RESUMEN
The nucleic acid test is still the standard assessment for the diagnosis of coronavirus disease 2019 (COVID-19), which is caused by human infection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In addition to supporting the confirmation of disease cases, serological assays are used for the analysis of antibody status and epidemiological surveys. In this study, a single Western blot strip (WBS) coated with multiple Escherichia coli (E. coli)-expressed SARS-CoV-2 antigens was developed for comprehensive studies of antibody profiles in COVID-19 patient sera. The levels of specific antibodies directed to SARS-CoV-2 spike (S), S2, and nucleocapsid (N) proteins were gradually increased with the same tendency as the disease progressed after hospitalization. The signal readouts of S, S2, and N revealed by the multi-antigen-coated WBS (mWBS)-based serological assay (mWBS assay) also demonstrated a positive correlation with the SARS-CoV-2 neutralizing potency of the sera measured by the plaque reduction neutralization test (PRNT) assays. Surprisingly, the detection signals against the unstructured receptor-binding domain (RBD) purified from E. coli inclusion bodies were not observed, although the COVID-19 patient sera exhibited strong neutralizing potency in the PRNT assays, suggesting that the RBD-specific antibodies in patient sera mostly recognize the conformational epitopes. Furthermore, the mWBS assay identified a unique and major antigenic epitope at the residues 1148, 1149, 1152, 1155, and 1156 located within the 1127-1167 fragment of the S2 subunit, which was specifically recognized by the COVID-19 patient serum. The mWBS assay can be finished within 14-16 min by using the automatic platform of Western blotting by thin-film direct coating with suction (TDCS WB). Collectively, the mWBS assay can be applied for the analysis of antibody responses, prediction of the protective antibody status, and identification of the specific epitope. KEY POINTS: ⢠A Western blot strip (WBS) coated with multiple SARS-CoV-2 antigens was developed for the serological assay. ⢠The multi-antigen-coated WBS (mWBS) can be utilized for the simultaneous detection of antibody responses to multiple SARS-CoV-2 antigens. ⢠The mWBS-based serological assay (mWBS assay) identified a unique epitope recognized by the COVID-19 patient serum.
Asunto(s)
COVID-19 , SARS-CoV-2 , Humanos , Formación de Anticuerpos , COVID-19/diagnóstico , Escherichia coli/genética , Western BlottingRESUMEN
Many cases of avian influenza A(H7N9) virus infection in humans have been reported since its first emergence in 2013. The disease is of concern because most patients have become severely ill with roughly 30% mortality rate. Because the threat in public health caused by H7N9 virus remains high, advance preparedness is essentially needed. In this study, the recombinant H7N9 hemagglutinin (HA) was expressed in insect cells and purified for generation of two monoclonal antibodies, named F3-2 and 1C6B. F3-2 can only recognize the H7N9 HA without having cross-reactivity with HA proteins of H1N1, H3N2, H5N1, and H7N7. 1C6B has the similar specificity with F3-2, but 1C6B can also bind to H7N7 HA. The binding epitope of F3-2 is mainly located in the region of H7N9 HA(299-307). The binding epitope of 1C6B is located in the region of H7N9 HA(489-506). F3-2 and 1C6B could not effectively inhibit the hemagglutination activity of H7N9 HA. However, F3-2 can prevent H7N9 HA from trypsin cleavage and can bind to H7N9 HA which has undergone pH-induced conformational change. F3-2 also has the ability of binding to H7N9 viral particles and inhibiting H7N9 virus infection to MDCK cells with the IC50 value of 22.18 µg/mL. In addition, F3-2 and 1C6B were utilized for comprising a lateral flow immunochromatographic test strip for specific detection of H7N9 HA. KEY POINTS: ⢠Two mouse monoclonal antibodies, F3-2 and 1C6B, were generated for recognizing the novel binding epitopes in H7N9 HA. ⢠F3-2 can prevent H7N9 HA from trypsin cleavage and inhibit H7N9 virus infection to MDCK cells. ⢠F3-2 and 1C6B were developed as a lateral flow immunochromatographic test for specific detection of H7N9 HA.
