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1.
Clin Immunol ; 231: 108850, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34506944

RESUMO

BACKGROUND: The coronavirus disease 2019 (COVID-19) pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, has resulted in considerable morbidity and mortality in humans. Little is known regarding the development of immunological memory following SARS-CoV-2 infection or whether immunological memory can provide long-lasting protection against reinfection. Urgent need for vaccines is a considerable issue for all governments worldwide. METHODS: A total of 39 patients were recruited in this study. Tonsillar mononuclear cells (MNCs) were co-cultured in RPMI medium and stimulated with the full-length SARS-CoV-2 spike protein in the presence and absence of a CpG-DNA adjuvant. An enzyme-linked immunosorbent assay (ELISA) was utilised to measure the specific antibody response to the spike protein in the cell culture supernatants. RESULTS: The SARS-CoV-2 spike protein primed a potent memory B cell-mediated immune response in nasal-associated lymphoid tissue (NALT) from patients previously infected with the virus. Additionally, spike protein combined with the CpG-DNA adjuvant induced a significantly increased level of specific anti-spike protein IgG antibody compared with the spike protein alone (p < 0.0001, n = 24). We also showed a strong positive correlation between the specific anti-spike protein IgG antibody level in a serum samples and that produced by MNCs derived from the same COVID-19-recovered patients following stimulation (r = 0.76, p = 0.0002, n = 24). CONCLUSION: Individuals with serological evidence of previous SARS-CoV-2 exposure showed a significant anti-spike protein-specific memory humoral immune response to the viral spike protein upon stimulation. Additionally, our results demonstrated the functional response of NALT-derived MNCs to the viral spike protein. CpG-DNA adjuvant combined with spike protein induced significantly stronger humoral immune responses than the spike protein alone. These data indicate that the S protein antigen combined with CpG-DNA adjuvant could be used as a future vaccine candidate.


Assuntos
COVID-19/imunologia , COVID-19/virologia , Memória Imunológica/fisiologia , Tecido Linfoide/fisiologia , SARS-CoV-2/imunologia , Anticorpos Antivirais/metabolismo , Linfócitos B , Células Cultivadas , DNA , Ensaio de Imunoadsorção Enzimática , Humanos , Imunidade Celular , Imunidade Humoral , Imunoglobulina G/metabolismo , Tecido Linfoide/virologia , Nariz , Oligodesoxirribonucleotídeos , Glicoproteína da Espícula de Coronavírus/imunologia
3.
Clin Chem Lab Med ; 59(11): 1878-1884, 2021 10 26.
Artigo em Inglês | MEDLINE | ID: mdl-34348424

RESUMO

OBJECTIVES: Numerous analytical systems, rapidly made available on the market throughout the SARS-CoV-2 pandemic, aim to detect COVID-19, and to continuously update and improve the same systems. Medical laboratory professionals have also developed in-house analytical procedures in order to satisfy the enormous volume of requests for tests. These developments have highlighted the need control the analytical procedures used in order to guarantee patient safety. The External Quality Assessment (EQA) Scheme, an important quality assurance tool, aims to guarantee high standard performance for laboratory and analytical procedures. The aim of the present study was to report on the results collected in an experimental EQA scheme for the serological diagnosis of SARS-CoV-2. METHODS: All qualitative results collected in the different EQA surveys were summarized in order to identify the percentage of laboratory results in relation to typology of antibodies, results and samples. RESULTS: A total of 4,867 data sets were collected. The analysis of EQA data made, demonstrates a better agreement among laboratories results for total Ig than single immunoglobulins (IgG, IgM, IgA) in the case samples positive for SARS-CoV-2, and a wide divergence between IgM results for positive samples (only 34.9% were correct). Results for negative controls and specificity controls demonstrated a better overall agreement than results for positive samples. CONCLUSIONS: Working in collaboration with the IVD manufacturers, laboratory professionals must strive to achieve harmonization of results, and to develop well-defined protocols complying with the ISO 15189 requirements.


