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Despite the high efficiency of current SARS-CoV-2 mRNA vaccines in reducing COVID-19 morbidity and mortality, waning immunity and the emergence of resistant variants underscore the need for novel vaccination strategies. This study explores a heterologous mRNA/Modified Vaccinia virus Ankara (MVA) prime/boost regimen employing a trimeric form of the receptor binding domain (RBD) of the SARS-CoV-2 spike (S) protein compared to a homologous MVA/MVA regimen. In C57BL/6 mice, the RBD was delivered during priming via an mRNA vector encapsulated in nanoemulsions (NE) or lipid nanoparticles (LNP), followed by a booster with a replication-deficient MVA-based recombinant virus (MVA-RBD). This heterologous mRNA/MVA regimen elicited strong anti-RBD binding and neutralizing antibodies (BAbs and NAbs) against both the ancestral SARS-CoV-2 strain and different variants of concern (VoCs). Additionally, this protocol induced robust and polyfunctional RBD-specific CD4 and CD8 T cell responses, particularly in animals primed with mLNP-RBD. In K18-hACE2 transgenic mice, the LNP-RBD/MVA combination provided complete protection from morbidity and mortality following a live SARS-CoV-2 challenge compared with the partial protection observed with mNE-RBD/MVA or MVA/MVA regimens. Although the mNE-RBD/MVA regimen only protects half of the animals, it was able to induce antibodies with Fc-mediated effector functions besides NAbs. Moreover, viral replication and viral load in the respiratory tract were markedly reduced and decreased pro-inflammatory cytokine levels were observed. These results support the efficacy of heterologous mRNA/MVA vaccine combinations over homologous MVA/MVA regimen, using alternative nanocarriers that circumvent intellectual property restrictions of current mRNA vaccine formulations.
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Anticorpos Neutralizantes , Anticorpos Antivirais , Vacinas contra COVID-19 , COVID-19 , Camundongos Endogâmicos C57BL , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , Vaccinia virus , Animais , Vacinas contra COVID-19/imunologia , Vacinas contra COVID-19/administração & dosagem , SARS-CoV-2/imunologia , SARS-CoV-2/genética , Camundongos , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/genética , COVID-19/prevenção & controle , COVID-19/imunologia , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/sangue , Anticorpos Neutralizantes/imunologia , Vaccinia virus/genética , Vaccinia virus/imunologia , Humanos , Feminino , Nanopartículas/administração & dosagem , Vacinação , Vacinas de mRNA/administração & dosagem , Camundongos Transgênicos , Vacinas Sintéticas/imunologia , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Linfócitos T CD8-Positivos/imunologia , Enzima de Conversão de Angiotensina 2/imunologia , Enzima de Conversão de Angiotensina 2/genética , LipossomosRESUMO
Introduction: It is now clear that coronavirus disease 19 (COVID-19) severity is associated with a dysregulated immune response, but the relative contributions of different immune cells is still not fully understood. SARS CoV-2 infection triggers marked changes in NK cell populations, but there are contradictory reports as to whether these effector lymphocytes play a protective or pathogenic role in immunity to SARS-CoV-2. Methods: To address this question we have analysed differences in the phenotype and function of NK cells in SARS-CoV-2 infected individuals who developed either very mild, or life-threatening COVID-19 disease. Results: Although NK cells from patients with severe disease appeared more activated and the frequency of adaptive NK cells was increased, they were less potent mediators of ADCC than NK cells from patients with mild disease. Further analysis of peripheral blood NK cells in these patients revealed that a population of NK cells that had lost expression of the activating receptor NKG2D were a feature of patients with severe disease and this correlated with elevated levels of cell free NKG2D ligands, especially ULBP2 and ULBP3 in the plasma of critically ill patients. In vitro, culture in NKG2DL containing patient sera reduced the ADCC function of healthy donor NK cells and this could be blocked by NKG2DL-specific antibodies. Discussion: These observations of reduced NK function in severe disease are consistent with the hypothesis that defects in immune surveillance by NK cells permit higher levels of viral replication, rather than that aberrant NK cell function contributes to immune system dysregulation and immunopathogenicity.
