mucosa Bovina Viral/inmunología , Diarrea Mucosa Bovina Viral/virología , Anticuerpos Neutralizantes/inmunología , Anticuerpos Neutralizantes/sangre , Ovinos , Proteínas del Envoltorio Viral/inmunología , Proteínas del Envoltorio Viral/genética , Citocinas/metabolismo , Virus de la Diarrea Viral Bovina/inmunología , Virus de la Diarrea Viral Bovina/genética , Virus de la Diarrea Viral Bovina Tipo 1/inmunología , Virus de la Diarrea Viral Bovina Tipo 1/genéticaRESUMEN
Assisted reproduction is a key aspect of modern animal breeding, providing valuable assistance in improving breeding programs. In this field, the administration of exogenous hormones, such as follicle-stimulating hormone (FSH), plays a crucial role in the induction of multiple ovulations. However, commercial FSH used in veterinary practice has been derived primarily from pituitary glands, obtained mostly from pigs for nearly four decades. Although these hormones have contributed significantly to the advancement of assisted reproductive techniques, they have certain limitations that warrant further improvements. These limitations include contamination with luteinizing hormone (LH), the potential risk of pathogen contamination, the potential to trigger an immune response in non-pig species, and the short half-life in circulation, requiring the implementation of complex 8-dose superovulation schedules. Our research team has developed and characterized a new variant of bovine follicle-stimulating hormone (bscrFSH) to address these limitations. The new hormone is produced recombinantly in CHO cell cultures, with a specific productivity of about 30 pg/cell/day. The bscrFSH can be purified to a high purity of 97 % using a single step of immobilized metal affinity chromatography (IMAC). N-glycan analysis of bscrFSH showed that approximately 74 % of the glycans corresponded to charged structures, including mono-, di-, tri-, and tetra-sialylated glycans. Superovulation trials conducted in cattle revealed that bscrFSH, administered at a total dose of about 0.5 µg per kg of body weight, using a decrescent schedule of 4 doses with 24-h intervals, resulted in an average yield of 8-12 transferable embryos per animal. Further research is required; however, the preliminary findings indicate that bscrFSH, currently packaged under the provisional brand name of Cebitropin B, holds potential as a commercial product for assisted reproduction in ruminants.
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Hormona Folículo Estimulante , Animales , Bovinos , Hormona Folículo Estimulante/farmacología , Hormona Folículo Estimulante/administración & dosificación , Femenino , Células CHO , Cricetulus , Proteínas Recombinantes , Superovulación/efectos de los fármacosRESUMEN
NK-lysin is a potent antimicrobial peptide (AMP) with antimicrobial activity against bacteria, fungi, viruses, and parasites. NK-lysin is a type of granulysin, a member of the saposin-like proteins family first isolated from a pig's small intestine. In previous work, for the first time, we identified four variants of nk-lysin from Atlantic salmon (Salmo salar) using EST sequences. In the present study, we reported and characterized two additional transcripts of NK-lysin from S. salar. Besides, we evaluated the tissue distribution of three NK-lysins from S. salar and assessed the antimicrobial, hemolytic, and immunomodulatory activities and signaling pathways of three NK-lysin-derived peptides. The synthetic peptides displayed antimicrobial activity against Piscirickettsia salmonis (LF-89) and Flavobacterium psychrophilum. These peptides induced the expression of immune genes related to innate and adaptive immune responses in vitro and in vivo. The immunomodulatory activity of the peptides involves the mitogen-activated protein kinases-mediated signaling pathway, including p38, extracellular signal-regulated kinase 1/2, and/or c-Jun N-terminal kinases. Besides, the peptides modulated the immune response induced by pathogen-associated molecular patterns (PAMPs). Our findings show that NK-lysin could be a highly effective immunostimulant or vaccine adjuvant for use in fish aquaculture.