Assuntos
Anticorpos Antivirais/imunologia , COVID-19/diagnóstico , SARS-CoV-2/imunologia , Testes Sorológicos/métodos , Anticorpos Antivirais/sangue , COVID-19/imunologia , Humanos , Imunoglobulina A/sangue , Imunoglobulina A/metabolismo , Imunoglobulina G/sangue , Imunoglobulina G/metabolismo , Imunoglobulina M/sangue , Imunoglobulina M/metabolismo , Projetos Piloto , Garantia da Qualidade dos Cuidados de Saúde , Estudos Retrospectivos , Sensibilidade e Especificidade , Índice de Gravidade de Doença
4.
J Immunol ; 207(5): 1310-1321, 2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-34380652

RESUMO

The respiratory tract is constantly exposed to various airborne pathogens. Most vaccines against respiratory infections are designed for the parenteral routes of administration; consequently, they provide relatively minimal protection in the respiratory tract. A vaccination strategy that aims to induce the protective mucosal immune responses in the airway is urgently needed. The FcRn mediates IgG Ab transport across the epithelial cells lining the respiratory tract. By mimicking this natural IgG transfer, we tested whether FcRn delivers vaccine Ags to induce a protective immunity to respiratory infections. In this study, we designed a monomeric IgG Fc fused to influenza virus hemagglutinin (HA) Ag with a trimerization domain. The soluble trimeric HA-Fc were characterized by their binding with conformation-dependent HA Abs or FcRn. In wild-type, but not FcRn knockout, mice, intranasal immunization with HA-Fc plus CpG adjuvant conferred significant protection against lethal intranasal challenge with influenza A/PR/8/34 virus. Further, mice immunized with a mutant HA-Fc lacking FcRn binding sites or HA alone succumbed to lethal infection. Protection was attributed to high levels of neutralizing Abs, robust and long-lasting B and T cell responses, the presence of lung-resident memory T cells and bone marrow plasma cells, and a remarkable reduction of virus-induced lung inflammation. Our results demonstrate for the first time, to our knowledge, that FcRn can effectively deliver a trimeric viral vaccine Ag in the respiratory tract and elicit potent protection against respiratory infection. This study further supports a view that FcRn-mediated mucosal immunization is a platform for vaccine delivery against common respiratory pathogens.


Assuntos
Antígenos de Histocompatibilidade Classe I/metabolismo , Vacinas contra Influenza/imunologia , Influenza Humana/imunologia , Infecções por Orthomyxoviridae/imunologia , Orthomyxoviridae/fisiologia , Receptores Fc/metabolismo , Mucosa Respiratória/metabolismo , Administração Intranasal , Animais , Anticorpos Antivirais/metabolismo , Modelos Animais de Doenças , Resistência à Doença , Feminino , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Antígenos de Histocompatibilidade Classe I/genética , Humanos , Fragmentos Fc das Imunoglobulinas/genética , Imunoglobulina G/metabolismo , Vacinas contra Influenza/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores Fc/genética , Mucosa Respiratória/imunologia , Vacinação
5.
Int J Mol Sci ; 22(15)2021 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-34360971

RESUMO

Interleukin-22 (IL-22) plays a role in epithelial barrier function and repair, and may provide benefits in conditions like inflammatory bowel disease. However, limited human data are available to assess the clinical effect of IL-22 administration. This study used a human intestinal cell line to identify an IL-22-dependent gene signature that could serve as a pharmacodynamic biomarker for IL-22 therapy. The response to IL-22Fc (UTTR1147A, an Fc-stabilized version of IL-22) was assessed in HT-29 cells by microarray, and the selected responsive genes were confirmed by qPCR. HT-29 cells demonstrated dose-dependent increases in STAT3 phosphorylation and multiple gene expression changes in response to UTTR1147A. Genes were selected that were upregulated by UTTR1147A, but to a lesser extent by IL-6, which also signals via STAT3. IL-1R1 was highly upregulated by UTTR1147A, and differential gene expression patterns were observed in response to IL-22Fc in the presence of IL-1ß. An IL-22-dependent gene signature was identified that could serve as a pharmacodynamic biomarker in intestinal biopsies to support the clinical development of an IL-22 therapeutic. The differential gene expression pattern in the presence of IL-1ß suggests that an inflammatory cytokine milieu in the disease setting could influence the clinical responses to IL-22.


