RESUMO
The COVID-19 pandemic caused by the SARS-CoV-2 virus has highlighted the need for serological assays that can accurately evaluate the neutralizing efficiency of antibodies produced during infection or induced by vaccines. However, conventional assays often require the manipulation of live viruses on a level-three biosafety (BSL3) facility, which presents practical and safety challenges. Here, we present a novel, alternative assay that measures neutralizing antibodies (NAbs) against SARS-CoV-2 in plasma using flow cytometry. This assay is based on antibody binding to the S protein and has demonstrated precision in both intra- and inter-assay measurements at a dilution of 1:50. The cut-off was determined using Receiver Operating Characteristic (ROC) analysis and the value of 36.01% has shown high sensitivity and specificity in distinguishing between pre-pandemic sera, COVID-19 patients, and vaccinated individuals. The efficiency significantly correlates with the gold standard test, PRNT. Our new assay offers a safe and efficient alternative to conventional assays for evaluating NAbs against SARS-CoV-2.
Assuntos
Anticorpos Neutralizantes , Anticorpos Antivirais , COVID-19 , Citometria de Fluxo , SARS-CoV-2 , Humanos , Citometria de Fluxo/métodos , SARS-CoV-2/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/sangue , COVID-19/imunologia , COVID-19/diagnóstico , COVID-19/virologia , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/sangue , Glicoproteína da Espícula de Coronavírus/imunologia , Testes de Neutralização/métodos , Teste Sorológico para COVID-19/métodos , Sensibilidade e Especificidade , Masculino , FemininoRESUMO
Human cytomegalovirus (CMV) is a widespread persistent herpes virus requiring lifelong immune surveillance to maintain latency. Such long-term interactions with the immune system may be associated with deleterious effects including immune exhaustion and senescence. Regarding the COVID-19 pandemic, we asked whether CMV-specific cellular and humoral activity could influence immune responses toward SARS-CoV-2 and/or disease severity. All adults with mild (n = 15) and severe (n = 14) COVID-19 were seropositive for anti-CMV IgG, but negative for IgM antibodies. Antibody titers did not correlate with COVID-19 severity. Six patients presented elevated frequencies of CMV-specific CD4 + and CD8 + T cells producing IFNγ, IL-17, and TNFα, designated as CMV high responders (hiT CMV). In comparison to low CMV responders, hiT CMV individuals exhibited higher frequencies of SARS-CoV-2-specific CD4 + IL-17 + and CD8 + IFNγ + , IL-17 + or TNFα + T cells. These results indicate that high frequencies of CMV-specific T cells may be associated with a SARS-CoV-2-reactive profile skewed toward Th17-dominated immunity.
Assuntos
COVID-19 , Infecções por Citomegalovirus , Adulto , Humanos , Fator de Necrose Tumoral alfa , SARS-CoV-2 , Linfócitos T CD4-Positivos , Interleucina-17 , Pandemias , Linfócitos T CD8-Positivos , Anticorpos AntiviraisRESUMO
This is the third year of the SARS-CoV-2 pandemic, and yet most children remain unvaccinated. COVID-19 in children manifests as mostly mild or asymptomatic, however high viral titers and strong cellular and humoral responses are observed upon acute infection. It is still unclear how long these responses persist, and if they can protect from re-infection and/or disease severity. Here, we analyzed immune memory responses in a cohort of children and adults with COVID-19. Important differences between children and adults are evident in kinetics and profile of memory responses. Children develop early N-specific cytotoxic T cell responses, that rapidly expand and dominate their immune memory to the virus. Children's anti-N, but not anti-S, antibody titers increase over time. Neutralization titers correlate with N-specific antibodies and CD8+T cells. However, antibodies generated by infection do not efficiently cross-neutralize variants Gamma or Delta. Our results indicate that mechanisms that protect from disease severity are possibly different from those that protect from reinfection, bringing novel insights for pediatric vaccine design. They also underline the importance of vaccination in children, who remain at risk for COVID-19 despite having been previously infected.
