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1.
mSphere ; 5(5)2020 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-32938700

RESUMO

As severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to spread around the world, there is an urgent need for new assay formats to characterize the humoral response to infection. Here, we present an efficient, competitive serological assay that can simultaneously determine an individual's seroreactivity against the SARS-CoV-2 Spike protein and determine the proportion of anti-Spike antibodies that block interaction with the human angiotensin-converting enzyme 2 (ACE2) required for viral entry. In this approach based on the use of enzyme-linked immunosorbent assays (ELISA), we present natively folded viral Spike protein receptor-binding domain (RBD)-containing antigens via avidin-biotin interactions. Sera are then competed with soluble ACE2-Fc, or with a higher-affinity variant thereof, to determine the proportion of ACE2 blocking anti-RBD antibodies. Assessment of sera from 144 SARS-CoV-2 patients ultimately revealed that a remarkably consistent and high proportion of antibodies in the anti-RBD pool targeted the epitope responsible for ACE2 engagement (83% ± 11%; 50% to 107% signal inhibition in our largest cohort), further underscoring the importance of tailoring vaccines to promote the development of such antibodies.IMPORTANCE With the emergence and continued spread of the SARS-CoV-2 virus, and of the associated disease, coronavirus disease 2019 (COVID-19), there is an urgent need for improved understanding of how the body mounts an immune response to the virus. Here, we developed a competitive SARS-CoV-2 serological assay that can simultaneously determine whether an individual has developed antibodies against the SARS-CoV-2 Spike protein receptor-binding domain (RBD) and measure the proportion of these antibodies that block interaction with the human angiotensin-converting enzyme 2 (ACE2) required for viral entry. Using this assay and 144 SARS-CoV-2 patient serum samples, we found that a majority of anti-RBD antibodies compete for ACE2 binding. These results not only highlight the need to design vaccines to generate such blocking antibodies but also demonstrate the utility of this assay to rapidly screen patient sera for potentially neutralizing antibodies.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Betacoronavirus/imunologia , Peptidil Dipeptidase A/imunologia , Testes Sorológicos/métodos , Glicoproteína da Espícula de Coronavírus/imunologia , Antígenos Virais/imunologia , Sítios de Ligação/imunologia , Infecções por Coronavirus/prevenção & controle , Ensaios de Triagem em Larga Escala/métodos , Humanos , Pandemias/prevenção & controle , Pneumonia Viral/prevenção & controle , Ligação Proteica , Domínios Proteicos/imunologia
2.
Emerg Microbes Infect ; 9(1): 2105-2113, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32893735

RESUMO

The global pandemic of coronavirus disease 2019 (COVID-19) is a disaster for human society. A convenient and reliable neutralization assay is very important for the development of vaccines and novel drugs. In this study, a G protein-deficient vesicular stomatitis virus (VSVdG) bearing a truncated spike protein (S with C-terminal 18 amino acid truncation) was compared to that bearing the full-length spike protein of SARS-CoV-2 and showed much higher efficiency. A neutralization assay was established based on VSV-SARS-CoV-2-Sdel18 pseudovirus and hACE2-overexpressing BHK21 cells (BHK21-hACE2 cells). The experimental results can be obtained by automatically counting the number of EGFP-positive cells at 12 h after infection, making the assay convenient and high-throughput. The serum neutralizing titer measured by the VSV-SARS-CoV-2-Sdel18 pseudovirus assay has a good correlation with that measured by the wild type SARS-CoV-2 assay. Seven neutralizing monoclonal antibodies targeting the receptor binding domain (RBD) of the SARS-CoV-2 S protein were obtained. This efficient and reliable pseudovirus assay model could facilitate the development of new drugs and vaccines.


Assuntos
Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Betacoronavirus/imunologia , Infecções por Coronavirus/diagnóstico , Testes de Neutralização/métodos , Pneumonia Viral/diagnóstico , Glicoproteína da Espícula de Coronavírus/imunologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Linhagem Celular , Chlorocebus aethiops , Cricetinae , Pandemias , Glicoproteína da Espícula de Coronavírus/genética , Células Vero , Vírus da Estomatite Vesicular Indiana/genética , Vírus da Estomatite Vesicular Indiana/imunologia
3.
Emerg Microbes Infect ; 9(1): 2076-2090, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32897177