Assuntos
Anti-Inflamatórios/farmacologia , Imunoglobulina G/genética , Doenças Inflamatórias Intestinais/metabolismo , Interleucinas/genética , Transcriptoma/efeitos dos fármacos , Biomarcadores/metabolismo , Células HT29 , Humanos , Imunoglobulina G/metabolismo , Interleucinas/metabolismo , Receptores de Interleucina-1/genética , Receptores de Interleucina-1/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Fator de Transcrição STAT3/metabolismo
6.
Sci Rep ; 11(1): 17325, 2021 08 30.
Artigo em Inglês | MEDLINE | ID: mdl-34462501

RESUMO

Two-dose messenger RNA vaccines against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are highly effective in preventing symptomatic COVID-19 infection. However, the durability of protection is not known, nor is the effectiveness against emerging viral variants. Additionally, vaccine responses may differ based on prior SARS-CoV-2 exposure history. To investigate protection against SARS-CoV-2 variants we measured binding and neutralizing antibody responses following both vaccine doses. We document significant declines in antibody levels three months post-vaccination, and reduced neutralization of emerging variants, highlighting the need to identify correlates of clinical protection to inform the timing of and indications for booster vaccination.


Assuntos
Enzima de Conversão de Angiotensina 2/metabolismo , Anticorpos Neutralizantes/análise , COVID-19/prevenção & controle , SARS-CoV-2/imunologia , Vacinas Sintéticas/administração & dosagem , Adulto , Anticorpos Neutralizantes/metabolismo , Anticorpos Antivirais/análise , Anticorpos Antivirais/metabolismo , COVID-19/imunologia , COVID-19/metabolismo , Teste de Ácido Nucleico para COVID-19 , Vacinas contra COVID-19/administração & dosagem , Vacinas contra COVID-19/imunologia , Feminino , Humanos , Imunoglobulina G/análise , Imunoglobulina G/metabolismo , Masculino , Pessoa de Meia-Idade , Glicoproteína da Espícula de Coronavírus/metabolismo , Fatores de Tempo , Vacinação , Vacinas Sintéticas/imunologia , Adulto Jovem
7.
EBioMedicine ; 71: 103561, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34455390

RESUMO

BACKGROUND: Assessment of the kinetics of SARS-CoV-2 antibodies is essential in predicting risk of reinfection and durability of vaccine protection. METHODS: This is a prospective, monocentric, longitudinal, cohort clinical study. Healthcare workers (HCW) from Strasbourg University Hospital were enrolled between April 6th and May 7th, 2020 and followed up to 422 days. Serial serum samples were tested for antibodies against the Receptor Binding Domain (RBD) of the spike protein and nucleocapsid protein (N) to characterize the kinetics of SARS-CoV-2 antibodies and the incidence of reinfection. Live-neutralization assays were performed for a subset of samples before and after vaccination to analyze sensitivity to SARS-CoV-2 variants. FINDINGS: A total of 4290 samples from 393 convalescent COVID-19 and 916 COVID-19 negative individuals were analyzed. In convalescent individuals, SARS-CoV-2 antibodies followed a triphasic kinetic model with half-lives at month (M) 11-13 of 283 days (95% CI 231-349) for anti-N and 725 days (95% CI 623-921) for anti-RBD IgG, which stabilized at a median of 1.54 log BAU/mL (95% CI 1.42-1.67). The incidence of SARS-CoV-2 infections was 12.22 and 0.40 per 100 person-years in COVID-19-negative and COVID-19-positive HCW, respectively, indicating a relative reduction in the incidence of SARS-CoV-2 reinfection of 96.7%. Live-virus neutralization assay revealed that after one year, variants D614G and B.1.1.7, but less so B.1.351, were sensitive to anti-RBD antibodies at 1.4 log BAU/mL, while IgG ≥ 2.0 log BAU/mL strongly neutralized all three variants. These latter anti-RBD IgG titers were reached by all vaccinated HCW regardless of pre-vaccination IgG levels and type of vaccine. INTERPRETATION: Our study demonstrates a long-term persistence of anti-RBD antibodies that may reduce risk of reinfection. By significantly increasing cross-neutralizing antibody titers, a single-dose vaccination strengthens protection against variants. FUN1DING: None.