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Péptidos Antimicrobianos , Proteínas de Peces , Proteolípidos , Salmo salar , Animales , Péptidos Antimicrobianos/metabolismo , Péptidos Antimicrobianos/farmacología , Enfermedades de los Peces/inmunología , Enfermedades de los Peces/microbiología , Proteínas de Peces/metabolismo , Proteínas de Peces/farmacología , Inmunidad Innata , Proteolípidos/metabolismo , Proteolípidos/farmacología , Salmo salar/inmunología , Transducción de SeñalRESUMEN
Toll-like receptor 5 (TLR5) responds to the monomeric form of flagellin and induces the MyD88-depending signaling pathway, activating proinflammatory transcription factors such as NF-κB and the consequent induction of cytokines. On the other hand, HMGB1 is a highly conserved non-histone chromosomal protein shown to interact with and activate TLR5. The present work aimed to design and characterize TLR5 agonist peptides derived from the acidic tail of Salmo salar HMGB1 based on the structural knowledge of the TLR5 surface using global molecular docking platforms. Peptide binding poses complexed on TLR5 ectodomain model from each algorithm were filtrated based on docking scoring functions and predicted theoretical binding affinity of the complex. Circular dichroism spectra were recorded for each peptide selected for synthesis. Only intrinsically disordered peptides (6W, 11W, and SsOri) were selected for experimental functional assay. The functional characterization of the peptides was performed by NF-κB activation assays, RT-qPCR gene expression assays, and Piscirickettsia salmonis challenge in SHK-1 cells. The 6W and 11W peptides increased the nuclear translation of p65 and phosphorylation. In addition, the peptides induced the expression of genes related to the TLR5 pathway activation, pro- and anti-inflammatory response, and differentiation and activation of T lymphocytes towards phenotypes such as TH1, TH17, and TH2. Finally, it was shown that the 11W peptide protects immune cells against infection with P. salmonis bacteria. Overall, the results indicate the usefulness of novel peptides as potential immunostimulants in salmonids.
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Proteína HMGB1 , Salmo salar , Animales , Receptor Toll-Like 5/genética , Receptor Toll-Like 5/metabolismo , FN-kappa B/genética , FN-kappa B/metabolismo , Salmo salar/genética , Salmo salar/metabolismo , Simulación del Acoplamiento Molecular , Péptidos/farmacología , Flagelina/farmacologíaRESUMEN
Pig is one of the most consumed meats worldwide. One of the main conditions for pig production is Porcine Enteropathy caused by Lawsonia intracellularis. Among the effects of this disease is chronic mild diarrhea, which affects the weight gain of pigs, generating economic losses. Vaccines available to prevent this condition do not have the desired effect, but this limitation can be overcome using adjuvants. Pro-inflammatory cytokines, such as interleukin 18 (IL-18), can improve an immune response, reducing the immune window of protection. In this study, recombinant porcine IL-18 was produced and expressed in Escherichia coli and Pichia pastoris. The protein's biological activity was assessed in vitro and in vivo, and we determined that the P. pastoris protein had better immunostimulatory activity. A vaccine candidate against L. intracellularis, formulated with and without IL-18, was used to determine the pigs' cellular and humoral immune responses. Animals injected with the candidate vaccine co-formulated with IL-18 showed a significant increase of Th1 immune response markers and an earlier increase of antibodies than those vaccinated without the cytokine. This suggests that IL-18 acts as an immunostimulant and vaccine adjuvant to boost the immune response against the antigens, reducing the therapeutic window of recombinant protein-based vaccines.
RESUMEN
Previously, we designed a subunit vaccine candidate based on three L. intracellularis antigens with promising results in pigs. In this study, antigens were produced individually to achieve an even antigen ratio in the formulation. The emulsion characterization included the drop size and the mechanical and thermal stability. Immune response was evaluated by indirect and sandwich ELISAs, qPCR, and flow cytometry. The vaccine candidate's safety was assessed by histopathology and monitoring the clinical behavior of animals. The average production yielded for the chimeric antigen as inclusion bodies was around 75 mg/L. The formulation showed mechanical and thermal stability, with a ratio Hu/Ho > 0.85 and a drop size under 0.15 nm. Antigens formulated at a ratio of 1:1:1 induced a significant immune response in inoculated pigs that persisted until the end of the experiment (week 14). The dose of 200 µg significantly activated cellular response measured by transcriptional and translational levels of cytokines. The cell proliferation assay revealed an increment of lymphocytes T CD4+ at the same dose. Animals gained weight constantly and showed proper clinical behavior during immunization assays. This research demonstrated the immunological robustness of the new subunit vaccine candidate against Porcine Proliferative Enteropathy evenly formulated with three chimeric antigens of L. intracellularis.