RESUMO

The current coronavirus disease 2019 (COVID-19) pandemic was the result of the rapid transmission of a highly pathogenic coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), for which there is no efficacious vaccine or therapeutic. Toward the development of a vaccine, here we expressed and evaluated as potential candidates four versions of the spike (S) protein using an insect cell expression system: receptor binding domain (RBD), S1 subunit, the wild-type S ectodomain (S-WT), and the prefusion trimer-stabilized form (S-2P). We showed that RBD appears as a monomer in solution, whereas S1, S-WT, and S-2P associate as homotrimers with substantial glycosylation. Cryo-electron microscopy analyses suggested that S-2P assumes an identical trimer conformation as the similarly engineered S protein expressed in 293 mammalian cells but with reduced glycosylation. Overall, the four proteins confer excellent antigenicity with convalescent COVID-19 patient sera in enzyme-linked immunosorbent assay (ELISA), yet show distinct reactivities in immunoblotting. RBD, S-WT and S-2P, but not S1, induce high neutralization titres (>3-log) in mice after a three-round immunization regimen. The high immunogenicity of S-2P could be maintained at the lowest dose (1 µg) with the inclusion of an aluminium adjuvant. Higher doses (20 µg) of S-2P can elicit high neutralization titres in non-human primates that exceed 40-times the mean titres measured in convalescent COVID-19 subjects. Our results suggest that the prefusion trimer-stabilized SARS-CoV-2 S-protein from insect cells may offer a potential candidate strategy for the development of a recombinant COVID-19 vaccine.


Assuntos
Antígenos Virais/imunologia , Betacoronavirus/imunologia , Infecções por Coronavirus/prevenção & controle , Imunogenicidade da Vacina/imunologia , Pandemias/prevenção & controle , Pneumonia Viral/prevenção & controle , Glicoproteína da Espícula de Coronavírus/imunologia , Vacinas Virais/imunologia , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Linhagem Celular , Infecções por Coronavirus/imunologia , Microscopia Crioeletrônica , Ensaio de Imunoadsorção Enzimática , Humanos , Macaca fascicularis , Camundongos , Camundongos Endogâmicos BALB C , Testes de Neutralização , Peptidil Dipeptidase A/metabolismo , Domínios Proteicos/genética , Domínios Proteicos/imunologia , Células Sf9 , Glicoproteína da Espícula de Coronavírus/genética , Spodoptera , Vacinação , Proteínas do Envelope Viral/imunologia
4.
Emerg Microbes Infect ; 9(1): 2091-2093, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32930052

RESUMO

We studied plasma antibody responses of 35 patients about 1 month after SARS-CoV-2 infection. Titers of antibodies binding to the viral nucleocapsid and spike proteins were significantly higher in patients with severe disease. Likewise, mean antibody neutralization titers against SARS-CoV-2 pseudovirus and live virus were higher in the sicker patients, by ∼5-fold and ∼7-fold, respectively. These findings have important implications for those pursuing plasma therapy, isolation of neutralizing monoclonal antibodies, and determinants of immunity.


Assuntos
Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Betacoronavirus/imunologia , Nucleocapsídeo/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Adulto , Idoso , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Antígenos Virais/imunologia , Infecções por Coronavirus/imunologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Testes de Neutralização , Pandemias , Pneumonia Viral/imunologia , Índice de Gravidade de Doença , Proteínas do Envelope Viral/imunologia
5.
Vet Res ; 51(1): 110, 2020 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-32883344

RESUMO

Canine parvovirus (CPV) can cause acute and highly contagious bloody enteritis in dog. To obtain antibodies against CPV, hens were immunized with virus-like particles (VLP) of CPV-VP2. The IgY single chain fragment variables (scFv) were generated by T7 phage display system and expressed in E. coli system. The titer of the primary scFv library reached to 1.5 × 106 pfu/mL, and 95% of the phages contained the target fragments. The CPV-VLP and CPV-VP2 protein showed similar reaction values to the purified scFv in the ELISA test, and the results of ELISA analysis using IgY-scFv toward CPV clinical samples were consistent with commercial immunochromatographic assay (ICA) and PCR detection, the scFv did not show cross reactivity with canine distemper virus (CDV) and canine coronavirus (CCV). IgY-scFv was successfully expressed in CRFK cells, and in the virus suppression assay, 55% of CPV infections were eliminated within 24 h. Docking results demonstrated that the number of amino acids of the binding sides between scFv and VP2 were AA37 and AA40, respectively. This study revealed the feasibility of a novel functional antibody fragment development strategy by generating diversified avian IgY-scFv libraries towards the pathogenic target of interest for both detection and therapeutic purposes in veterinary medicine.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Proteínas do Capsídeo/imunologia , Doenças do Cão/virologia , Imunoglobulinas/imunologia , Infecções por Parvoviridae/veterinária , Parvovirus Canino/imunologia , Anticorpos de Cadeia Única/imunologia , Animais , Galinhas/imunologia , Doenças do Cão/diagnóstico , Doenças do Cão/imunologia , Cães , Ensaio de Imunoadsorção Enzimática/veterinária , Feminino , Simulação de Acoplamento Molecular , Infecções por Parvoviridae/diagnóstico , Infecções por Parvoviridae/imunologia , Infecções por Parvoviridae/virologia , Anticorpos de Cadeia Única/genética
6.
Nat Commun ; 11(1): 4528, 2020 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-32913273