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COVID-19 , Citotoxicidade Imunológica , Humanos , COVID-19/patologia , Células Matadoras Naturais , Subfamília K de Receptores Semelhantes a Lectina de Células NK/metabolismo , SARS-CoV-2/metabolismoRESUMO
The constant appearance of new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VoCs) has jeopardized the protective capacity of approved vaccines against coronavirus disease-19 (COVID-19). For this reason, the generation of new vaccine candidates adapted to the emerging VoCs is of special importance. Here, we developed an optimized COVID-19 vaccine candidate using the modified vaccinia virus Ankara (MVA) vector to express a full-length prefusion-stabilized SARS-CoV-2 spike (S) protein, containing 3 proline (3P) substitutions in the S protein derived from the beta (B.1.351) variant, termed MVA-S(3Pbeta). Preclinical evaluation of MVA-S(3Pbeta) in head-to-head comparison to the previously generated MVA-S(3P) vaccine candidate, expressing a full-length prefusion-stabilized Wuhan S protein (with also 3P substitutions), demonstrated that two intramuscular doses of both vaccine candidates fully protected transgenic K18-hACE2 mice from a lethal challenge with SARS-CoV-2 beta variant, reducing mRNA and infectious viral loads in the lungs and in bronchoalveolar lavages, decreasing lung histopathological lesions and levels of proinflammatory cytokines in the lungs. Vaccination also elicited high titers of anti-S Th1-biased IgGs and neutralizing antibodies against ancestral SARS-CoV-2 Wuhan strain and VoCs alpha, beta, gamma, delta, and omicron. In addition, similar systemic and local SARS-CoV-2 S-specific CD4+ and CD8+ T-cell immune responses were elicited by both vaccine candidates after a single intranasal immunization in C57BL/6 mice. These preclinical data support clinical evaluation of MVA-S(3Pbeta) and MVA-S(3P), to explore whether they can diversify and potentially increase recognition and protection of SARS-CoV-2 VoCs.
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COVID-19 , Vacinas , Camundongos , Animais , Humanos , SARS-CoV-2/genética , Vaccinia virus/genética , Vacinas contra COVID-19 , Anticorpos Antivirais , COVID-19/prevenção & controle , Camundongos Endogâmicos C57BLRESUMO
Ebola virus (EBOV) is a single-strand RNA virus belonging to the Filoviridae family, which has been associated to most Ebola virus disease outbreaks to date, including the West African and the North Kivu epidemics between 2013 and 2022. This unprecedented health emergency prompted the search for effective medical countermeasures. Following up on the carbazole hit identified in our previous studies, we synthetized a new series of compounds, which demonstrated to prevent EBOV infection in cells by acting as virus entry inhibitors. The in vitro inhibitory activity was evaluated through the screening against surrogate models based on viral pseudotypes and further confirmed using replicative EBOV. Docking and molecular dynamics simulations joined to saturation transfer difference-nuclear magnetic resonance (STD-NMR) and mutagenesis experiments to elucidate the biological target of the most potent compounds. Finally, in vitro metabolic stability and in vivo pharmacokinetic studies were performed to confirm their therapeutic potential.
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Ebolavirus , Doença pelo Vírus Ebola , Humanos , Simulação de Dinâmica Molecular , Mutagênese , Replicação ViralRESUMO
A method development aimed for high-throughput and automated antibody screening holds great potential for areas ranging from fundamental molecular interactions to the discovery of novel disease markers, therapeutic targets, and monoclonal antibody engineering. Surface display techniques enable efficient manipulation of large molecular libraries in small volumes. Specifically, phage display appeared as a powerful technology for selecting peptides and proteins with enhanced, target-specific binding affinities. Here, we present a phage-selection microfluidic device wherein electrophoresis was performed under two orthogonal electric fields through an agarose gel functionalized with the respective antigen. This microdevice was capable of screening and sorting in a single round high-affinity phage-displayed antibodies against virus glycoproteins, including human immunodeficiency virus-1 glycoprotein 120 or the Ebola virus glycoprotein (EBOV-GP). Phages were differentially and laterally swept depending on their antigen affinity; the high-affinity phages were recovered at channels proximal to the application site, whereas low-affinity phages migrated distal after electrophoresis. These experiments proved that the microfluidic device specifically designed for phage-selection is rapid, sensitive, and effective. Therefore, this is an efficient and cost-effective method that allowed highly controlled assay conditions for isolating and sorting high-affinity ligands displayed in phages.