RESUMEN
Neuropeptides are a group of peptides derived from precursor proteins synthesized in neuronal and nonneuronal cells. The classical functions of neuropeptides have been extensively studied in mammals, including neuromodulation in the central nervous system, molecular signaling in the peripheral nervous system, and immunomodulation associated mainly with anti-inflammatory activity. In contrast, in teleosts, studies of the immunomodulatory function of these neuropeptides are limited. In Oncorhynchus mykiss, vasoactive intestinal peptide (VIP) mRNA sequences have not been cloned, and the role of VIP in modulating the immune system has not been studied. Furthermore, in relation to other neuropeptides with possible immunomodulatory function, such as ghrelin, there are also few studies. Therefore, in this work, we performed molecular cloning, identification, and phylogenetic analysis of three VIP precursor sequences (prepro-VIP1, VIP2 and VIP3) in rainbow trout. In addition, the immunomodulatory function of both neuropeptides was evaluated in an in vitro model using the VIP1 sequence identified in this work and a ghrelin sequence already studied in O. mykiss. The results suggest that the prepro-VIP2 sequence has the lowest percentage of identity with respect to the other homologous sequences and is more closely related to mammalian orthologous sequences. VIP1 induces significant expression of both pro-inflammatory (IFN-γ, IL-1ß) and anti-inflammatory (IL-10 and TGF-ß) cytokines, whereas ghrelin only induces significant expression of proinflammatory cytokines such as IL-6 and TNF-α.
RESUMEN
Vascular endothelial growth factor (VEGF) has essential functions in angiogenesis, endothelial cell proliferation, migration, and tumor invasion. Different approaches have been developed to suppress tumor angiogenesis, which is considered a hallmark of cancer. Anti-VEGF monoclonal antibodies constitute an important strategy for cancer immunotherapy, which has been produced on several platforms. In this study, a novel single-chain anti-VEGF monoclonal antibody (scVEGFmAb) was produced in the goat mammary gland by adenoviral transduction. scVEGFmAb was purified by affinity chromatography. N-glycans were analyzed by exoglycosidase digestion and hydrophilic interaction ultra-performance liquid chromatography coupled to electrospray ionization mass spectrometry. The biological activity of scVEGFmAb was assessed by scratch and mouse aortic ring assays. scVEGFmAb was produced at 0.61 g/L in the goat milk, and its purification rendered 95 % purity. N-glycans attached to scVEGFmAb backbone were mainly neutral biantennary core fucosylated with Galß1,4GlcNAc motif, and charged structures were capped with Neu5Ac and Neu5Gc. The chimeric molecule significantly prevented cell migration and suppressed microvessel sprouting. These results demonstrated for the first time the feasibility of producing an anti-VEGF therapeutic antibody in the milk of non-transgenic goats with the potential to counteract tumor angiogenesis.
Asunto(s)
Leche , Factor A de Crecimiento Endotelial Vascular , Animales , Proliferación Celular , Cabras , Ratones , Polisacáridos , Factor A de Crecimiento Endotelial Vascular/genéticaRESUMEN
Complex recombinant glycoproteins produced as potential biopharmaceuticals in goat's milk have an aberrant pattern of N-glycosylation due to the lack of multi-antennary structures. Overexpression of glycosyltransferases may increase oligosaccharide branching of the desired glycoproteins. Here, human erythropoietin fused to human IgG Fc (EPO-Fc) was co-expressed with N-acetyl-glucosaminyltransferase-IVa (GnT-IVa) by adenoviral transduction in goat mammary gland to evaluate the in vivo modification of N-glycosylation pattern in this tissue. Adenoviral vectors, containing the EPO-Fc and GnT-IVa sequences were assembled for in vitro and in vivo expression in mammalian cell culture or in goat mammary gland. Protein detection was assessed by gel electrophoresis and western blot, and N-glycans were identified by HPLC and mass spectrometry. GnT-IVa overexpression and its colocalization with EPO-Fc in the Golgi apparatus of SiHa cells were demonstrated. N-glycan analysis of in vitro and in vivo expression of EPO-Fc modified by GnT-IVa (EPO-Fc/GnT-IVa) showed an increase in high molecular weight structures, which corresponded to tri- and tetra-antennary N-glycans in SiHa cells and mostly tri-antennary N-glycans in goat's milk from transformed mammary tissue. The results confirmed that successful modification of the goat mammary gland secretion pathway could be achieved by co-expressing glycoenzymes together with the glycoprotein of interest. This is the first report of modification of the N-glycosylation pattern in the goat mammary gland in vivo, and constitutes a step forward for improving the use of the mammary gland as a bioreactor for the production of complex recombinant proteins.