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spreads worldwide and leads to an unprecedented medical burden and lives lost. Neutralizing antibodies provide efficient blockade for viral infection and are a promising category of biological therapies. Here, using SARS-CoV-2 spike receptor-binding domain (RBD) as a bait, we generate a panel of humanized single domain antibodies (sdAbs) from a synthetic library. These sdAbs reveal binding kinetics with the equilibrium dissociation constant (KD) of 0.99-35.5 nM. The monomeric sdAbs show half maximal neutralization concentration (EC50) of 0.0009-0.07 µg/mL and 0.13-0.51 µg/mL against SARS-CoV-2 pseudotypes, and authentic SARS-CoV-2, respectively. Competitive ligand-binding experiments suggest that the sdAbs either completely block or significantly inhibit the association between SARS-CoV-2 RBD and viral entry receptor ACE2. Fusion of the human IgG1 Fc to sdAbs improve their neutralization activity by up to ten times. These results support neutralizing sdAbs as a potential alternative for antiviral therapies.


Assuntos
Anticorpos Neutralizantes/imunologia , Infecções por Coronavirus/virologia , Pneumonia Viral/virologia , Anticorpos de Domínio Único/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Animais , Anticorpos Neutralizantes/farmacologia , Chlorocebus aethiops , Infecções por Coronavirus/tratamento farmacológico , Células HEK293 , Humanos , Imunoglobulina G , Modelos Moleculares , Pandemias , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/tratamento farmacológico , Ligação Proteica , Receptores Virais/imunologia , Anticorpos de Domínio Único/farmacologia , Glicoproteína da Espícula de Coronavírus/metabolismo , Células Vero
7.
PLoS Pathog ; 16(9): e1008796, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32913364

RESUMO

There is an urgent need for effective treatment and preventive vaccine to contain this devastating global pandemic, which requires a comprehensive understanding of humoral responses specific to SARS-CoV-2 during the disease progression and convalescent phase of COVID-19 patients. We continuously monitored the serum IgM and IgG responses specific to four SARS-CoV-2 related antigens, including the nucleoprotein (NP), receptor binding domain (RBD), S1 protein, and ectodomain (ECD) of the spike protein among non-severe and severe COVID-19 patients for seven weeks since disease onset. Most patients generated humoral responses against NP and spike protein-related antigens but with their distinct kinetics profiles. Combined detection of NP and ECD antigens as detecting antigen synergistically improved the sensitivity of the serological assay, compared to that of using NP or RBD as detection antigen. 80.7% of convalescent sera from COVID-19 patients revealed that the varying extents of neutralization activities against SARS-CoV-2. S1-specific and ECD-specific IgA responses were strongly correlated with the neutralization activities in non-severe patients, but not in severe patients. Moreover, the neutralizing activities of the convalescent sera were shown to significantly decline during the period between 21 days to 28 days after hospital discharge, accompanied by a substantial drop in RBD-specific IgA response. Our data provide evidence that are crucial for serological testing, antibody-based intervention, and vaccine design of COVID-19.