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Bacteriófagos , Biblioteca de Peptídeos , Humanos , Anticorpos Monoclonais/metabolismo , Bacteriófagos/genética , Bacteriófagos/metabolismo , Antígenos , Eletroforese , Dispositivos Lab-On-A-ChipRESUMO
Current coronavirus disease-19 (COVID-19) vaccines are administered by the intramuscular route, but this vaccine administration failed to prevent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus infection in the upper respiratory tract, mainly due to the absence of virus-specific mucosal immune responses. It is hypothesized that intranasal (IN) vaccination could induce both mucosal and systemic immune responses that blocked SARS-CoV-2 transmission and COVID-19 progression. Here, we evaluated in mice IN administration of three modified vaccinia virus Ankara (MVA)-based vaccine candidates expressing the SARS-CoV-2 spike (S) protein, either the full-length native S or a prefusion-stabilized [S(3P)] protein; SARS-CoV-2-specific immune responses and efficacy were determined after a single IN vaccine application. Results showed that in C57BL/6 mice, MVA-based vaccine candidates elicited S-specific IgG and IgA antibodies in serum and bronchoalveolar lavages, respectively, and neutralizing antibodies against parental and SARS-CoV-2 variants of concern (VoC), with MVA-S(3P) being the most immunogenic vaccine candidate. IN vaccine administration also induced polyfunctional S-specific Th1-skewed CD4+ and cytotoxic CD8+ T-cell immune responses locally (in lungs and bronchoalveolar lymph nodes) or systemically (in spleen). Remarkably, a single IN vaccine dose protected susceptible K18-hACE2 transgenic mice from morbidity and mortality caused by SARS-CoV-2 infection, with MVA-S(3P) being the most effective candidate. Infectious SARS-CoV-2 viruses were undetectable in lungs and nasal washes, correlating with high titers of S-specific IgGs and neutralizing antibodies against parental SARS-CoV-2 and several VoC. Moreover, low histopathological lung lesions and low levels of pro-inflammatory cytokines in lungs and nasal washes were detected in vaccinated animals. These results demonstrated that a single IN inoculation of our MVA-based vaccine candidates induced potent immune responses, either locally or systemically, and protected animal models from COVID-19. These results also identified an effective vaccine administration route to induce mucosal immunity that should prevent SARS-CoV-2 host-to-host transmission.
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COVID-19 , Vacinas Virais , Administração Intranasal , Animais , Anticorpos Neutralizantes , Anticorpos Antivirais , Formação de Anticorpos , COVID-19/prevenção & controle , Vacinas contra COVID-19 , Citocinas , Imunoglobulina A , Imunoglobulina G , Camundongos , Camundongos Endogâmicos C57BL , SARS-CoV-2 , Vaccinia virus/genéticaRESUMO
The emergence of SARS-CoV-2 variants that escape from immune neutralization are challenging vaccines and antibodies developed to stop the COVID-19 pandemic. Thus, it is important to establish therapeutics directed toward multiple or specific SARS-CoV-2 variants. The envelope spike (S) glycoprotein of SARS-CoV-2 is the key target of neutralizing antibodies (Abs). We selected a panel of nine nanobodies (Nbs) from dromedary camels immunized with the receptor-binding domain (RBD) of the S, and engineered Nb fusions as humanized heavy chain Abs (hcAbs). Nbs and derived hcAbs bound with subnanomolar or picomolar affinities to the S and its RBD, and S-binding cross-competition clustered them in two different groups. Most of the hcAbs hindered RBD binding to its human ACE2 (hACE2) receptor, blocked cell entry of viruses pseudotyped with the S protein and neutralized SARS-CoV-2 infection in cell cultures. Four potent neutralizing hcAbs prevented the progression to lethal SARS-CoV-2 infection in hACE2-transgenic mice, demonstrating their therapeutic potential. Cryo-electron microscopy identified Nb binding epitopes in and out the receptor binding motif (RBM), and showed different ways to prevent virus binding to its cell entry receptor. The Nb binding modes were consistent with its recognition of SARS-CoV-2 RBD variants; mono and bispecific hcAbs efficiently bound all variants of concern except omicron, which emphasized the immune escape capacity of this latest variant.
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COVID-19 , Anticorpos de Domínio Único , Animais , Microscopia Crioeletrônica , Epitopos/química , Humanos , Camundongos , Pandemias , SARS-CoV-2 , Glicoproteína da Espícula de CoronavírusRESUMO
Multiple questions about SARS-CoV-2 humoral and cellular immunity remain unanswered. One key question is whether preexisting memory T or B cells, specific for related coronaviruses in SARS-CoV-2-unexposed individuals, can recognize and suppress COVID-19, but this issue remains unclear. Here, we demonstrate that antibody responses to SARS-CoV-2 antigens are restricted to serum samples from COVID-19 convalescent individuals. In contrast, cross-reactive T cell proliferation and IFN-γ production responses were detected in PBMCs of around 30% of donor samples collected prepandemic, although we found that these prepandemic T cell responses only elicited weak cTFH activation upon stimulation with either HCoV-OC43 or SARS-CoV-2 NP protein. Overall, these observations confirm that T cell cross-reactive with SARS-CoV-2 antigens are present in unexposed people, but suggest that the T cell response to HCoV-OC43 could be deficient in some important aspects, like TFH expansion, that might compromise the generation of cross-reactive TFH cells and antibodies. Understanding these differences in cellular responses may be of critical importance to advance in our knowledge of immunity against SARS-CoV-2.