Asunto(s)
Glicoproteínas/metabolismo , Glándulas Mamarias Animales/metabolismo , Animales , Células Cultivadas , Eritropoyetina , Femenino , Glicosilación , Cabras , Humanos , N-Acetilglucosaminiltransferasas , Transducción GenéticaRESUMEN
TNFα is a pro-inflammatory cytokine that is a therapeutic target for inflammatory autoimmune disorders. Thus, TNFα antagonists are successfully used for the treatment of these disorders. Here, new association patterns of rhTNFα and its antagonists Adalimumab and Etanercept are disclosed. Active rhTNFα was purified by IMAC from the soluble fraction of transformed Escherichia coli. Protein detection was assessed by SDS-PAGE and Western blot. The KD values for rhTNFα interactions with their antagonists were obtained by non-competitive ELISA and by microscale thermophoresis (MST). Molecular sizes of the complexes were evaluated by size-exclusion chromatography-high performance liquid chromatography (SEC-HPLC). Surprisingly, both antagonists recognized the monomeric form of rhTNFα under reducing and non-reducing conditions, indicating unexpected bindings of the antagonists to linear epitopes and to rhTNFα monomers. For the first time, the interactions of rhTNFα with Adalimumab and Etanercept were assessed by MST, which allows evaluating molecular interactions in solution with a wide range of concentrations. Biphasic binding curves with low and high KD values (<10-9â M and >10-8â M) were observed during thermophoresis experiments, suggesting the generation of complexes with different stoichiometry, which were confirmed by SEC-HPLC. Our results demonstrated the binding of TNFα-antagonists with rhTNFα monomers and linear epitopes. Also, complexes of high molecular mass were observed. This pioneer investigation constitutes valuable data for future approaches into the study of the interaction mechanism of TNFα and its antagonists.
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Adalimumab/química , Etanercept/química , Factor de Necrosis Tumoral alfa , Humanos , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/químicaRESUMEN
Antimicrobial peptides (AMPs) are amphipathic peptides, which play an important role in innate defence. These peptides are gene-encoded and either constitutively expressed and/or upregulated during an infection. NK-lysins are AMPs with a three-dimensional globular structure. They are larger molecules, which comprise 74-78 amino acid residues and six conserved cysteine residues forming three disulphide bonds. Cathelicidins are a family of antimicrobial peptides that act as important components of the innate immune system with a broad spectrum of antimicrobial activity and immunomodulatory properties. Although they are widely studied in mammals, little is known about their immunomodulatory function. In the present study, we identified and characterized for the first time four NK-lysin-like transcripts from Atlantic salmon (Salmo salar) based on EST reported sequences. In vitro, NK-lysin derived peptides were able to induce the expression of IL-1ß and IL-8 in Salmo salar head kidney leukocytes. We also tested Salmo salar cathelicidin 1 derived peptide in a similar assay, showing its ability to induce the expression of IFN-γ. These results indicate that NK-lysin and cathelicidin 1 derived peptides are able to modulated immune response, suggesting their potential use to enhance immune response in fish.