Assuntos
COVID-19/patologia , Imunidade Humoral , Reinfecção/patologia , Adulto , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/metabolismo , COVID-19/imunologia , COVID-19/virologia , Vacinas contra COVID-19/administração & dosagem , Vacinas contra COVID-19/imunologia , Proteínas do Nucleocapsídeo de Coronavírus/imunologia , Feminino , Humanos , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Imunoglobulina G/metabolismo , Cinética , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Fosfoproteínas/imunologia , Estudos Prospectivos , SARS-CoV-2/isolamento & purificação , SARS-CoV-2/metabolismo , Glicoproteína da Espícula de Coronavírus/imunologia , Fatores de Tempo
8.
Sci Rep ; 11(1): 14390, 2021 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-34257394

RESUMO

The coronavirus disease 2019 (COVID-19) pandemic affected people at all ages. Whereas pregnant women seemed to have a worse course of disease than age-matched non-pregnant women, the risk of feto-placental infection is low. Using a cohort of 66 COVID-19-positive women in late pregnancy, we correlated clinical parameters with disease severity, placental histopathology, and the expression of viral entry and Interferon-induced transmembrane (IFITM) antiviral transcripts. All newborns were negative for SARS-CoV-2. None of the demographic parameters or placental histopathological characteristics were associated with disease severity. The fetal-maternal transfer ratio for IgG against the N or S viral proteins was commonly less than one, as recently reported. We found that the expression level of placental ACE2, but not TMPRSS2 or Furin, was higher in women with severe COVID-19. Placental expression of IFITM1 and IFITM3, which have been implicated in antiviral response, was higher in participants with severe disease. We also showed that IFITM3 protein expression, which localized to early and late endosomes, was enhanced in severe COVID-19. Our data suggest an association between disease severity and placental SARS-CoV-2 processing and antiviral pathways, implying a role for these proteins in placental response to SARS-CoV-2.


Assuntos
COVID-19/metabolismo , Placenta/metabolismo , SARS-CoV-2/patogenicidade , Adulto , Enzima de Conversão de Angiotensina 2/metabolismo , Feminino , Furina/metabolismo , Humanos , Imunoglobulina G/metabolismo , Transmissão Vertical de Doenças Infecciosas , Masculino , Proteínas do Nucleocapsídeo/metabolismo , Gravidez , Complicações Infecciosas na Gravidez/metabolismo , Complicações Infecciosas na Gravidez/virologia , Serina Endopeptidases/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , Adulto Jovem
9.
Int J Mol Sci ; 22(12)2021 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-34205578

RESUMO

Surface plasmon resonance (SPR)-based optical biosensors offer real-time and label-free analysis of protein interactions, which has extensively contributed to the discovery and development of therapeutic monoclonal antibodies (mAbs). As the biopharmaceutical market for these biologics and their biosimilars is rapidly growing, the role of SPR biosensors in drug discovery and quality assessment is becoming increasingly prominent. One of the critical quality attributes of mAbs is the N-glycosylation of their Fc region. Other than providing stability to the antibody, the Fc N-glycosylation influences immunoglobulin G (IgG) interactions with the Fcγ receptors (FcγRs), modulating the immune response. Over the past two decades, several studies have relied on SPR-based assays to characterize the influence of N-glycosylation upon the IgG-FcγR interactions. While these studies have unveiled key information, many conclusions are still debated in the literature. These discrepancies can be, in part, attributed to the design of the reported SPR-based assays as well as the methodology applied to SPR data analysis. In fact, the SPR biosensor best practices have evolved over the years, and several biases have been pointed out in the development of experimental SPR protocols. In parallel, newly developed algorithms and data analysis methods now allow taking into consideration complex biomolecular kinetics. In this review, we detail the use of different SPR biosensing approaches for characterizing the IgG-FcγR interactions, highlighting their merit and inherent experimental complexity. Furthermore, we review the latest SPR-derived conclusions on the influence of the N-glycosylation upon the IgG-FcγR interactions and underline the differences and similarities across the literature. Finally, we explore new avenues taking advantage of novel computational analysis of SPR results as well as the latest strategies to control the glycoprofile of mAbs during production, which could lead to a better understanding and modelling of the IgG-FcγRs interactions.