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COVID-19 , Coronavirus Humano OC43 , Anticorpos Antivirais , Reações Cruzadas , Humanos , Imunidade Humoral , SARS-CoV-2RESUMO
Two doses of the MVA-CoV2-S vaccine candidate expressing the SARS-CoV-2 spike (S) protein protected K18-hACE2 transgenic mice from a lethal dose of SARS-CoV-2. This vaccination regimen prevented virus replication in the lungs, reduced lung pathology, and diminished levels of pro-inflammatory cytokines. High titers of IgG antibodies against S and receptor-binding domain (RBD) proteins and of neutralizing antibodies were induced against parental virus and variants of concern, markers that correlated with protection. Similar SARS-CoV-2-specific antibody responses were observed at prechallenge and postchallenge in the two-dose regimen, while the single-dose treatment does not avoid vaccine breakthrough infection. All vaccinated animals survived infection and were also protected to SARS-CoV-2 reinfection. Furthermore, two MVA-CoV2-S doses induced long-term memory S-specific humoral and cellular immune responses in C57BL/6 mice, 6 months after immunization. The efficacy and immunological benefits of the MVA-CoV2-S vaccine candidate against COVID-19 supports its consideration for human clinical trials.
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Dendritic cells (DCs) are the most potent antigen-presenting cells, and their function is essential to configure adaptative immunity and avoid excessive inflammation. DCs are predicted to play a crucial role in the clinical evolution of the infection by the severe acute respiratory syndrome (SARS) coronavirus (CoV)-2. DCs interaction with the SARS-CoV-2 Spike protein, which mediates cell receptor binding and subsequent fusion of the viral particle with host cell, is a key step to induce effective immunity against this virus and in the S protein-based vaccination protocols. Here we evaluated human DCs in response to SARS-CoV-2 S protein, or to a fragment encompassing the receptor binding domain (RBD) challenge. Both proteins increased the expression of maturation markers, including MHC molecules and costimulatory receptors. DCs interaction with the SARS-CoV-2 S protein promotes activation of key signaling molecules involved in inflammation, including MAPK, AKT, STAT1, and NFκB, which correlates with the expression and secretion of distinctive proinflammatory cytokines. Differences in the expression of ACE2 along the differentiation of human monocytes to mature DCs and inter-donor were found. Our results show that SARS-CoV-2 S protein promotes inflammatory response and provides molecular links between individual variations and the degree of response against this virus.
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Células Dendríticas/patologia , Células Dendríticas/virologia , Receptores Virais/metabolismo , SARS-CoV-2/metabolismo , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/metabolismo , Enzima de Conversão de Angiotensina 2/metabolismo , Moléculas de Adesão Celular/metabolismo , Diferenciação Celular , Citocinas/biossíntese , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Inflamação/patologia , Lectinas Tipo C/metabolismo , Domínios Proteicos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores de Superfície Celular/metabolismo , Fatores de Transcrição STAT/metabolismo , Transdução de Sinais , Doadores de TecidosRESUMO
Here, we describe a new, simple, highly multiplexed serological test that generates a more complete picture of seroconversion than single antigen-based assays. Flow cytometry is used to detect multiple Ig isotypes binding to four SARS-CoV-2 antigens: the Spike glycoprotein, its RBD fragment (the main target for neutralizing antibodies), the nucleocapsid protein, and the main cysteine-like protease in a single reaction. Until now, most diagnostic serological tests measured antibodies to only one antigen and in some laboratory-confirmed patients no SARS-CoV-2-specific antibodies could be detected. Our data reveal that while most patients respond against all the viral antigens tested, others show a marked bias to make antibodies against either proteins exposed on the viral particle or those released after cellular infection. With this assay, it was possible to discriminate between patients and healthy controls with 100% confidence. Analysing the response of multiple Ig isotypes to the four antigens in combination may also help to establish a correlation with the severity degree of disease. A more detailed description of the immune responses of different patients to SARS-CoV-2 virus might provide insight into the wide array of clinical presentations of COVID-19.