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Catelicidinas/genética , Proteínas de Peces/inmunología , Factores Inmunológicos/inmunología , Proteolípidos/genética , Salmo salar/inmunología , Animales , Catelicidinas/inmunología , Enfermedades de los Peces/inmunología , Riñón Cefálico/citología , Riñón Cefálico/inmunología , Inmunidad Innata , Interferón gamma/inmunología , Leucocitos/inmunología , Proteolípidos/inmunologíaRESUMEN
Analytical techniques are essential in the process of standardizing and validating vaccines. In this study we described a methodology to establish an ELISA sandwich for the quantification of a new vaccine against avian influenza virus H5N1 based on the main antigenic determinant of the virus, the extracellular domain of the glycoprotein hemagglutinin (HA), fused to the extracellular domain of the chicken CD154 glycoprotein (HACD). The chimerical proteins HA and HACD were produced in SiHa cells and the experiments were performed by using three monoclonal antibodies (MAb-HA1, MAb-HA2 and MAb-HA3), alone or conjugated to horseradish peroxidase (HRP-HA1, HRP-HA2 and HRP-HA3). The hemagglutination inhibition assay was carried out with a negative and a positive H5N2 reference serum, together with the antigen H5N1 A/Mallard/Italy/3401/05, all purchased from the "Istituto Zooprofilattico delle Venezie", Italy. After demonstrating the similar recognition pattern between the HA and the HACD proteins, the MAb-HA2 at a concentration of 2,5⯵g/mL was selected as the capture antibody and the HRP-HA3 at a dilution of 1/20000 was selected as the detection antibody due to their optimal values of optical density at these conditions. The best dynamic range of the standard curve using the protein HACD was achieved at concentrations from 100 to 1,56â¯ng/mL. There were no significant differences when five batches of HACD were quantified by the ELISA sandwich and the bicinchoninic acid method linked to densitometry. In conclusion, the final parameters for the quantification of the chimeric protein HACD using an ELISA sandwich were described, which could contribute to develop a large-scale process for the final vaccine production.
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Anticuerpos Antivirales/sangre , Ensayo de Inmunoadsorción Enzimática/métodos , Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Subtipo H5N1 del Virus de la Influenza A/inmunología , Vacunas contra la Influenza/inmunología , Animales , Pollos/inmunología , Epítopos/inmunología , Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Vacunas contra la Influenza/normas , Gripe AviarRESUMEN
Contact with the urticating setae from the abdomen of adult females of the neo-tropical moth Hylesia metabus gives rise to an urticating dermatitis, characterized by intense pruritus, generalized malaise and occasionally ocular lesions (lepidopterism). The setae contain a pro-inflammatory glycosylated protease homologous to other S1A serine proteases of insects. Deglycosylation with PNGase F in the presence of a buffer prepared with 40% H2 (18)O allowed the assignment of an N-glycosylation site. Five main paucimannosidic N-glycans were identified, three of which were exclusively α(1-6)-fucosylated at the proximal GlcNAc. A considerable portion of these N-glycans are anionic species sulfated on either the 4- or the 6-position of the α(1-6)-mannose residue of the core. The application of chemically and enzymatically modified variants of the toxin in an animal model in guinea pigs showed that the pro-inflammatory and immunological reactions, e.g. disseminated fibrin deposition and activation of neutrophils, are due to the presence of sulfate-linked groups and not on disulfide bonds, as demonstrated by the reduction and S-alkylation of the toxin. On the other hand, the hemorrhagic vascular lesions observed are attributed to the proteolytic activity of the toxin. Thus, N-glycan sulfation may constitute a defense mechanism against predators.
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Péptido-N4-(N-acetil-beta-glucosaminil) Asparagina Amidasa/química , Polisacáridos/química , Serina Proteasas/química , Animales , Glicosilación , Mariposas Nocturnas/enzimología , Péptido-N4-(N-acetil-beta-glucosaminil) Asparagina Amidasa/metabolismo , Polisacáridos/metabolismo , Serina Proteasas/metabolismo , Sulfatos/química , Sulfatos/metabolismoRESUMEN
BACKGROUND: Recombinant erythropoietin (EPO) has been marketed as biopharmaceutical for anemia and chronic renal failure. Long-acting EPO variants that aimed at achieving less frequent dosing have been generated, either by the addition of glycosylation sites or increasing its molecular weight. METHODS: The hEPO cDNA linked to the human IgG Fc fragment was cloned as a single codifying gene on the pAdtrack-CMV vector, yielding the recombinant adenoviral genome. For in vitro and in vivo expression assays cervical cancer cell line (SiHa) and nulliparous goats were used, respectively. The hematopoietic activity of EPO-Fc, expressed as the differential increment of hematocrit was evaluated in B6D2F1 mice. NP-HPLC of the 2AB-labeled N-glycan was carried out to profile analysis. RESULTS: The direct transduction of mammary secretory cells with adenoviral vector is a robust methodology to obtain high levels of EPO of up to 3.5mg/mL in goat's milk. SiHa-derived EPO-Fc showed significant improvement in hematopoietic activity compared to the commercial hEPO counterpart or with the homologous milk-derived EPO-Fc. The role of the molecular weight seemed to be important in enhancing the hematopoietic activity of SiHa-derived EPO-Fc. However, the lack of sialylated multi-antennary glycosylation profile in milk-derived EPO-Fc resulted in lower biological activity. CONCLUSIONS: The low content of tri- or tetra-antennary sialylated N-glycans linked to the chimeric EPO-Fc hormone, expressed in the goat mammary gland epithelial cells, defined its in vivo hematopoietic activity. GENERAL SIGNIFICANCE: The sialylated N-glycan content plays a more significant role in the in vivo biological activity of hEPO than its increased molecular weight.