Assuntos
Imunoglobulina G/metabolismo , Receptores de IgG/metabolismo , Ressonância de Plasmônio de Superfície
11.
ACS Chem Biol ; 16(7): 1142-1146, 2021 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-34152722

RESUMO

While natural protein-protein interactions have evolved to be induced by complex stimuli, rational design of interactions that can be switched-on-demand still remain challenging in the protein design world. Here, we demonstrate that a computationally redesigned natural interface for improved binding affinity could further be mutated to adopt a pH switchable interaction. The redesigned interface of Protein G/human IgG Fc domain (referred to as PrG/hIgG), when incorporated with histidine and glutamic acid on PrG (PrG-EHHE), showed a switch in binding affinity by 50-fold when the pH was altered from mild acidic to mild basic. The wild-type (WT) interface showed a negligible switch. The overall binding affinity under mild acidic pH for PrG-EHHE outperformed the wild-type PrG (PrG-WT) interaction. The new reagent PrG-EHHE can be revolutionary in IgG purification, since the standard method of using an extreme acidic pH for elution can be circumvented.


Assuntos
Proteínas de Bactérias/metabolismo , Fragmentos Fc das Imunoglobulinas/metabolismo , Imunoglobulina G/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Ácido Glutâmico/química , Histidina/química , Humanos , Concentração de Íons de Hidrogênio , Fragmentos Fc das Imunoglobulinas/química , Fragmentos Fc das Imunoglobulinas/genética , Imunoglobulina G/química , Mutação , Ligação Proteica , Domínios Proteicos , Engenharia de Proteínas , Streptococcus/química
12.
Cell ; 184(15): 3936-3948.e10, 2021 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-34192529

RESUMO

In this study we profiled vaccine-induced polyclonal antibodies as well as plasmablast-derived mAbs from individuals who received SARS-CoV-2 spike mRNA vaccine. Polyclonal antibody responses in vaccinees were robust and comparable to or exceeded those seen after natural infection. However, the ratio of binding to neutralizing antibodies after vaccination was greater than that after natural infection and, at the monoclonal level, we found that the majority of vaccine-induced antibodies did not have neutralizing activity. We also found a co-dominance of mAbs targeting the NTD and RBD of SARS-CoV-2 spike and an original antigenic-sin like backboost to spikes of seasonal human coronaviruses OC43 and HKU1. Neutralizing activity of NTD mAbs but not RBD mAbs against a clinical viral isolate carrying E484K as well as extensive changes in the NTD was abolished, suggesting that a proportion of vaccine-induced RBD binding antibodies may provide substantial protection against viral variants carrying single E484K RBD mutations.


Assuntos
Anticorpos Antivirais/imunologia , Vacinas contra COVID-19/imunologia , RNA Mensageiro/imunologia , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/imunologia , Vacinação , Substituição de Aminoácidos , Enzima de Conversão de Angiotensina 2/imunologia , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Neutralizantes/imunologia , Formação de Anticorpos/imunologia , Ligação Competitiva , Humanos , Imunoglobulina G/metabolismo , Mutação/genética , Domínios Proteicos , Hipermutação Somática de Imunoglobulina/genética
13.
Int J Mol Sci ; 22(10)2021 May 12.
Artigo em Inglês | MEDLINE | ID: mdl-34065791

RESUMO

Intestinal homeostasis encompasses a complex and balanced interplay among a wide array of components that collaborate to maintain gut barrier integrity. The appropriate function of the gut barrier requires the mucus layer, a sticky cushion of mucopolysaccharides that overlays the epithelial cell surface. Mucus plays a critical anti-inflammatory role by preventing direct contact between luminal microbiota and the surface of the epithelial cell monolayer. Moreover, mucus is enriched with pivotal effectors of intestinal immunity, such as immunoglobulin A (IgA). A fragile and delicate equilibrium that supports proper barrier function can be disturbed by stress. The impact of stress upon intestinal homeostasis results from neuroendocrine mediators of the brain-gut axis (BGA), which comprises a nervous branch that includes the enteric nervous system (ENS) and the sympathetic and parasympathetic nervous systems, as well as an endocrine branch of the hypothalamic-pituitary-adrenal axis. This review is the first to discuss the experimental animal models that address the impact of stress on components of intestinal homeostasis, with special emphasis on intestinal mucus and IgA. Basic knowledge from animal models provides the foundations of pharmacologic and immunological interventions to control disturbances associated with conditions that are exacerbated by emotional stress, such as irritable bowel syndrome.


