RESUMEN
The 501Y.V2 variants of SARS-CoV-2 containing multiple mutations in spike are now dominant in South Africa and are rapidly spreading to other countries. Here, experiments with 18 pseudotyped viruses showed that the 501Y.V2 variants do not confer increased infectivity in multiple cell types except for murine ACE2-overexpressing cells, where a substantial increase in infectivity was observed. Notably, the susceptibility of the 501Y.V2 variants to 12 of 17 neutralizing monoclonal antibodies was substantially diminished, and the neutralization ability of the sera from convalescent patients and immunized mice was also reduced for these variants. The neutralization resistance was mainly caused by E484K and N501Y mutations in the receptor-binding domain of spike. The enhanced infectivity in murine ACE2-overexpressing cells suggests the possibility of spillover of the 501Y.V2 variants to mice. Moreover, the neutralization resistance we detected for the 501Y.V2 variants suggests the potential for compromised efficacy of monoclonal antibodies and vaccines.
Asunto(s)
COVID-19/inmunología , COVID-19/virología , Evasión Inmune , SARS-CoV-2/patogenicidad , Enzima Convertidora de Angiotensina 2/metabolismo , Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/inmunología , Antígenos Virales/inmunología , Línea Celular Tumoral , Células HEK293 , Humanos , Mutación/genética , SARS-CoV-2/genéticaRESUMEN
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has had a major impact on global human health. During the spread of SARS-CoV-2, weakened host immunity and the use of vaccines with low efficacy may result in the development of more-virulent strains or strains with resistance to existing vaccines and antibodies. The prevalence of SARS-CoV-2 mutant strains differs between regions, and this variation may have an impact on the effectiveness of vaccines. In this study, an epidemiological investigation of SARS-CoV-2 in Portugal was performed, and the VSV-ΔG-G* pseudovirus system was used to construct 12 spike protein epidemic mutants, D614G, A222V+D614G, B.1.1.7, S477N+D614G, P1162R+D614G+A222V, D839Y+D614G, L176F+D614G, B.1.1.7+L216F, B.1.1.7+M740V, B.1.258, B.1.258+L1063F, and B.1.258+N751Y. The mutant pseudoviruses were used to infect four susceptible cell lines (Huh7, hACE2-293T-293T, Vero, and LLC-MK2) and 14 cell lines overexpressing ACE2 from different species. Mutant strains did not show increased infectivity or cross-species transmission. Neutralization activity against these pseudoviruses was evaluated using mouse serum and 11 monoclonal antibodies. The neutralizing activity of immunized mouse serum was not significantly reduced with the mutant strains, but the mutant strains from Portugal could evade nine of the 11 monoclonal antibodies tested. Neutralization resistance was mainly caused by the mutations S477N, N439K, and N501Y in the spike-receptor binding domain. These findings emphasize the importance of SARS-CoV-2 mutation tracking in different regions for epidemic prevention and control.
Asunto(s)
COVID-19 , SARS-CoV-2 , Animales , Anticuerpos Neutralizantes , Humanos , Ratones , Mutación , Portugal/epidemiología , Glicoproteína de la Espiga del Coronavirus/genéticaRESUMEN
The ubiquitously-expressed proteolytic enzyme furin is closely related to the pathogenesis of SARS-CoV-2 and therefore represents a key target for antiviral therapy. Based on bioinformatic analysis and pseudovirus tests, we discovered a second functional furin site located in the spike protein. Furin still increased the infectivity of mutated SARS-CoV-2 pseudovirus in 293T-ACE2 cells when the canonical polybasic cleavage site (682-686) was deleted. However, K814A mutation eliminated the enhancing effect of furin on virus infection. Furin inhibitor prevented infection by 682-686-deleted SARS-CoV-2 in 293T-ACE2-furin cells, but not the K814A mutant. K814A mutation did not affect the activity of TMPRSS2 and cathepsin L but did impact the cleavage of S2 into S2' and cell-cell fusion. Additionally, we showed that this functional furin site exists in RaTG13 from bat and PCoV-GD/GX from pangolin. Therefore, we discovered a new functional furin site that is pivotal in promoting SARS-CoV-2 infection.