Assuntos
Anticorpos Neutralizantes/farmacologia , Anticorpos Antivirais/farmacologia , Betacoronavirus/efeitos dos fármacos , Infecções por Coronavirus/tratamento farmacológico , Pneumonia Viral/tratamento farmacológico , Glicoproteína da Espícula de Coronavírus/antagonistas & inibidores , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Betacoronavirus/patogenicidade , Humanos , Imunoglobulina G/sangue , Estudos Longitudinais , Pandemias , Testes Sorológicos , Glicoproteína da Espícula de Coronavírus/imunologia
8.
Nat Commun ; 11(1): 4566, 2020 09 11.
Artigo em Inglês | MEDLINE | ID: mdl-32917903

RESUMO

Influenza virus exposures in childhood can establish long-lived memory B cell responses that can be recalled later in life. Here, we complete a large serological survey to elucidate the specificity of antibodies against contemporary H3N2 viruses in differently aged individuals who were likely primed with different H3N2 strains in childhood. We find that most humans who were first infected in childhood with H3N2 viral strains from the 1960s and 1970s possess non-neutralizing antibodies against contemporary 3c2.A H3N2 viruses. We find that 3c2.A H3N2 virus infections boost non-neutralizing H3N2 antibodies in middle-aged individuals, potentially leaving many of them in a perpetual state of 3c2.A H3N2 viral susceptibility.


Assuntos
Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Vírus da Influenza A Subtipo H3N2/imunologia , Influenza Humana/imunologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Neutralizantes/imunologia , Antígenos Virais/imunologia , Criança , Pré-Escolar , Suscetibilidade a Doenças , Feminino , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Modelos Biológicos , Philadelphia , Proteínas Recombinantes , Estações do Ano , Adulto Jovem
9.
Sci Rep ; 10(1): 14991, 2020 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-32929138

RESUMO

Here we have generated 3D structures of glycoforms of the spike (S) glycoprotein from SARS-CoV-2, based on reported 3D structures and glycomics data for the protein produced in HEK293 cells. We also analyze structures for glycoforms representing those present in the nascent glycoproteins (prior to enzymatic modifications in the Golgi), as well as those that are commonly observed on antigens present in other viruses. These models were subjected to molecular dynamics (MD) simulation to determine the extent to which glycan microheterogeneity impacts the antigenicity of the S glycoprotein. Lastly, we have identified peptides in the S glycoprotein that are likely to be presented in human leukocyte antigen (HLA) complexes, and discuss the role of S protein glycosylation in potentially modulating the innate and adaptive immune response to the SARS-CoV-2 virus or to a related vaccine. The 3D structures show that the protein surface is extensively shielded from antibody recognition by glycans, with the notable exception of the ACE2 receptor binding domain, and also that the degree of shielding is largely insensitive to the specific glycoform. Despite the relatively modest contribution of the glycans to the total molecular weight of the S trimer (17% for the HEK293 glycoform) they shield approximately 40% of the protein surface.


Assuntos
Betacoronavirus/metabolismo , Infecções por Coronavirus/patologia , Pneumonia Viral/patologia , Polissacarídeos/química , Glicoproteína da Espícula de Coronavírus/metabolismo , Imunidade Adaptativa , Sequência de Aminoácidos , Anticorpos Neutralizantes/imunologia , Complexo Antígeno-Anticorpo , Betacoronavirus/imunologia , Betacoronavirus/isolamento & purificação , Sítios de Ligação , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/virologia , Glicosilação , Células HEK293 , Antígenos HLA/metabolismo , Humanos , Imunidade Inata , Simulação de Dinâmica Molecular , Pandemias , Peptidil Dipeptidase A/química , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/imunologia , Pneumonia Viral/virologia , Ligação Proteica , Estrutura Terciária de Proteína , Alinhamento de Sequência , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologia
10.
Nat Commun ; 11(1): 4420, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32887876

RESUMO

SARS-CoV-2 enters host cells through an interaction between the spike glycoprotein and the angiotensin converting enzyme 2 (ACE2) receptor. Directly preventing this interaction presents an attractive possibility for suppressing SARS-CoV-2 replication. Here, we report the isolation and characterization of an alpaca-derived single domain antibody fragment, Ty1, that specifically targets the receptor binding domain (RBD) of the SARS-CoV-2 spike, directly preventing ACE2 engagement. Ty1 binds the RBD with high affinity, occluding ACE2. A cryo-electron microscopy structure of the bound complex at 2.9 Å resolution reveals that Ty1 binds to an epitope on the RBD accessible in both the 'up' and 'down' conformations, sterically hindering RBD-ACE2 binding. While fusion to an Fc domain renders Ty1 extremely potent, Ty1 neutralizes SARS-CoV-2 spike pseudovirus as a 12.8 kDa nanobody, which can be expressed in high quantities in bacteria, presenting opportunities for manufacturing at scale. Ty1 is therefore an excellent candidate as an intervention against COVID-19.