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Anticorpos Antivirais/sangue , Teste Sorológico para COVID-19/métodos , COVID-19/diagnóstico , Citometria de Fluxo/métodos , Antígenos Virais/imunologia , COVID-19/imunologia , Ensaios de Triagem em Larga Escala , Humanos , SARS-CoV-2 , Sensibilidade e Especificidade , Testes SorológicosRESUMO
Vaccines against SARS-CoV-2, the causative agent of the COVID-19 pandemic, are urgently needed. We developed two COVID-19 vaccines based on modified vaccinia virus Ankara (MVA) vectors expressing the entire SARS-CoV-2 spike (S) protein (MVA-CoV2-S); their immunogenicity was evaluated in mice using DNA/MVA or MVA/MVA prime/boost immunizations. Both vaccines induced robust, broad and polyfunctional S-specific CD4+ (mainly Th1) and CD8+ T-cell responses, with a T effector memory phenotype. DNA/MVA immunizations elicited higher T-cell responses. All vaccine regimens triggered high titers of IgG antibodies specific for the S, as well as for the receptor-binding domain; the predominance of the IgG2c isotype was indicative of Th1 immunity. Notably, serum samples from vaccinated mice neutralized SARS-CoV-2 in cell cultures, and those from MVA/MVA immunizations showed a higher neutralizing capacity. Remarkably, one or two doses of MVA-CoV2-S protect humanized K18-hACE2 mice from a lethal dose of SARS-CoV-2. In addition, two doses of MVA-CoV2-S confer full inhibition of virus replication in the lungs. These results demonstrate the robust immunogenicity and full efficacy of MVA-based COVID-19 vaccines in animal models and support its translation to the clinic.IMPORTANCE The continuous dissemination of the novel emerging SARS-CoV-2 virus, with more than 78 million infected cases worldwide and higher than 1,700,000 deaths as of December 23, 2020, highlights the urgent need for the development of novel vaccines against COVID-19. With this aim, we have developed novel vaccine candidates based on the poxvirus modified vaccinia virus Ankara (MVA) strain expressing the full-length SARS-CoV-2 spike (S) protein, and we have evaluated their immunogenicity in mice using DNA/MVA or MVA/MVA prime/boost immunization protocols. The results showed the induction of a potent S-specific T-cell response and high titers of neutralizing antibodies. Remarkably, humanized K18-hACE2 mice immunized with one or two doses of the MVA-based vaccine were 100% protected from SARS-CoV-2 lethality. Moreover, two doses of the vaccine prevented virus replication in lungs. Our findings prove the robust immunogenicity and efficacy of MVA-based COVID-19 vaccines in animal models and support its translation to the clinic.
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The immune response promoted by SARS-CoV-2 vaccination is relevant to develop novel vaccines and optimized prevention strategies. We analyzed the adaptive immunity in healthy donors (HD) and convalescent individuals (CD), before and after administering BNT162b2 vaccine. Our results revealed specific changes in CD4+ T cell reactivity profile in vaccinated HD and CD, with an increase in S1 and S2 positive individuals, proportionally higher for S2. On the contrary, NCAP reactivity observed in HD and CD patients was no longer detectable after vaccination. Despite the substantial antibody response in CD, MPro-derived peptides did not elicit CD4+ lymphocyte activation in our assay in either condition. HD presented an increment in anti-S and anti-RBD IgG after first dose vaccination, which increased after the second vaccination. Conversely, anti-S and anti-RBD IgG and IgA titers increased in already positive CD after first dose administration, remaining stable after second dose inoculation. Interestingly, we found a strong significant correlation between S1-induced CD4+ response and anti-S IgA pre-vaccination, which was lost after vaccine administration.
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Vacina BNT162/imunologia , Linfócitos T CD4-Positivos/imunologia , COVID-19/imunologia , SARS-CoV-2/fisiologia , Adulto , Células Cultivadas , Convalescença , Feminino , Voluntários Saudáveis , Humanos , Imunização Secundária , Imunoglobulina A/metabolismo , Imunoglobulina G/metabolismo , Masculino , Pessoa de Meia-Idade , Glicoproteína da Espícula de Coronavírus/imunologia , Especificidade do Receptor de Antígeno de Linfócitos T , VacinaçãoRESUMO
We generated an optimized COVID-19 vaccine candidate based on the modified vaccinia virus Ankara (MVA) vector expressing a full-length prefusion-stabilized SARS-CoV-2 spike (S) protein, termed MVA-CoV2-S(3P). The S(3P) protein was expressed at higher levels (2-fold) than the non-stabilized S in cells infected with the corresponding recombinant MVA viruses. One single dose of MVA-CoV2-S(3P) induced higher IgG and neutralizing antibody titers against parental SARS-CoV-2 and variants of concern than MVA-CoV2-S in wild-type C57BL/6 and in transgenic K18-hACE2 mice. In immunized C57BL/6 mice, two doses of MVA-CoV2-S or MVA-CoV2-S(3P) induced similar levels of SARS-CoV-2-specific B- and T-cell immune responses. Remarkably, a single administration of MVA-CoV2-S(3P) protected all K18-hACE2 mice from morbidity and mortality caused by SARS-CoV-2 infection, reducing SARS-CoV-2 viral loads, histopathological lesions, and levels of pro-inflammatory cytokines in the lungs. These results demonstrated that expression of a novel full-length prefusion-stabilized SARS-CoV-2 S protein by the MVA poxvirus vector enhanced immunogenicity and efficacy against SARS-CoV-2 in animal models, further supporting MVA-CoV2-S(3P) as an optimized vaccine candidate for clinical trials.