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Eritropoyetina/farmacología , Hematopoyesis/efectos de los fármacos , Fragmentos Fc de Inmunoglobulinas/farmacología , Procesamiento Proteico-Postraduccional , Proteínas Recombinantes de Fusión/farmacología , Animales , Relación Dosis-Respuesta a Droga , Células HEK293 , Humanos , Ratones , Polisacáridos/farmacologíaRESUMEN
Nimotuzumab (TheraCIM, CIMAher, h-R3, humanized anti-EGF-R antibody), monoclonal antibody (mAb) manufactured at the Center of Molecular Immunology (Havana, Cuba) is currently being tested in several clinical trials. Nimotuzumab has a single N-glycosylation site in the Fc-CH2 fragment but no N-glycosylation site in the Fab region. The current study reports the full characterization of the mAb N-glycosylation and the consistency observed in several production batches from a perfusion mode culturing system that lasted between 68 and 150 days. It confirms that the N-glycan structures of Nimotuzumab expressed in the NS0 murine myeloma cell line are of the murine type. They consist mainly of fucosylated G0, G1 and G2 oligosaccharides, which are normally found in the CH2 region of IgG. Other minor species found were high mannose and sialylated structures. A small portion of the glycans were sialylated (â¼12%) and the only type of sialic acid detected was N-glycolyl-sialic acid, α2,6-linked to Gal. No Galα1-3Gal moieties were detected.
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Anticuerpos Monoclonales Humanizados/química , Oligosacáridos/química , Conformación de Carbohidratos , Cromatografía Líquida de Alta Presión , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Espectrometría de Masas en TándemRESUMEN
Classical swine fever virus (CSFV) outer surface E2 glycoprotein represents an important target to induce protective immunization during infection but the influence of N-glycosylation pattern in antigenicity is yet unclear. In the present work, the N-glycosylation of the E2-CSFV extracellular domain expressed in goat milk was determined. Enzymatic N-glycans releasing, 2-aminobenzamide (2AB) labeling, weak anion-exchange and normal-phase HPLC combined with exoglycosidase digestions and mass spectrometry of 2AB-labeled and unlabeled N-glycans showed a heterogenic population of oligomannoside, hybrid and complex-type structures. The detection of two Man(8)GlcNAc(2) isomers indicates an alternative active pathway in addition to the classical endoplasmic reticulum processing. N-acetyl or N-glycolyl monosialylated species predominate over neutral complex-type N-glycans. Asn207 site-specific micro-heterogeneity of the E2 most relevant antigenic and virulence site was determined by HPLC-mass spectrometry of glycopeptides. The differences in N-glycosylation with respect to the native E2 may not disturb the main antigenic domains when expressed in goat milk.