Asunto(s)
Enzima Convertidora de Angiotensina 2/metabolismo , Catepsina L/metabolismo , Furina/metabolismo , SARS-CoV-2/genética , Serina Endopeptidasas/metabolismo , Glicoproteína de la Espiga del Coronavirus/genética , Secuencia de Aminoácidos , Enzima Convertidora de Angiotensina 2/genética , Animales , Catepsina L/genética , Fusión Celular , Quirópteros , Furina/genética , Expresión Génica , Células HEK293 , Humanos , Ratones , Ratones Transgénicos , Mutación , Receptores Virales/genética , Receptores Virales/metabolismo , SARS-CoV-2/crecimiento & desarrollo , SARS-CoV-2/metabolismo , SARS-CoV-2/patogenicidad , Serina Endopeptidasas/genética , Glicoproteína de la Espiga del Coronavirus/metabolismo , VermilinguaRESUMEN
Emerging SARS-CoV-2 variants are the most serious problem for COVID-19 prophylaxis and treatment. To determine whether the SARS-CoV-2 vaccine strain should be updated following variant emergence like seasonal flu vaccine, the changed degree on antigenicity of SARS-CoV-2 variants and H3N2 flu vaccine strains was compared. The neutralization activities of Alpha, Beta and Gamma variants' spike protein-immunized sera were analysed against the eight current epidemic variants and 20 possible variants combining the top 10 prevalent RBD mutations based on the Delta variant, which were constructed using pseudotyped viruses. Meanwhile, the neutralization activities of convalescent sera and current inactivated and recombinant protein vaccine-elicited sera were also examined against all possible Delta variants. Eight HA protein-expressing DNAs elicited-animal sera were also tested against eight pseudotyped viruses of H3N2 flu vaccine strains from 2011-2019. Our results indicate that the antigenicity changes of possible Delta variants were mostly within four folds, whereas the antigenicity changes among different H3N2 vaccine strains were approximately 10-100-fold. Structural analysis of the antigenic characterization of the SARS-CoV-2 and H3N2 mutations supports the neutralization results. This study indicates that the antigenicity changes of the current SARS-CoV-2 may not be sufficient to require replacement of the current vaccine strain.
Asunto(s)
Anticuerpos Neutralizantes/metabolismo , Anticuerpos Antivirales/metabolismo , Vacunas contra la COVID-19/metabolismo , COVID-19/prevención & control , Inmunogenicidad Vacunal , SARS-CoV-2/inmunología , Glicoproteína de la Espiga del Coronavirus/inmunología , Sustitución de Aminoácidos , Anticuerpos Neutralizantes/química , Anticuerpos Neutralizantes/genética , Anticuerpos Antivirales/química , Anticuerpos Antivirales/genética , Sitios de Unión , COVID-19/inmunología , COVID-19/virología , Vacunas contra la COVID-19/administración & dosificación , Vacunas contra la COVID-19/química , Epítopos/química , Epítopos/genética , Epítopos/inmunología , Expresión Génica , Humanos , Sueros Inmunes/química , Subtipo H3N2 del Virus de la Influenza A/química , Subtipo H3N2 del Virus de la Influenza A/genética , Subtipo H3N2 del Virus de la Influenza A/inmunología , Vacunas contra la Influenza/administración & dosificación , Vacunas contra la Influenza/química , Vacunas contra la Influenza/metabolismo , Gripe Humana/inmunología , Gripe Humana/prevención & control , Gripe Humana/virología , Modelos Moleculares , Mutación , Pruebas de Neutralización , Unión Proteica , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , SARS-CoV-2/química , SARS-CoV-2/patogenicidad , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/genética , Pseudotipado ViralRESUMEN
SARS-CoV-2 has caused the COVID-19 pandemic. B.1.617 variants (including Kappa and Delta) have been transmitted rapidly in India. The transmissibility, pathogenicity, and neutralization characteristics of these variants have received considerable interest. In this study, 22 pseudotyped viruses were constructed for B.1.617 variants and their corresponding single amino acid mutations. B.1.617 variants did not exhibit significant enhanced infectivity in human cells, but mutations T478K and E484Q in the receptor binding domain led to enhanced infectivity in mouse ACE2-overexpressing cells. Furin activities were slightly increased against B.1.617 variants and cell-cell fusion after infection of B.1.617 variants were enhanced. Furthermore, B.1.617 variants escaped neutralization by several mAbs, mainly because of mutations L452R, T478K, and E484Q in the receptor binding domain. The neutralization activities of sera from convalescent patients, inactivated vaccine-immunized volunteers, adenovirus vaccine-immunized volunteers, and SARS-CoV-2 immunized animals against pseudotyped B.1.617 variants were reduced by approximately twofold, compared with the D614G variant.