Assuntos
Imunoglobulina G/metabolismo , Mucosa Intestinal/imunologia , Estresse Psicológico/imunologia , Animais , Homeostase , Humanos , Muco/imunologia
14.
Front Immunol ; 12: 695230, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34177962

RESUMO

A detailed understanding of the antibody response against SARS-CoV-2 is of high importance, especially with the emergence of novel vaccines. A multiplex-based assay, analyzing IgG, IgM, and IgA antibodies against the receptor binding domain (RBD), spike 1 (S1), and nucleocapsid proteins of the SARS-CoV-2 virus was set up. The multiplex-based analysis was calibrated against the Elecsys® Anti-SARS-CoV-2 assay on a Roche Cobas® instrument, using positive and negative samples. The calibration of the multiplex based assay yielded a sensitivity of 100% and a specificity of 97.7%. SARS-CoV-2 specific antibody levels were analyzed by multiplex in 251 samples from 221 patients. A significant increase in all antibody types (IgM, IgG, and IgA) against RBD was observed between the first and the third weeks of disease. Additionally, the S1 IgG antibody response increased significantly between weeks 1, 2, and 3 of disease. Class switching appeared to occur earlier for IgA than for IgG. Patients requiring hospital admission and intensive care had higher levels of SARS-CoV-2 specific IgA levels than outpatients. These findings describe the initial antibody response during the first weeks of disease and demonstrate the importance of analyzing different antibody isotypes against multiple antigens and include IgA when examining the immunological response to COVID-19.


Assuntos
Anticorpos Antivirais/metabolismo , COVID-19/imunologia , Imunoglobulina A/metabolismo , Imunoglobulina G/metabolismo , Imunoglobulina M/metabolismo , SARS-CoV-2/imunologia , Adulto , Idoso , Formação de Anticorpos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Domínios Proteicos/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia
15.
ACS Appl Mater Interfaces ; 13(24): 27972-27982, 2021 Jun 23.
Artigo em Inglês | MEDLINE | ID: mdl-34105952

RESUMO

Nicotine vaccines have been investigated to assist with smoking cessation. Because smoking cessation is a long process, past nicotine vaccines required multiple injections to achieve long-term efficacy. It would be of great significance if extended efficacy can be achieved with fewer injections. Here, we report the assembly of lipid-polylactic acid (PLA) and lipid-poly(lactic-co-glycolic acid) (PLGA) hybrid nanoparticle (NP) based nicotine vaccines. Mice immunized with the lipid-PLGA vaccine produced higher titers of nicotine-specific antibodies than the lipid-PLA vaccine in short-term. However, the lipid-PLA vaccine was found to induce long-lasting antibodies. Three months after the immunization, only mice that received first two injections of the lipid-PLGA vaccine and a third injection of the lipid-PLA vaccine achieved a significantly lower brain nicotine concentration of 65.13 ± 20.59 ng/mg than 115.88 ± 37.62 ng/mg from the negative controls. The results indicate that not only the stability of the vaccines but also the combination of the vaccines impacted the long-term efficacy of the immunization. Lastly, both the body weight and the histopathology study suggest that the vaccines were safe to mice. These findings suggest that long-term immunity against nicotine can be realized by a rational administration of nanovaccines of different levels of stability.