Assuntos
Inibidores da Enzima Conversora de Angiotensina/farmacologia , Betacoronavirus/efeitos dos fármacos , Camelídeos Americanos/imunologia , Infecções por Coronavirus/tratamento farmacológico , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/tratamento farmacológico , Anticorpos de Domínio Único/farmacologia , Glicoproteína da Espícula de Coronavírus/antagonistas & inibidores , Sequência de Aminoácidos , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/farmacologia , Anticorpos Antivirais/química , Anticorpos Antivirais/imunologia , Betacoronavirus/imunologia , Betacoronavirus/metabolismo , Sítios de Ligação , Chlorocebus aethiops , Infecções por Coronavirus/virologia , Microscopia Crioeletrônica , Epitopos/imunologia , Epitopos/metabolismo , Células HEK293 , Humanos , Masculino , Modelos Moleculares , Pandemias , Peptidil Dipeptidase A/química , Pneumonia Viral/virologia , Ligação Proteica , Anticorpos de Domínio Único/imunologia , Anticorpos de Domínio Único/isolamento & purificação , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/metabolismo , Células Vero
11.
JCI Insight ; 5(19)2020 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-32870820

RESUMO

Most of the patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mount a humoral immune response to the virus within a few weeks of infection, but the duration of this response and how it correlates with clinical outcomes has not been completely characterized. Of particular importance is the identification of immune correlates of infection that would support public health decision-making on treatment approaches, vaccination strategies, and convalescent plasma therapy. While ELISA-based assays to detect and quantitate antibodies to SARS-CoV-2 in patient samples have been developed, the detection of neutralizing antibodies typically requires more demanding cell-based viral assays. Here, we present a safe and efficient protein-based assay for the detection of serum and plasma antibodies that block the interaction of the SARS-CoV-2 spike protein receptor binding domain (RBD) with its receptor, angiotensin-converting enzyme 2 (ACE2). The assay serves as a surrogate neutralization assay and is performed on the same platform and in parallel with an ELISA for the detection of antibodies against the RBD, enabling a direct comparison. The results obtained with our assay correlate with those of 2 viral-based assays, a plaque reduction neutralization test (PRNT) that uses live SARS-CoV-2 virus and a spike pseudotyped viral vector-based assay.


Assuntos
Anticorpos Neutralizantes/imunologia , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/terapia , Pneumonia Viral/imunologia , Pneumonia Viral/terapia , Glicoproteína da Espícula de Coronavírus/imunologia , Anticorpos Antivirais/sangue , Área Sob a Curva , Ensaio de Imunoadsorção Enzimática , Humanos , Imunização Passiva/métodos , Testes de Neutralização , Pandemias , Análise de Regressão , Amostragem , Resultado do Tratamento , Proteínas do Envelope Viral/imunologia
12.
Cell Mol Immunol ; 17(10): 1095-1097, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32895485
13.
PLoS Pathog ; 16(9): e1008857, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32936838

RESUMO

An outbreak of the hand-foot-mouth disease with severe neurological cases, mainly caused by the genotype C1 enterovirus A71 (EV-A71), occurred in Taiwan between 2018 and early 2019. In the recent decade, the most dominant EV-A71 genotypes in Taiwan were B5 and C4 but changed to C1 in 2018. Antibody-mediated immunity plays a key role in limiting the EV-A71 illness in humans. However, the level of neutralizing activities against genotype C1 virus by human polyclonal and monoclonal antibodies (MAbs) remains largely unclear. In the study, we demonstrated that that 39% (9 in 23) of post-infection sera from the genotype B5- or C4-infected patients in 2014-2017 exhibit reduced titers with the 2018-2019 genotype C1 viruses than with the earlier B5 and C4 viruses tested. This finding with polyclonal sera is confirmed with human MAbs derived from genotype B5 virus-infected individuals. The 2018-2019 genotype C1 virus is resistant to the majority of canyon-targeting human MAbs, which may be associated with the residue change near or at the bottom of the canyon region on the viral capsid. The remaining three antibodies (16-2-11B, 16-3-4D, and 17-1-12A), which target VP1 S241 on the 5-fold vertex, VP3 E81 on the 3-fold plateau and VP2 D84 on the 2-fold plateau of genotype C1 viral capsid, respectively, retained neutralizing activities with variable potencies. These neutralizing antibodies were also found to be protective against a lethal challenge of the 2018-2019 genotype C1 virus in an hSCARB2-transgenic mice model. These results indicate that the EV-A71-specific antibody response may consist of a fraction of poorly neutralizing antibodies against 2018-2019 genotype C1 viruses among a subset of previously infected individuals. Epitope mapping of protective antibodies that recognize the emerging genotype C1 virus has implications for anti-EV-A71 MAbs and the vaccine field.