Asunto(s)
COVID-19 , SARS-CoV-2 , Animales , Anticuerpos Neutralizantes , Fusión Celular , Humanos , Ratones , Mutación , Pandemias , SARS-CoV-2/genética , Glicoproteína de la Espiga del Coronavirus , Tropismo ViralRESUMEN
To determine whether the neutralization activity of monoclonal antibodies, convalescent sera and vaccine-elicited sera was affected by the top five epidemic SARS-CoV-2 variants in the UK, including D614G+L18F+A222V, D614G+A222V, D614G+S477N, VOC-202012/01(B.1.1.7) and D614G+69-70del+N439K, a pseudovirus-neutralization assay was performed to evaluate the relative neutralization titers against the five SARS-CoV-2 variants and 12 single deconvolution mutants based on the variants. In this study, 18 monoclonal antibodies, 10 sera from convalescent COVID-19 patients, 10 inactivated-virus vaccine-elicited sera, 14 mRNA vaccine-elicited sera, nine RBD-immunized mouse sera, four RBD-immunized horse sera, and four spike-encoding DNA-immunized guinea pig sera were tested and analyzed. The N501Y, N439K, and S477N mutations caused immune escape from nine of 18 mAbs. However, the convalescent sera, inactivated virus vaccine-elicited sera, mRNA vaccine-elicited sera, spike DNA-elicited sera, and recombinant RBD protein-elicited sera could still neutralize these variants (within three-fold changes compared to the reference D614G variant). The neutralizing antibody responses to different types of vaccines were different, whereby the response to inactivated-virus vaccine was similar to the convalescent sera.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , COVID-19/terapia , SARS-CoV-2/inmunología , Glicoproteína de la Espiga del Coronavirus/inmunología , Animales , COVID-19/inmunología , Vacunas contra la COVID-19/inmunología , Línea Celular , Células HEK293 , Humanos , Inmunización Pasiva , Ratones , Pruebas de Neutralización/métodos , Reino Unido , Vacunación , Sueroterapia para COVID-19RESUMEN
Emerging mutations in SARS-CoV-2 cause several waves of COVID-19 pandemic. Here we investigate the infectivity and antigenicity of ten emerging SARS-CoV-2 variants-B.1.1.298, B.1.1.7(Alpha), B.1.351(Beta), P.1(Gamma), P.2(Zeta), B.1.429(Epsilon), B.1.525(Eta), B.1.526-1(Iota), B.1.526-2(Iota), B.1.1.318-and seven corresponding single amino acid mutations in the receptor-binding domain using SARS-CoV-2 pseudovirus. The results indicate that the pseudovirus of most of the SARS-CoV-2 variants (except B.1.1.298) display slightly increased infectivity in human and monkey cell lines, especially B.1.351, B.1.525 and B.1.526 in Calu-3 cells. The K417N/T, N501Y, or E484K-carrying variants exhibit significantly increased abilities to infect mouse ACE2-overexpressing cells. The activities of furin, TMPRSS2, and cathepsin L are increased against most of the variants. RBD amino acid mutations comprising K417T/N, L452R, Y453F, S477N, E484K, and N501Y cause significant immune escape from 11 of 13 monoclonal antibodies. However, the resistance to neutralization by convalescent serum or vaccines elicited serum is mainly caused by the E484K mutation. The convalescent serum from B.1.1.7- and B.1.351-infected patients neutralized the variants themselves better than other SARS-CoV-2 variants. Our study provides insights regarding therapeutic antibodies and vaccines, and highlights the importance of E484K mutation.