Asunto(s)
Subtipo H7N9 del Virus de la Influenza A , Gripe Humana , Animales , Anticuerpos Monoclonales , Anticuerpos Antivirales , Glicoproteínas Hemaglutininas del Virus de la Influenza , Hemaglutininas , Humanos , Subtipo H1N1 del Virus de la Influenza A , Subtipo H3N2 del Virus de la Influenza A , Subtipo H5N1 del Virus de la Influenza A , Subtipo H7N7 del Virus de la Influenza A , Gripe Humana/prevención & control , RatonesRESUMEN
Neutralizing antibodies (NAbs) are believed to be promising prophylactic and therapeutic treatment against the coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here, we reported two mouse monoclonal antibodies 7 Eb-4G and 1Ba-3H that specifically recognized the receptor-binding domain (RBD) of SARS-CoV-2 spike (S) protein without exhibiting cross-reactivity with the S proteins of SARS-CoV and MERS-CoV. The binding epitopes of 7 Eb-4G and 1Ba-3H were respectively located in the regions of residues 457-476 and 477-496 in the S protein. Only 1Ba-3H exhibited the neutralizing activity for preventing the pseudotyped lentivirus from binding to the angiotensin-converting enzyme 2 (ACE2)-transfected HEK293T cells. The competitive ELISA further showed that 1Ba-3H interfered with the binding between RBD and ACE2. Epitope mapping experiments demonstrated that a single alanine replacement at residues 480, 482, 484, 485, and 488-491 in the RBD abrogated 1Ba-3H binding. 1Ba-3H exhibited the neutralizing activity against the wild-type, Alpha, Delta, and Epsilon variants of SARS-CoV-2, but lost the neutralizing activity against Gamma variant in the plaque reduction assay. On the contrary, 1Ba-3H enhanced the cellular infection of Gamma variant in a dose-dependent manner. Our findings suggest that the antibody-dependent enhancement of infection mediated by the RBD-specific antibody for different SARS-CoV-2 variants must be considered while developing the NAb.
Asunto(s)
COVID-19 , SARS-CoV-2 , Animales , Anticuerpos Monoclonales , Epítopos , Células HEK293 , Humanos , Ratones , Glicoproteína de la Espiga del CoronavirusRESUMEN
Most of SARS-CoV-2 neutralizing antibodies (nAbs) targeted the receptor binding domain (RBD) of the SARS-CoV-2 spike (S) protein. However, mutations at RBD sequences found in the emerging SARS-CoV-2 variants greatly reduced the effectiveness of nAbs. Here we showed that four nAbs, S2-4D, S2-5D, S2-8D, and S2-4A, which recognized a conserved epitope in the S2 subunit of the S protein, can inhibit SARS-CoV-2 infection through blocking the S protein-mediated membrane fusion. Notably, these four nAbs exhibited broadly neutralizing activity against SARS-CoV-2 Alpha, Gamma, Delta, and Epsilon variants. Antisera collected from mice immunized with the identified epitope peptides of these four nAbs also exhibited potent virus neutralizing activity. Discovery of the S2-specific nAbs and their unique antigenic epitopes paves a new path for development of COVID-19 therapeutics and vaccines. IMPORTANCE The spike (S) protein on the surface of SARS-CoV-2 mediates receptor binding and virus-host cell membrane fusion during virus entry. Many neutralizing antibodies (nAbs), which targeted the receptor binding domain (RBD) of S protein, lost the neutralizing activity against the newly emerging SARS-CoV-2 variants with sequence mutations at the RBD. In contrast, the nAb against the highly conserved S2 subunit, which plays the key role in virus-host cell membrane fusion, was poorly discovered. We showed that four S2-specific nAbs, S2-4D, S2-5D, S2-8D, and S2-4A, inhibited SARS-CoV-2 infection through blocking the S protein-mediated membrane fusion. These nAbs exhibited broadly neutralizing activity against Alpha, Gamma, Delta, and Epsilon variants. Antisera induced by the identified epitope peptides also possessed potent neutralizing activity. This work not only unveiled the S2-specific nAbs but also discovered an immunodominant epitope in the S2 subunit that can be rationally designed as the broad-spectrum vaccine against the SARS-like coronaviruses.