Assuntos
Antígenos Virais/genética , Enterovirus Humano A/genética , Variação Genética , Genoma Viral , Genótipo , Doença de Mão, Pé e Boca/genética , Animais , Anticorpos Neutralizantes/genética , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/genética , Anticorpos Antivirais/imunologia , Antígenos Virais/imunologia , Criança , Pré-Escolar , Enterovirus Humano A/imunologia , Enterovirus Humano A/isolamento & purificação , Feminino , Doença de Mão, Pé e Boca/epidemiologia , Doença de Mão, Pé e Boca/imunologia , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Taiwan
14.
BMC Infect Dis ; 20(1): 641, 2020 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-32867698

RESUMO

BACKGROUND: Maternal poliovirus antibodies could provide passive immunity to the newborns from poliovirus infection during their first few months of life, but they may impair the immune responses of infants to the poliovirus vaccine as well. In our study, we pooled the data from three clinical trials of the inactivated poliovirus vaccine (IPV) based on Sabin strains to investigate the effect of maternal poliovirus antibodies on the immune responses of infants to poliovirus vaccines. METHODS: There were five groups in the pooled analysis, including low-dose Sabin IPV, medium-dose Sabin IPV, high-dose Sabin IPV, control Sabin IPV, and control Salk IPV groups. We reclassified the infants in different groups according to their maternal poliovirus antibodies by two methods, the first one included maternal antibody negative (< 1:8) and maternal antibody positive (≥1:8), and the second one included maternal antibody titer < 1:8, 1:8 ~ < 1:32 and ≥ 1:32. Then, we compared the geometric mean titers (GMTs), geometric mean antibody fold increases (GMIs) and seroconversion rates of poliovirus type-specific neutralizing antibodies after vaccination among participants with different maternal poliovirus antibody levels. RESULTS: The GMTs and GMIs of three types of poliovirus antibodies after vaccination in maternal antibody negative participants were significantly higher than those in maternal antibody positive participants. The seroconversion rates of type II and type III poliovirus antibodies in maternal antibody positive participants were significantly lower than those in maternal antibody negative participants. Among participants with maternal antibody titer < 1:8, 1:8 ~ < 1:32 and ≥ 1:32, the GMTs and GMIs of three types of poliovirus antibodies after vaccination showed a tendency to decline with the increasing of maternal antibody levels. The seroconversion rates of three types of poliovirus antibodies in participants with maternal antibody titer ≥1:32 were significantly lower than those in participants with maternal antibody titer < 1:8 and 1:8 ~ < 1:32. CONCLUSIONS: Maternal poliovirus antibodies interfered with the immune responses of infants to poliovirus vaccines, and a high level of maternal antibodies exhibited a greater dampening effect. TRIAL REGISTRATION: ClinicalTrials.gov NCT04264598 February 11, 2020; ClinicalTrials.gov NCT04264546 February 11, 2020; ClinicalTrials.gov NCT03902054 April 3, 2019. Retrospectively registered.


Assuntos
Anticorpos Antivirais/imunologia , Imunidade Materno-Adquirida/imunologia , Imunogenicidade da Vacina , Poliomielite/prevenção & controle , Vacina Antipólio de Vírus Inativado/uso terapêutico , Poliovirus/imunologia , Vacinação/métodos , Anticorpos Neutralizantes/imunologia , China , Feminino , Humanos , Lactente , Masculino , Poliomielite/virologia , Vacina Antipólio de Vírus Inativado/efeitos adversos , Soroconversão
15.
PLoS Pathog ; 16(8): e1008736, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32745149