Assuntos
Imunidade Humoral/efeitos dos fármacos , Nanopartículas/química , Nicotina/imunologia , Vacinas/imunologia , Animais , Encéfalo/metabolismo , Feminino , Imunoglobulina G/imunologia , Imunoglobulina G/metabolismo , Camundongos Endogâmicos BALB C , Nanopartículas/toxicidade , Nicotina/metabolismo , Poliésteres/química , Poliésteres/toxicidade , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/química , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/toxicidade , Vacinas/química , Vacinas/toxicidade
16.
Diagn Microbiol Infect Dis ; 100(4): 115403, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34058541

RESUMO

Since the worldwide outbreak of the novel coronavirus (SARS-CoV-2), the question raised whether infected patients would elicit long-lasting protective immunity. Several companies developed serological assays for the detection of SARS-CoV-2 antibodies. In this study, we compared 4 different serology assays in convalescents up to 7 months post-infection. Both Abbott assays showed a significative decrease of IgG antibodies over time. Whereas the Elecsys Anti­SARS­CoV­2 N assay (Roche) initially showed a significant increase, antibody titers significantly decreased at the latest timepoint. Although not significant, the Elecsys Anti­SARS­CoV­2 S assay (Roche) showed tendency towards increasing titers overtime. Our data showed that results of SARS-CoV-2 serology should be interpreted with caution.


Assuntos
Anticorpos Antivirais/sangue , Teste Sorológico para COVID-19/métodos , COVID-19/sangue , Imunoglobulina G/sangue , SARS-CoV-2/imunologia , Anticorpos Antivirais/metabolismo , COVID-19/imunologia , Humanos , Imunoglobulina G/metabolismo , Sensibilidade e Especificidade , Fatores de Tempo
17.
Biotechnol J ; 16(7): e2100098, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34014036

RESUMO

BACKGROUND: Ammonia concentrations typically increase during mammalian cell cultures, mainly due to glutamine and other amino acid consumption. An early ammonia stress indicator is a metabolic shift with respect to alanine. To determine the underlying mechanisms of this metabolic shift, a Chinese hamster ovary (CHO) cell line with two distinct ages (standard and young) was cultured in parallel fed-batch bioreactors with 0 mM or 10 mM ammonia added at 12 h. Reduced viable cell densities were observed for the stressed cells, while viability was not significantly affected. The stressed cultures had higher alanine, lactate, and glutamate accumulation. Interestingly, the ammonia concentrations were similar by Day 8.5 for all cultures. We hypothesized the ammonia was converted to alanine as a coping mechanism. Interestingly, no significant differences were observed for metabolite profiles due to cell age. Glycosylation analysis showed the ammonia stress reduced galactosylation, sialylation, and fucosylation. Transcriptome analysis of the standard-aged cultures indicated the ammonia stress had a limited impact on the transcriptome, where few of the significant changes were directly related metabolite or glycosylation reactions. These results indicate that mechanisms used to alleviate ammonia stress are most likely controlled post-transcriptionally, and this is where future research should focus.


Assuntos
Amônia , Imunoglobulina G , Alanina , Animais , Células CHO , Cricetinae , Cricetulus , Glicosilação , Imunoglobulina G/metabolismo
18.
J Med Chem ; 64(11): 7809-7838, 2021 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-34043358

RESUMO

We report on the design, synthesis, and biological evaluation of a series of nucleotide-binding oligomerization-domain-containing protein 2 (NOD2) desmuramylpeptide agonists with improved in vitro and in vivo adjuvant properties. We identified two promising compounds: 68, a potent nanomolar in vitro NOD2 agonist, and the more lipophilic 75, which shows superior adjuvant activity in vivo. Both compounds had immunostimulatory effects on peripheral blood mononuclear cells at the protein and transcriptional levels, and augmented dendritic-cell-mediated activation of T cells, while 75 additionally enhanced the cytotoxic activity of peripheral blood mononuclear cells against malignant cells. The C18 lipophilic tail of 75 is identified as a pivotal structural element that confers in vivo adjuvant activity in conjunction with a liposomal delivery system. Accordingly, liposome-encapsulated 75 showed promising adjuvant activity in mice, surpassing that of muramyl dipeptide, while achieving a more balanced Th1/Th2 immune response, thus highlighting its potential as a vaccine adjuvant.