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Vacinas contra COVID-19/imunologia , COVID-19/prevenção & controle , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Vacinas de DNA/imunologia , Vacinas Virais/imunologia , Idoso , Enzima de Conversão de Angiotensina 2/genética , Enzima de Conversão de Angiotensina 2/metabolismo , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , COVID-19/mortalidade , Vacinas contra COVID-19/genética , Linhagem Celular Tumoral , Embrião de Galinha , Chlorocebus aethiops , Citocinas/análise , Feminino , Células HeLa , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Plasmídeos/genética , Glicoproteína da Espícula de Coronavírus/genética , Eficácia de Vacinas , Vacinas de DNA/genética , Vaccinia virus/imunologia , Células Vero , Vacinas Virais/genéticaRESUMO
SARS-CoV-2 infection causes an abrupt response by the host immune system, which is largely responsible for the outcome of COVID-19. We investigated whether the specific immune responses in the peripheral blood of 276 patients were associated with the severity and progression of COVID-19. At admission, dramatic lymphopenia of T, B, and NK cells is associated with severity. Conversely, the proportion of B cells, plasmablasts, circulating follicular helper T cells (cTfh) and CD56- CD16+ NK-cells increased. Regarding humoral immunity, levels of IgM, IgA, and IgG were unaffected, but when degrees of severity were considered, IgG was lower in severe patients. Compared to healthy donors, complement C3 and C4 protein levels were higher in mild and moderate, but not in severe patients, while the activation peptide of C5 (C5a) increased from the admission in every patient, regardless of their severity. Moreover, total IgG, the IgG1 and IgG3 isotypes, and C4 decreased from day 0 to day 10 in patients who were hospitalized for more than two weeks, but not in patients who were discharged earlier. Our study provides important clues to understand the immune response observed in COVID-19 patients, associating severity with an imbalanced humoral response, and identifying new targets for therapeutic intervention.
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Linfócitos B/imunologia , COVID-19/patologia , Imunoglobulinas/sangue , Células Matadoras Naturais/imunologia , SARS-CoV-2/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Idoso , COVID-19/imunologia , Complemento C3/análise , Complemento C4/análise , Complemento C5/análise , Feminino , Humanos , Imunoglobulina A/sangue , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Contagem de Linfócitos , Linfopenia/imunologia , Masculino , Pessoa de Meia-Idade , Síndrome do Desconforto Respiratório/imunologia , Síndrome do Desconforto Respiratório/patologiaRESUMO
Currently, there is a need for reliable tests that allow identification of individuals that have been infected with SARS-CoV-2 even if the infection was asymptomatic. To date, the vast majority of the serological tests for SARS-CoV-2-specific Abs are based on serum detection of Abs to either the viral spike glycoprotein (the major target for neutralizing Abs) or the viral nucleocapsid protein that is known to be highly immunogenic in other coronaviruses. Conceivably, exposure of Ags released from infected cells could stimulate Ab responses that might correlate with tissue damage and, hence, they may have some value as a prognostic indicator. We addressed whether other nonstructural viral proteins, not incorporated into the infectious viral particle, specifically the viral cysteine-like protease, might also be potent immunogens. Using ELISA tests, coating several SARS-CoV-2 proteins produced in vitro, we describe that COVID-19 patients make high titer IgG, IgM, and IgA Ab responses to the Cys-like protease from SARS-CoV-2, also known as 3CLpro or Mpro, and it can be used to identify individuals with positive serology against the coronavirus. Higher Ab titers in these assays associated with more-severe disease, and no cross-reactive Abs against prior betacoronavirus were found. Remarkably, IgG Abs specific for Mpro and other SARS-CoV-2 Ags can also be detected in saliva. In conclusion, Mpro is a potent Ag in infected patients that can be used in serological tests, and its detection in saliva could be the basis for a rapid, noninvasive test for COVID-19 seropositivity.