RESUMO

Human cytomegalovirus (HCMV) is one of the main causative agents of congenital viral infection in neonates. HCMV infection also causes serious morbidity and mortality among organ transplant patients. Glycoprotein B (gB) is a major target for HCMV neutralizing antibodies, yet the underlying neutralization mechanisms remain largely unknown. Here we report that 3-25, a gB-specific monoclonal antibody previously isolated from a healthy HCMV-positive donor, efficiently neutralized 14 HCMV strains in both ARPE-19 cells and MRC-5 cells. The core epitope of 3-25 was mapped to a highly conserved linear epitope on antigenic domain 2 (AD-2) of gB. A 1.8 Å crystal structure of 3-25 Fab in complex with the peptide epitope revealed the molecular determinants of 3-25 binding to gB at atomic resolution. Negative-staining electron microscopy (EM) 3D reconstruction of 3-25 Fab in complex with de-glycosylated postfusion gB showed that 3-25 Fab fully occupied the gB trimer at the N-terminus with flexible binding angles. Functionally, 3-25 efficiently inhibited HCMV infection at a post-attachment step by interfering with viral membrane fusion, and restricted post-infection viral spreading in ARPE-19 cells. Interestingly, bivalency was required for HCMV neutralization by AD-2 specific antibody 3-25 but not the AD-4 specific antibody LJP538. In contrast, bivalency was not required for HCMV binding by both antibodies. Taken together, our results reveal the structural basis of gB recognition by 3-25 and demonstrate that inhibition of viral membrane fusion and a requirement of bivalency may be common for gB AD-2 specific neutralizing antibody.


Assuntos
Anticorpos Antivirais/imunologia , Infecções por Citomegalovirus/imunologia , Citomegalovirus/imunologia , Epitopos/imunologia , Proteínas do Envelope Viral/imunologia , Motivos de Aminoácidos , Anticorpos Neutralizantes/imunologia , Sequência Conservada , Citomegalovirus/química , Citomegalovirus/genética , Citomegalovirus/fisiologia , Infecções por Citomegalovirus/virologia , Epitopos/química , Epitopos/genética , Humanos , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/genética , Internalização do Vírus
16.
Nat Commun ; 11(1): 4198, 2020 08 21.
Artigo em Inglês | MEDLINE | ID: mdl-32826914

RESUMO

COVID-19 caused by SARS-CoV-2 has become a global pandemic requiring the development of interventions for the prevention or treatment to curtail mortality and morbidity. No vaccine to boost mucosal immunity, or as a therapeutic, has yet been developed to SARS-CoV-2. In this study, we discover and characterize a cross-reactive human IgA monoclonal antibody, MAb362. MAb362 binds to both SARS-CoV and SARS-CoV-2 spike proteins and competitively blocks ACE2 receptor binding, by overlapping the ACE2 structural binding epitope. Furthermore, MAb362 IgA neutralizes both pseudotyped SARS-CoV and SARS-CoV-2 in 293 cells expressing ACE2. When converted to secretory IgA, MAb326 also neutralizes authentic SARS-CoV-2 virus while the IgG isotype shows no neutralization. Our results suggest that SARS-CoV-2 specific IgA antibodies, such as MAb362, may provide effective immunity against SARS-CoV-2 by inducing mucosal immunity within the respiratory system, a potentially critical feature of an effective vaccine.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Betacoronavirus/imunologia , Imunoglobulina A/imunologia , Peptidil Dipeptidase A/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , Animais , Anticorpos Monoclonais/metabolismo , Anticorpos Neutralizantes/metabolismo , Chlorocebus aethiops , Reações Cruzadas , Epitopos , Células HEK293 , Humanos , Imunoglobulina A/metabolismo , Imunoglobulina A Secretora/imunologia , Imunoglobulina A Secretora/metabolismo , Imunoglobulina G/imunologia , Imunoglobulina G/metabolismo , Modelos Moleculares , Mutação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Vírus da SARS/imunologia , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologia , Células Vero
17.
Indian J Med Res ; 152(1 & 2): 82-87, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32859866

RESUMO

Background & objectives: The global pandemic caused by SARS-CoV-2 virus has challenged public health system worldwide due to the unavailability of approved preventive and therapeutic options. Identification of neutralizing antibodies (NAb) and understanding their role is important. However, the data on kinetics of NAb response among COVID-19 patients are unclear. To understand the NAb response in COVID-19 patients, we compared the findings of microneutralization test (MNT) and plaque reduction neutralization test (PRNT) for the SARS-CoV-2. Further, the kinetics of NAb response among COVID-19 patients was assessed. Methods: A total of 343 blood samples (89 positive, 58 negative for SARS-CoV-2 and 17 cross-reactive and 179 serum from healthy individuals) were collected and tested by MNT and PRNT. SARS-CoV-2 virus was prepared by propagating the virus in Vero CCL-81 cells. The intra-class correlation was calculated to assess the correlation between MNT and PRNT. The neutralizing endpoint as the reduction in the number of plaque count by 90 per cent (PRNT90) was also calculated. Results: The analysis of MNT and PRNT quantitative results indicated that the intra-class correlation was 0.520. Of the 89 confirmed COVID-19 patients, 64 (71.9%) showed NAb response. Interpretation & conclusions: The results of MNT and PRNT were specific with no cross-reactivity. In the early stages of infection, the NAb response was observed with variable antibody kinetics. The neutralization assays can be used for titration of NAb in recovered/vaccinated or infected COVID-19 patients.