Assuntos
COVID-19 , SARS-CoV-2 , Humanos , Adulto , Criança , Memória Imunológica , Linfócitos T CD8-Positivos , Nucleocapsídeo , AnticorposRESUMO
Purinergic signaling modulates immune function and is involved in the immunopathogenesis of several viral infections. This study aimed to investigate alterations in purinergic pathways in coronavirus disease 2019 (COVID-19) patients. Mild and severe COVID-19 patients had lower extracellular adenosine triphosphate and adenosine levels, and higher cytokines than healthy controls. Mild COVID-19 patients presented lower frequencies of CD4+ CD25+ CD39+ (activated/memory regulatory T cell [mTreg]) and increased frequencies of high-differentiated (CD27- CD28- ) CD8+ T cells compared with healthy controls. Severe COVID-19 patients also showed higher frequencies of CD4+ CD39+ , CD4+ CD25- CD39+ (memory T effector cell), and high-differentiated CD8+ T cells (CD27- CD28- ), and diminished frequencies of CD4+ CD73+ , CD4+ CD25+ CD39+ mTreg cell, CD8+ CD73+ , and low-differentiated CD8+ T cells (CD27+ CD28+ ) in the blood in relation to mild COVID-19 patients and controls. Moreover, severe COVID-19 patients presented higher expression of PD-1 on low-differentiated CD8+ T cells. Both severe and mild COVID-19 patients presented higher frequencies of CD4+ Annexin-V+ and CD8+ Annexin-V+ T cells, indicating increased T-cell apoptosis. Plasma samples collected from severe COVID-19 patients were able to decrease the expression of CD73 on CD4+ and CD8+ T cells of a healthy donor. Interestingly, the in vitro incubation of peripheral blood mononuclear cell from severe COVID-19 patients with adenosine reduced the nuclear factor-κB activation in T cells and monocytes. Together, these data add new knowledge to the COVID-19 immunopathology through purinergic regulation.
Assuntos
5'-Nucleotidase , Apirase , COVID-19 , Linfócitos T , 5'-Nucleotidase/metabolismo , Adenosina/sangue , Trifosfato de Adenosina/sangue , Anexinas , Apirase/metabolismo , Antígenos CD28/metabolismo , COVID-19/imunologia , Citocinas/sangue , Proteínas Ligadas por GPI/metabolismo , Humanos , Leucócitos Mononucleares/metabolismo , Receptores Purinérgicos , Transdução de Sinais , Linfócitos T/imunologiaRESUMO
COVID-19 manifests as a milder disease in children than adults, but the underlying mechanisms are not fully characterized. Here we assess the difference in cellular or humoral immune responses of pediatric and adult COVID-19 patients to see if these factors contribute to the severity dichotomy. Children's non-specific immune profile is dominated by naive lymphocytes and HLA-DRhighCX3CR1low dendritic cells; meanwhile, children show strong specific antibody and T cell responses for viral structural proteins, with their T cell responses differing from adults by having weaker CD8+TNF+ T cells responses to S peptide pool but stronger responses to N and M peptide pools. Finally, viral mRNA is more abundant in pediatric patients. Our data thus support a scenario in which SARS-CoV-2 infected children contribute to transmission yet are less susceptible to COVID-19 symptoms due to strong and differential responses to the virus.
Assuntos
Anticorpos Antivirais/imunologia , COVID-19/imunologia , Imunidade Humoral , RNA Viral , SARS-CoV-2/genética , Vacinas Sintéticas/imunologia , Adolescente , Adulto , Idoso , Anticorpos Antivirais/sangue , Brasil , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos , COVID-19/prevenção & controle , Vacinas contra COVID-19 , Criança , Pré-Escolar , Citocinas/sangue , Feminino , Humanos , Imunidade Inata , Masculino , Pessoa de Meia-Idade , RNA Mensageiro , Glicoproteína da Espícula de Coronavírus/imunologia , Linfócitos T , Proteínas Estruturais Virais/imunologia , Adulto Jovem , Vacinas de mRNARESUMO
Respiratory syncytial virus (RSV) is the major cause of lower respiratory tract infections in children under 1 year. RSV vaccines are currently unavailable, and children suffering from multiple reinfections by the same viral strain fail to develop protective responses. Although RSV-specific antibodies can be detected upon infection, these have limited neutralizing capacity. Follicular helper T (Tfh) cells are specialized in providing signals to B cells and help the production and affinity maturation of antibodies, mainly via interleukin (IL) 21 secretion. In this study, we evaluated whether RSV could inhibit Tfh responses. We observed that Tfh cells fail to upregulate IL-21 production upon RSV infection. In the lungs, RSV infection downregulated the expression of IL-21/interleukin-21 receptor (IL-21R) in Tfh cells and upregulated programmed death-ligand 1 (PD-L1) expression in dendritic cells (DCs) and B cells. PD-L1 blockade during infection recovered IL-21R expression in Tfh cells and increased the secretion of IL-21 in a DC-dependent manner. IL-21 treatment decreased RSV viral load and lung inflammation, inducing the formation of tertiary lymphoid organs in the lung. It also decreased regulatory follicular T cells, and increased Tfh cells, B cells, antibody avidity and neutralization capacity, leading to an overall improved anti-RSV humoral response in infected mice. Passive immunization with purified immunoglobulin G from IL-21-treated RSV-infected mice protected against RSV infection. Our results unveil a pathway by which RSV affects Tfh cells by increasing PD-L1 expression on antigen-presenting cells, highlighting the importance of an IL-21-PD-L1 axis for the generation of protective responses to RSV infection.