Assuntos
Acetilmuramil-Alanil-Isoglutamina/química , Adjuvantes Imunológicos/química , Proteína Adaptadora de Sinalização NOD2/agonistas , Acetilmuramil-Alanil-Isoglutamina/metabolismo , Acetilmuramil-Alanil-Isoglutamina/farmacologia , Adjuvantes Imunológicos/metabolismo , Adjuvantes Imunológicos/farmacologia , Animais , Formação de Anticorpos/efeitos dos fármacos , Linhagem Celular , Desenho de Fármacos , Humanos , Imunoglobulina G/metabolismo , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Lipossomos/química , Ativação Linfocitária/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Proteína Adaptadora de Sinalização NOD2/metabolismo , Ovalbumina/imunologia , Relação Estrutura-Atividade , Células Th1/citologia , Células Th1/imunologia , Células Th1/metabolismo , Células Th2/citologia , Células Th2/imunologia , Células Th2/metabolismo
19.
J Immunol Methods ; 495: 113082, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34051226

RESUMO

The development of new diagnostic assays become a priority for managing COVID-19. To this aim, we presented here an in-house ELISA based on the production of two major recombinant and high-quality antigens from SARS-CoV-2. Full-length N and S-RBD fragment proteins fused to mouse IgG2a-Fc were produced in the CHO cell line. Secreted recombinant proteins were easily purified with standard Protein A chromatography and were used in an in-house ELISA to detect anti-N and anti-RBD IgGs in the plasma of COVID-19 RTPCR-positive patients. High reactivity against recombinant antigens was readily detected in all positive plasma samples, whereas no recognition was observed with control healthy subject's plasmas. Remarkably, unpurified recombinant N protein obtained from cell culture supernatant was also suitable for the monitoring by ELISA of IgG levels in positive patients. This work provides an early prospection for low price but high-quality serological kit development.


Assuntos
Teste Sorológico para COVID-19/métodos , COVID-19/imunologia , Ensaio de Imunoadsorção Enzimática/métodos , Fragmentos Fc das Imunoglobulinas/metabolismo , Imunoglobulina G/metabolismo , Proteínas Recombinantes/metabolismo , SARS-CoV-2/fisiologia , Animais , Anticorpos Antivirais/sangue , Células CHO , Teste Sorológico para COVID-19/economia , Custos e Análise de Custo , Cricetulus , Humanos , Fragmentos Fc das Imunoglobulinas/genética , Imunoglobulina G/genética , Proteínas Recombinantes/genética , Sensibilidade e Especificidade , Glicoproteína da Espícula de Coronavírus/imunologia
20.
Sci Rep ; 11(1): 11046, 2021 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-34040046

RESUMO

Among the therapies against the pandemic SARS-CoV-2 virus, monoclonal Antibodies (mAbs) targeting the Spike glycoprotein represent good candidates to interfere in the Spike/ACE2 interaction, preventing virus cell entry. Since anti-spike mAbs, used individually, might be unable to block the virus entry in the case of resistant mutations, we designed an innovative strategy for the isolation of multiple novel human scFvs specific for the binding domain (RBD) of Spike. By panning a large phage display antibody library on immobilized RBD, we obtained specific binders by eluting with ACE2 in order to identify those scFvs recognizing the epitope of Spike interacting with its receptor. We converted the novel scFvs into full size IgG4, differently from the previously isolated IgG1 mAbs, to avoid unwanted potential side effects of IgG1 potent effector functions on immune system. The novel antibodies specifically bind to RBD in a nanomolar range and interfere in the interaction of Spike with ACE2 receptor, either used as purified protein or when expressed on cells in its native conformation. Furthermore, some of them have neutralizing activity for virus infection in cell cultures by using two different SARS-CoV-2 isolates including the highly contagious VOC 202012/01 variant and could become useful therapeutic tools to fight against the SARS-CoV-2 virus.


Assuntos
Anticorpos Neutralizantes/metabolismo , Anticorpos Antivirais/metabolismo , COVID-19/terapia , Imunoglobulina G/metabolismo , Imunoterapia/métodos , SARS-CoV-2/fisiologia , Glicoproteína da Espícula de Coronavírus/metabolismo , Enzima de Conversão de Angiotensina 2/metabolismo , Anticorpos Monoclonais , Anticorpos Neutralizantes/genética , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/genética , Anticorpos Antivirais/imunologia , COVID-19/imunologia , Células Cultivadas , Epitopos , Humanos , Imunoglobulina G/imunologia , Pandemias , Ligação Proteica , Domínios Proteicos/genética , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologia
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