RESUMO
Despite that more than one hundred vaccines against SARS-CoV-2 have been developed and that some of them were evaluated in clinical trials, the latest results revealed that these vaccines still face great challenges. Among the components of the virus, the N-protein constitutes an attractive target for a subunit vaccine because it is the most abundant, highly conserved and immunogenic protein. In the present work, a chimeric protein (N-CD protein) was constructed by the fusion of the N-protein to the extracellular domain of human CD154 as the molecular adjuvant. HEK-293 cells were transduced with lentiviral vector bearing the N-CD gene and polyclonal cell populations were obtained. The N-CD protein was purified from cell culture supernatant and further characterized by several techniques. Immunogenicity studies in mice and non-human primates showed the N-CD protein induced high IgG titers in both models after two doses. Moreover, overall health monitoring of non-human primates demonstrated that animals were healthy during 228 days after first immunization. Data obtained support further investigation in order to develop this chimeric protein as vaccine candidate against COVID-19 and other coronavirus diseases.
Assuntos
COVID-19 , Vacinas , Humanos , Animais , Camundongos , SARS-CoV-2/genética , COVID-19/prevenção & controle , Células HEK293 , Vacinas contra COVID-19 , Nucleocapsídeo , Ligante de CD40/genética , Proteínas Recombinantes de Fusão/genéticaRESUMO
COVID-19 is a respiratory viral disease caused by a new coronavirus called SARS-CoV-2. This disease has spread rapidly worldwide with a high rate of morbidity and mortality. The receptor-binding domain (RBD) of protein spike (S) mediates the attachment of the virus to the host's cellular receptor. The RBD domain constitutes a very attractive target for subunit vaccine development due to its ability to induce a neutralizing antibody response against the virus. With the aim of boosting the immunogenicity of RBD, it was fused to the extracellular domain of CD154, an immune system modulator molecule. To obtain the chimeric protein, stable transduction of HEK-293 was carried out with recombinant lentivirus and polyclonal populations and cell clones were obtained. RBD-CD was purified from culture supernatant and further characterized by several techniques. RBD-CD immunogenicity evaluated in mice and non-human primates (NHP) indicated that recombinant protein was able to induce a specific and high IgG response after two doses. NHP sera also neutralize SARS-CoV-2 infection of Vero E6 cells. RBD-CD could improve the current vaccines against COVID-19, based in the enhancement of the host humoral and cellular response. Further experiments are necessary to confirm the utility of RBD-CD as a prophylactic vaccine and/or booster purpose.
RESUMO
Plant cells are able to perform most of the post-translational modifications that are required by recombinant proteins to achieve adequate bioactivity and pharmacokinetics. However, regarding N-glycosylation the processing of plant N-glycans in the Golgi apparatus displays major differences when compared with that of mammalian cells. These differences in N-glycosylation are expected to influence serum clearance rate of plant-derived monoclonal antibodies. The monoclonal antibody against the hepatitis B virus surface antigen expressed in Nicotiana tabacum leaves without KDEL endoplasmic reticulum (ER) retention signal (CB.Hep1(-)KDEL) and with a KDEL (Lys-Asp-Glu-Leu) fused to both IgG light and heavy chains (CB.Hep1(+)KDEL) were tested for in vivo stability in mice. Full characterization of N-glycosylation and aggregate formation in each monoclonal antibody batch was determined. The mouse counterpart (CB.Hep1) was used as control. Both (CB.Hep1(-)KDEL) and (CB.Hep1(+)KDEL) showed a faster initial clearance rate (first 24 h) compared with the analogous murine antibody while the terminal phase was similar in the three antibodies. Despite the differences between CB.Hep1(+)KDEL and CB.Hep1(-)KDEL N-glycans, the in vivo elimination in mice was indistinguishable from each other and higher than the murine monoclonal antibody. Molecular modelling confirmed that N-glycans linked to plantibodies were oriented away from the interdomain region, increasing the accessibility of the potential glycan epitopes by glycoprotein receptors that might be responsible for the difference in stability of these molecules.