Assuntos
Anticorpos Neutralizantes , Infecções por Vírus Respiratório Sincicial , Animais , Anticorpos Antivirais , Interleucinas , Camundongos , Infecções por Vírus Respiratório Sincicial/terapia , Células T Auxiliares FolicularesAssuntos
Proliferação de Células , Sinapses Imunológicas/imunologia , Ativação Linfocitária , Linfócitos do Interstício Tumoral/imunologia , Neoplasias/imunologia , Linfócitos T/imunologia , Animais , Humanos , Inibidores de Checkpoint Imunológico/uso terapêutico , Sinapses Imunológicas/efeitos dos fármacos , Sinapses Imunológicas/metabolismo , Sinapses Imunológicas/patologia , Linfócitos do Interstício Tumoral/efeitos dos fármacos , Linfócitos do Interstício Tumoral/metabolismo , Linfócitos do Interstício Tumoral/patologia , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Neoplasias/patologia , Transdução de Sinais , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , Linfócitos T/patologia , Viroses/tratamento farmacológico , Viroses/imunologia , Viroses/metabolismo , Viroses/patologiaRESUMO
The use of model organisms for recombinant protein production results in the addition of model-specific post-translational modifications (PTMs) that can affect the structure, charge, and function of the protein. The 70-kDa heat shock proteins (Hsp70) were originally described as intracellular chaperones, with ATPase and foldase activity. More recently, new extracellular activities of Hsp70 proteins (e.g. as immunomodulators) have been identified. While some studies indicate an inflammatory potential for extracellular Hsp70 proteins, others suggest an immunosuppressive activity. We hypothesized that the production of recombinant Hsp70 in different expression systems would result in the addition of different PTMs, perhaps explaining at least some of these opposing immunological outcomes. We produced and purified Mycobacterium tuberculosis DnaK from two different systems, Escherichia coli and Pichia pastoris, and analyzed by mass spectrometry the protein preparations, investigating the impact of PTMs in an in silico and in vitro perspective. The comparisons of DnaK structures in silico highlighted that electrostatic and topographical differences exist that are dependent upon the expression system. Production of DnaK in the eukaryotic system dramatically affected its ATPase activity, and significantly altered its ability to downregulate MHC II and CD86 expression on murine dendritic cells (DCs). Phosphatase treatment of DnaK indicated that some of these differences related specifically to phosphorylation. Altogether, our data indicate that PTMs are an important characteristic of the expression system, with differences that impact interactions of Hsps with their ligands and subsequent functional activities.
RESUMO
In vitro drug screening is widely used in the development of new drugs, because they constitute a cost-effective approach to select compounds with more potential for therapy. They are also an attractive alternative to in vivo testing. However, most of these assays are done in two-dimensional culture models, where cells are grown on a polystyrene or glass flat surface. In order to develop in vitro models that would more closely resemble physiological conditions, three-dimensional models have been developed. Here, we introduce two novel fully synthetic scaffolds produced using the polymer polyhydroxybutyrate (PHB): a Solvent-Casting Particle-Leaching (SCPL) membrane; and an electrospun membrane, to be used for 3D cultures of B16 F10 murine melanoma cells and 4T1 murine breast cancer cells. A 2D cell culture system in regular tissue culture plates and a classical 3D model where cells are grown on a commercially available gel derived from Engelbreth-Holm Swarm (EHS) tumor were used for comparison with the synthetic scaffolds. Cells were also collected from in vivo tumors grown as grafts in syngeneic mice. Morphology, cell viability, response to chemotherapy and gene expression analysis were used to compare all systems. In the electrospun membrane model, cells were grown on nanometer-scale fibers and in the SCPL membrane, which provides a foam-like structure for cell growth, pore sizes varied. Cells grown on all 3D models were able to form aggregates and spheroids, allowing for increased cell-cell contact when compared with the 2D system. Cell morphology was also more similar between 3D systems and cells collected from the in vivo tumors. Cells grown in 3D models showed an increase in resistance to dacarbazine, and cisplatin. Gene expression analysis also revealed similarities among all 3D platforms. The similarities between the two synthetic systems to the classic EHS gel model highlight their potential application as cost effective substitutes in drug screening, in which fully synthetic models could represent a step towards higher reproducibility. We conclude PHB synthetic membranes offer a valuable alternative for 3D cultures.