Assuntos
Anticorpos Neutralizantes/isolamento & purificação , Infecções por Coronavirus/sangue , Testes de Neutralização , Pandemias , Pneumonia Viral/sangue , Adolescente , Adulto , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/sangue , Betacoronavirus/imunologia , Betacoronavirus/patogenicidade , Criança , Chlorocebus aethiops/imunologia , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/virologia , Feminino , Humanos , Imunoglobulina G/sangue , Masculino , Pessoa de Meia-Idade , Pneumonia Viral/epidemiologia , Pneumonia Viral/imunologia , Pneumonia Viral/virologia , Células Vero/imunologia , Adulto Jovem
18.
Cell ; 182(5): 1271-1283.e16, 2020 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-32795413

RESUMO

There is an urgent need for vaccines against coronavirus disease 2019 (COVID-19) because of the ongoing SARS-CoV-2 pandemic. Among all approaches, a messenger RNA (mRNA)-based vaccine has emerged as a rapid and versatile platform to quickly respond to this challenge. Here, we developed a lipid nanoparticle-encapsulated mRNA (mRNA-LNP) encoding the receptor binding domain (RBD) of SARS-CoV-2 as a vaccine candidate (called ARCoV). Intramuscular immunization of ARCoV mRNA-LNP elicited robust neutralizing antibodies against SARS-CoV-2 as well as a Th1-biased cellular response in mice and non-human primates. Two doses of ARCoV immunization in mice conferred complete protection against the challenge of a SARS-CoV-2 mouse-adapted strain. Additionally, ARCoV is manufactured as a liquid formulation and can be stored at room temperature for at least 1 week. ARCoV is currently being evaluated in phase 1 clinical trials.


Assuntos
RNA Mensageiro/genética , RNA Viral/genética , Vacinas Sintéticas/imunologia , Vacinas Virais/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Sítios de Ligação , Chlorocebus aethiops , Infecções por Coronavirus/genética , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/prevenção & controle , Feminino , Células HEK293 , Células HeLa , Humanos , Imunogenicidade da Vacina , Injeções Intramusculares , Macaca fascicularis , Masculino , Camundongos , Camundongos Endogâmicos ICR , Nanopartículas/química , RNA Mensageiro/metabolismo , RNA Viral/metabolismo , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo , Células Th1/imunologia , Potência de Vacina , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Células Vero , Vacinas Virais/administração & dosagem , Vacinas Virais/genética
19.
Tidsskr Nor Laegeforen ; 140(11)2020 08 18.
Artigo em Inglês, Norueguês | MEDLINE | ID: mdl-32815342

RESUMO

BACKGROUND: Robust serological assays for SARS-CoV-2 are essential for determining prior infection and the suitability of donated convalescent plasma for plasma therapy. We compared two in-house and three commercial serological assays in a family cohort with SARS-CoV-2-infected members. CASE PRESENTATION: Three individuals in a family of five developed COVID-19 confirmed by PCR, following a trip abroad. Three to four weeks after the onset of symptoms, three commercial ELISAs and an in-house immunofluorescence test demonstrated antibodies to SARS-CoV-2. An in-house neutralisation test also demonstrated neutralising antibodies. INTERPRETATION: The in-house assays and one commercial assay gave congruent results, which were also consistent with the initial PCR and/or clinical evaluation, indicating prior infection in three of the five family members. The other commercial assays indicated possible infection in a fourth family member, but this result was likely due to cross-reactivity. The neutralising antibodies suggest complete or partial immunity against reinfection.


Assuntos
Anticorpos Antivirais/imunologia , Formação de Anticorpos , Infecções por Coronavirus/imunologia , Pneumonia Viral/imunologia , Anticorpos Neutralizantes/imunologia , Betacoronavirus , Ensaio de Imunoadsorção Enzimática , Saúde da Família , Imunofluorescência , Humanos , Testes de Neutralização , Pandemias , Testes Sorológicos
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