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Anticorpos Antivirais/sangue , Betacoronavirus/metabolismo , Infecções por Coronavirus/sangue , Cisteína Proteases/metabolismo , Proteínas do Nucleocapsídeo/metabolismo , Pneumonia Viral/sangue , Saliva/metabolismo , Adulto , Idoso , COVID-19 , Feminino , Células HEK293 , Humanos , Masculino , Pessoa de Meia-Idade , Pandemias , SARS-CoV-2RESUMO
BACKGROUND: Although the role of Th17 and regulatory T cells in the progression of atherosclerosis has been highlighted in recent years, their molecular mediators remain elusive. We aimed to evaluate the association between the CD69 receptor, a regulator of Th17/regulatory T cell immunity, and atherosclerosis development in animal models and in patients with subclinical disease. METHODS: Low-density lipoprotein receptor-deficient chimeric mice expressing or not expressing CD69 on either myeloid or lymphoid cells were subjected to a high fat diet. In vitro functional assays with human T cells were performed to decipher the mechanism of the observed phenotypes. Expression of CD69 and NR4A nuclear receptors was evaluated by reverse transcription-polymerase chain reaction in 305 male participants of the PESA study (Progression of Early Subclinical Atherosclerosis) with extensive (n=128) or focal (n=55) subclinical atherosclerosis and without disease (n=122). RESULTS: After a high fat diet, mice lacking CD69 on lymphoid cells developed large atheroma plaque along with an increased Th17/regulatory T cell ratio in blood. Oxidized low-density lipoprotein was shown to bind specifically and functionally to CD69 on human T lymphocytes, inhibiting the development of Th17 cells through the activation of NR4A nuclear receptors. Participants of the PESA study with evidence of subclinical atherosclerosis displayed a significant CD69 and NR4A1 mRNA downregulation in peripheral blood leukocytes compared with participants without disease. The expression of CD69 remained associated with the risk of subclinical atherosclerosis in an adjusted multivariable logistic regression model (odds ratio, 0.62; 95% CI, 0.40-0.94; P=0.006) after adjustment for traditional risk factors, the expression of NR4A1, the level of oxidized low-density lipoprotein, and the counts of different leucocyte subsets. CONCLUSIONS: CD69 depletion from the lymphoid compartment promotes a Th17/regulatory T cell imbalance and exacerbates the development of atherosclerosis. CD69 binding to oxidized low-density lipoprotein on T cells induces the expression of anti-inflammatory transcription factors. Data from a cohort of the PESA study with subclinical atherosclerosis indicate that CD69 expression in PBLs inversely correlates with the presence of disease. The expression of CD69 remained an independent predictor of subclinical atherosclerosis after adjustment for traditional risk factors.
Assuntos
Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos T/metabolismo , Aterosclerose/prevenção & controle , Imunidade Celular , Lectinas Tipo C/metabolismo , Lipoproteínas LDL/metabolismo , Receptores de LDL Oxidado/metabolismo , Linfócitos T Reguladores/metabolismo , Células Th17/metabolismo , Adulto , Animais , Antígenos CD/genética , Antígenos de Diferenciação de Linfócitos T/genética , Doenças Assintomáticas , Aterosclerose/imunologia , Aterosclerose/metabolismo , Aterosclerose/patologia , Modelos Animais de Doenças , Feminino , Predisposição Genética para Doença , Humanos , Células Jurkat , Lectinas Tipo C/deficiência , Lectinas Tipo C/genética , Masculino , Camundongos Knockout , Pessoa de Meia-Idade , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/genética , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/metabolismo , Fenótipo , Placa Aterosclerótica , Estudos Prospectivos , Ratos , Receptores de LDL/genética , Receptores de LDL/metabolismo , Fatores de Risco , Transdução de Sinais , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/patologia , Células Th17/imunologia , Células Th17/patologiaRESUMO
Acute hemorrhagic conjunctivitis (AHC) is a painful, contagious eye disease, with millions of cases in the last decades. Coxsackievirus A24 (CV-A24) was not originally associated with human disease, but in 1970 a pathogenic "variant" (CV-A24v) emerged, which is now the main cause of AHC. Initially, this variant circulated only in Southeast Asia, but it later spread worldwide, accounting for numerous AHC outbreaks and two pandemics. While both CV-A24 variant and nonvariant strains still circulate in humans, only variant strains cause AHC for reasons that are yet unknown. Since receptors are important determinants of viral tropism, we set out to map the CV-A24 receptor repertoire and establish whether changes in receptor preference have led to the increased pathogenicity and rapid spread of CV-A24v. Here, we identify ICAM-1 as an essential receptor for both AHC-causing and non-AHC strains. We provide a high-resolution cryo-EM structure of a virus-ICAM-1 complex, which revealed critical ICAM-1-binding residues. These data could help identify a possible conserved mode of receptor engagement among ICAM-1-binding enteroviruses and rhinoviruses. Moreover, we identify a single capsid substitution that has been adopted by all pandemic CV-A24v strains and we reveal that this adaptation enhances the capacity of CV-A24v to bind sialic acid. Our data elucidate the CV-A24v receptor repertoire and point to a role of enhanced receptor engagement in the adaptation to the eye, possibly enabling pandemic spread.