Asunto(s)
COVID-19/virología , SARS-CoV-2/genética , SARS-CoV-2/inmunología , Glicoproteína de la Espiga del Coronavirus/genética , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/genética , Anticuerpos Neutralizantes/inmunología , COVID-19/inmunología , COVID-19/terapia , Línea Celular , Células HEK293 , Humanos , Inmunización Pasiva/métodos , Mamíferos/inmunología , Ratones , Mutación , Pandemias , Primates/inmunología , Unión Proteica , Tropismo/genética , Sueroterapia para COVID-19RESUMEN
Increasing evidence suggests that dysregulated immune responses are associated with the clinical outcome of coronavirus disease 2019 (COVID-19). Nucleocapsid protein (NP)-, spike (S)-, receptor binding domain (RBD)- specific immunoglobulin (Ig) isotypes, IgG subclasses and neutralizing antibody (NAb) were analyzed in 123 serum from 63 hospitalized patients with severe, moderate, mild or asymptomatic COVID-19. Mild to modest correlations were found between disease severity and antigen specific IgG subclasses in serum, of which IgG1 and IgG3 were negatively associated with viral load in nasopharyngeal swab. Multiple cytokines were significantly related with antigen-specific Ig isotypes and IgG subclasses, and IL-1ß was positively correlated with most antibodies. Furthermore, the old patients (≥ 60 years old) had higher levels of chemokines, increased NAb activities and SARS-CoV-2 specific IgG1, and IgG3 responses and compromised T cell responses compared to the young patients (≤ 18 years old), which are related with more severe cases. Higher IgG1 and IgG3 were found in COVID-19 patients with comorbidities while biological sex had no effect on IgG subclasses. Overall, we have identified diseases severity was related to higher antibodies, of which IgG subclasses had weakly negative correlation with viral load, and cytokines were significantly associated with antibody response. Further, advancing age and comorbidities had obvious effect on IgG1 and IgG3.
Asunto(s)
Anticuerpos Antivirales/inmunología , COVID-19/inmunología , Inmunoglobulina G/inmunología , SARS-CoV-2/fisiología , Adolescente , Adulto , COVID-19/patología , COVID-19/virología , Niño , China , Citocinas/inmunología , Femenino , Humanos , Inmunoglobulina A/inmunología , Inmunoglobulina M/inmunología , Masculino , Persona de Mediana Edad , SARS-CoV-2/inmunología , Índice de Severidad de la Enfermedad , Linfocitos T/inmunología , Adulto JovenRESUMEN
Antibody-dependent cellular cytotoxicity (ADCC) responses to viral infection are a form of antibody regulated immune responses mediated through the Fc fragment. Whether severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) triggered ADCC responses contributes to COVID-19 disease development is currently not well understood. To understand the potential correlation between ADCC responses and COVID-19 disease development, we analyzed the ADCC activity and neutralizing antibody response in 255 individuals ranging from asymptomatic to fatal infections over 1 year post disease. ADCC was elicited by 10 days post-infection, peaked by 11-20 days, and remained detectable until 400 days post-infection. In general, patients with severe disease had higher ADCC activities. Notably, patients who had severe disease and recovered had higher ADCC activities than patients who had severe disease and deceased. Importantly, ADCC activities were mediated by a diversity of epitopes in SARS-COV-2-infected mice and induced to comparable levels against SARS-CoV-2 variants of concern (VOCs) (B.1.1.7, B.1.351, and P.1) as that against the D614G mutant in human patients and vaccinated mice. Our study indicates anti-SARS-CoV-2 ADCC as a major trait of COVID-19 patients with various conditions, which can be applied to estimate the extra-neutralization level against COVID-19, especially lethal COVID-19.