Assuntos
Anticorpos Monoclonais/biossíntese , Retículo Endoplasmático/metabolismo , Anticorpos Anti-Hepatite B/biossíntese , Nicotiana/imunologia , Planticorpos/metabolismo , Sinais Direcionadores de Proteínas , Agrobacterium tumefaciens/genética , Agrobacterium tumefaciens/metabolismo , Animais , Anticorpos Monoclonais/imunologia , Clonagem Molecular , Epitopos/imunologia , Epitopos/metabolismo , Feminino , Regulação da Expressão Gênica de Plantas , Glicosilação , Anticorpos Anti-Hepatite B/imunologia , Antígenos da Hepatite B/imunologia , Vírus da Hepatite B/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Folhas de Planta/imunologia , Folhas de Planta/metabolismo , Planticorpos/imunologia , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/imunologia , Plantas Geneticamente Modificadas/metabolismo , Polissacarídeos/imunologia , Polissacarídeos/isolamento & purificação , Polissacarídeos/metabolismo , Estabilidade Proteica , Coelhos , Nicotiana/genética , Nicotiana/metabolismoRESUMO
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.
Assuntos
Vírus da Febre Suína Clássica/imunologia , Vírus da Febre Suína Clássica/metabolismo , Leite/imunologia , Leite/virologia , Proteínas do Envelope Viral/imunologia , Proteínas do Envelope Viral/metabolismo , Animais , Antígenos Virais/genética , Antígenos Virais/metabolismo , Cromatografia Líquida de Alta Pressão , Vírus da Febre Suína Clássica/genética , Feminino , Glicosilação , Cabras , Polissacarídeos/química , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em Tandem , Transdução Genética , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/genética , Vacinas Virais/genética , Vacinas Virais/imunologiaRESUMO
Modern vaccines based on purified recombinant antigens have improved their safety; however they induce a suboptimal immune response without the help of adjuvants. Consequently, the development of new adjuvants to enhance the immunogenicity of purified subunit antigens and modulate resulting immune responses is of great interest. In the present study, we evaluated the ability of antimicrobial peptides Oreochromicins previously isolated from tilapia Oreochromis niloticus to enhance adaptive immune responses in mice and tilapia. When co-administrated with ovalbumin in mice, Oreochromicin-1 induced a TH1 humoral immune response. Oreochromicin-2 and 3 induce a TH1 cellular immune response characterized by the induction of interferon-γ in a dose depend manner. Additionally, co-administration of Oreochromicin-1 with the sea lice my32 from Lepeophtheirus salmonis antigen (my32-Ls) increases the humoral immune response in mice and tilapia. We also tested different combinations of these Oreochromicins with the sea lice antigen my32-Ls in mice. Humoral and cellular TH1 responses were enhanced by co-administration of my32-Ls/Oreochromicin-3 and the combination my32-Ls/Oreochromicin-2/3. In agreement with these results, Oreochromicin-1 and 3 enhanced in vitro TH1 cytokine IFN-γ production in Concanavalin A primed splenocytes from naïve mice after a 48h incubation period. In summary, the results showed that tilapia alpha-helical antimicrobial peptides Oreochromicins are able to boost immune response in mammals and fish, encouraging their use as TH1 molecular adjuvants to subunit antigens.
Assuntos
Adjuvantes Imunológicos/administração & dosagem , Antígenos/administração & dosagem , Peptídeos Catiônicos Antimicrobianos/administração & dosagem , Ciclídeos/imunologia , Células Th1/imunologia , Vacinas/imunologia , Imunidade Adaptativa , Adjuvantes Imunológicos/farmacologia , Animais , Antígenos/imunologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Células Cultivadas , Copépodes , Feminino , Imunidade Celular , Imunidade Humoral , Interferon gama/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Baço/citologia , Baço/imunologiaRESUMO
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.