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Virus de la Fiebre Porcina Clásica/inmunología , Virus de la Fiebre Porcina Clásica/metabolismo , Leche/inmunología , Leche/virología , Proteínas del Envoltorio Viral/inmunología , Proteínas del Envoltorio Viral/metabolismo , Animales , Antígenos Virales/genética , Antígenos Virales/metabolismo , Cromatografía Líquida de Alta Presión , Virus de la Fiebre Porcina Clásica/genética , Femenino , Glicosilación , Cabras , Polisacáridos/química , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismo , Espectrometría de Masa por Ionización de Electrospray , Espectrometría de Masas en Tándem , Transducción Genética , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/genética , Vacunas Virales/genética , Vacunas Virales/inmunologíaRESUMEN
The extracellular domain of E2 glycoprotein outer surface of the classical swine fever virus was expressed in epithelial kidney pig cells. The N-glycosylation determined by combination of Normal Phase-HPLC, Weak Anion Exchange-HPLC, exoglycosidase digestions and Mass Spectrometry revealed a complex mixture of neutral and monosialylated multiantennary N-glycans with variable number of alpha1-3-Gal-Gal antennae terminals. The most abundant neutral N-glycan has a composition of Hex(7)HexNAc(4)dHex(1), Negative ion ESI-MS/MS confirmed the presence of the alpha1-3-Gal-Gal motif on each arm of the fucosylated biantennary N-glycan. The most abundant monosialylated glycan was Hex(6)HexNAc(4)dHex(1)Neu(5)Ac(1), with the sialic acid linked to the terminal beta1-4-Gal-GlcNAc. Sialic acid on the antenna capping position was predominantly of the N-acetyl form.
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Polisacáridos/análisis , Proteínas del Envoltorio Viral/química , Animales , Conformación de Carbohidratos , Secuencia de Carbohidratos , Línea Celular , Células Epiteliales/citología , Células Epiteliales/metabolismo , Glicósido Hidrolasas/metabolismo , Glicosilación , Riñón/citología , Espectrometría de Masas/métodos , Datos de Secuencia Molecular , Sus scrofa , Proteínas del Envoltorio Viral/metabolismoRESUMEN
Classical swine fever virus produces a huge mortality in infected herds during recurrent outbreaks, predominantly in tropical and subtropical areas. In this scenario, it is common that cold-chain related issues affect the efficacy of virus attenuated-derived vaccines, which are frequently used in eradication programs. In the present work, the stability and protective capacity of a recombinant vaccine preparation, based on goat milk derived E2 glycoprotein extracellular domain, were both analyzed after incubation at 4 degrees C or 37 degrees C for 1 week. Differences in the viscosity and in the homodimeric form of the antigen were observed after comparing physicochemical properties of stressed and not stressed vaccine formulations. However, these differences did not affect the immunogenicity and protective capacity of such preparations. Noticeably, pigs immunized with the E2-based vaccine subjected to thermal stress became totally protected from the viral infection, after a challenge with 10(5) PLD(50) of a high virulent classical swine fever strain. This result supports the practical value of this vaccine preparation mostly for those regions in which cold-chain related failures tend to affect the protective capability of conventional virus attenuated vaccines.
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Virus de la Fiebre Porcina Clásica/inmunología , Peste Porcina Clásica/inmunología , Calor , Vacunas Virales/inmunología , Animales , Anticuerpos Antivirales/sangre , Cabras , Leche , PorcinosRESUMEN
E2 is the major envelope glycoprotein present on the outer surface of the classical swine fever virus (CSFV). It is exposed as a homodimer originated by disulfide linkage and represents an important target for the induction of neutralizing immune responses against the viral infection. The E2his glycoprotein nucleotide sequence used in this work contains the CSFV E2 extracellular domain preceded by the tissue plasminogen signal peptide and a hexa-histidine tag in the 3' terminus. The recombinant antigen was produced at a range of 120-150 microg/mL in the culture media of epithelial kidney pig cells, transduced with a replication defective adenoviral vector (Ad-E2his) generated by means of cloning the E2his sequence in the vector genome. The glycoprotein was obtained from clarified culture media as a homodimer of 110 kDa with purity over 95% after a single affinity chromatography step in Ni-NTA Agarose column. The E2his characterization by lectin-specific binding assay showed the presence of N-linked oligosaccharides of both hybrid and complex types. The protective capacity of E2his was demonstrated in two immunization and challenge experiments in pigs using doses of 15 or 30 microg of the glycoprotein, emulsified in Freund's adjuvant. The intramuscular immunization followed by a unique boost three weeks later, elicited high titers of neutralizing antibodies between the second and the fourth week after the primary vaccination. The immunized animals were fully protected from the viral infection after challenge with 10(5) PLD(50) of homologous CSFV "Margarita" strain administered by intramuscular injection. Consequently, no clinical signs of the disease or viral isolation from lymphocytes were detected in the vaccinated pigs. These results suggest that the E2his antigen produced in mammalian cells may be a feasible vaccine candidate for CSF prevention.