Assuntos
Conjuntivite Hemorrágica Aguda/metabolismo , Enterovirus Humano C/metabolismo , Molécula 1 de Adesão Intercelular/metabolismo , Receptores Virais/metabolismo , Sequência de Aminoácidos , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Conjuntivite Hemorrágica Aguda/epidemiologia , Conjuntivite Hemorrágica Aguda/virologia , Microscopia Crioeletrônica , Surtos de Doenças , Enterovirus Humano C/genética , Enterovirus Humano C/fisiologia , Humanos , Molécula 1 de Adesão Intercelular/química , Mutação , Ácido N-Acetilneuramínico/metabolismo , Pandemias , Filogenia , Ligação Proteica , Receptores Virais/química , Homologia de Sequência de Aminoácidos , Tropismo Viral/fisiologiaRESUMO
Measles virus (MV) remains a leading cause of vaccine-preventable deaths in children. Protection against MV is associated with neutralizing antibodies that preferentially recognize the viral hemagglutinin (MV-H), and to a lesser extent, the fusion protein (MV-F). Although MV is serologically monotypic, 24 genotypes have been identified. Here we report three neutralization epitopes conserved in the more prevalent circulating MV genotypes, two located in the MV-H receptor binding site (RBS) (antigenic site III) and a third in MV-H/MV-F interphase (antigenic site Ia) which are essential for MV multiplication. In contrast, two MV-H neutralization epitopes, showed a genotype-specific neutralization escape due to a single amino acid change, that we mapped in the "noose" antigenic site, or an enhanced neutralization epitope (antigenic site IIa). The monoclonal antibody (mAb) neutralization potency correlated with its binding affinity and was mainly driven by kinetic dissociation rate (koff). We developed an immunoassay for mAb binding to MV-H in its native hetero-oligomeric structure with MV-F on the surface of a MV productive steady-state persistently infected (p.i.) human cell lines, and a competitive-binding assay with serum from individuals with past infection by different MV genotypes. Binding assays revealed that a broad neutralization epitope, in RBS antigenic site, a genotype specific neutralization epitopes, in noose and IIa sites, were immunogenic in natural infection and vaccination and may elicit long-lasting humoral immunity that might contribute to explain MV immunogenic stability. These results support the design of improved measles vaccines, broad-spectrum prophylactic or therapeutic antibodies and MV-used in oncolytic therapies.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Hemaglutininas Virais/imunologia , Vírus do Sarampo/imunologia , Sarampo/imunologia , Anticorpos Neutralizantes/imunologia , Epitopos/administração & dosagem , Epitopos/imunologia , Genótipo , Hemaglutininas Virais/administração & dosagem , Hemaglutininas Virais/genética , Humanos , Sarampo/prevenção & controle , Sarampo/virologia , Vacina contra Sarampo/administração & dosagem , Vacina contra Sarampo/imunologia , Vírus do Sarampo/classificação , Vírus do Sarampo/genética , Vírus do Sarampo/isolamento & purificação , Testes de Neutralização , VacinaçãoRESUMO
Cell surface aminopeptidase N (APN) is a membrane-bound ectoenzyme that hydrolyzes proteins and peptides and regulates numerous cell functions. APN participates in tumor cell expansion and motility, and is a target for cancer therapies. Small drugs that bind to the APN active site inhibit catalysis and suppress tumor growth. APN is also a major cell entry receptor for coronavirus, which binds to a region distant from the active site. Three crystal structures that we determined of human and pig APN ectodomains defined the dynamic conformation of the protein. These structures offered snapshots of closed, intermediate and open APN, which represent distinct functional states. Coronavirus envelope proteins specifically recognized the open APN form, prevented ectodomain progression to the closed form and substrate hydrolysis. In addition, drugs that bind the active site inhibited both coronavirus binding to cell surface APN and infection; the drugs probably hindered APN transition to the virus-specific open form. We conclude that allosteric inhibition of APN functions occurs by ligand suppression of ectodomain motions necessary for catalysis and virus cell entry, as validated by locking APN with disulfides. Blocking APN dynamics can thus be a valuable approach to development of drugs that target this ectoenzyme.