The COVID-19 pandemic, caused by SARS-CoV-2, necessitates the development of effective preventative and therapeutic interventions. Mucosal immunity is a crucial aspect of protection against respiratory viruses, and currently, there is a lack of vaccines or therapies that effectively boost this type of immunity against SARS-CoV-2. This research focuses on the identification and characterization of human monoclonal antibodies (mAbs), specifically IgA isotype, as potential therapeutic agents. Previous studies have demonstrated the potential of mAbs targeting the viral spike (S) glycoprotein, specifically the receptor-binding domain (RBD), to neutralize coronaviruses. The RBD facilitates viral entry into human cells via binding to the human angiotensin-converting enzyme 2 (ACE2) receptor. While many studies have focused on IgG mAbs, there is growing interest in IgA's potential for mucosal immunity and its possible role in preventing or mitigating COVID-19. Concerns about antibody-dependent enhancement (ADE) also underscore the importance of careful investigation into isotype-specific effects. This study aims to investigate whether IgA mAbs offer superior neutralizing activity compared to their IgG counterparts and evaluate their potential as a component of a novel vaccine.

The literature review extensively examines previous studies on neutralizing antibodies against SARS-CoV and other coronaviruses. It highlights the importance of targeting the RBD of the spike protein to block ACE2 receptor binding and prevent viral entry. The review also discusses the limitations of previous approaches, including concerns regarding ADE, and the potential advantages of IgA antibodies for mucosal immunity. Several relevant studies involving human monoclonal antibodies, specifically those targeting SARS-CoV and MERS-CoV, are discussed and referenced. The review emphasizes the lack of effective vaccines that induce robust mucosal immunity against SARS-CoV-2 and the need for novel therapeutic strategies.

The study employed a comprehensive methodology encompassing several key steps. First, a panel of human mAbs targeting the RBD of the SARS-CoV S protein was generated from transgenic mice expressing human immunoglobulin genes. ELISA assays were used to screen these mAbs for cross-reactivity against the SARS-CoV-2 spike protein, leading to the identification of MAb362. The variable sequences of MAb362 were cloned into expression vectors as IgG and monomeric IgA isotypes. ELISA and biolayer interferometry were used to assess the binding affinity of both isotypes to SARS-CoV and SARS-CoV-2 spike proteins and their RBDs. A receptor-blocking assay with Vero E6 cells was conducted to evaluate the ability of MAb362 to block SARS-CoV-2 RBD binding to ACE2. Mutational scanning was performed to map the binding epitope. Structural modeling, utilizing known co-crystal and cryo-electron microscopy complexes, was used to predict the MAb362 binding epitope and its overlap with the ACE2 binding site. Pseudotyped virus neutralization assays using lentiviral pseudoviruses and authentic virus neutralization assays using live SARS-CoV-2 were performed to assess the neutralizing potency of MAb362 IgG and IgA. The study also involved the generation of dimeric IgA (dIgA) and secretory IgA (sIgA) forms of MAb362 to further evaluate the impact of IgA polymerization and secretory component on neutralization efficacy. Detailed protocols for ELISA, biolayer interferometry, receptor binding inhibition assay, pseudotyped virus neutralization assay, plaque reduction neutralization assay, structural modeling and mutational scanning are provided.

The key findings of the study demonstrate the superior neutralizing activity of MAb362 IgA compared to its IgG counterpart. MAb362, in its IgA form, demonstrated potent neutralization of both pseudotyped and authentic SARS-CoV-2 viruses. The study revealed that MAb362 binds to a cross-reactive epitope within the core receptor-binding interface of both SARS-CoV and SARS-CoV-2 spike proteins, competitively inhibiting ACE2 binding. Structural modeling predicted that MAb362 overlaps with the ACE2 epitope on the RBD, effectively blocking receptor interaction. The IgA isotype exhibited significantly higher binding affinity (0.3 nM) compared to the IgG isotype (13 nM). Further, isotype switching to IgA dramatically improved neutralization potency. The IC50 value for MAb362 IgA in the pseudotyped virus neutralization assay was 1.26 mg/ml, substantially lower than that for MAb362 IgG (5.867 mg/ml). Moreover, dimeric and secretory IgA forms of MAb362 exhibited even greater neutralization efficacy, with IC50 values of 30 ng/ml and 10 ng/ml respectively. The authentic virus neutralization assay confirmed the potent neutralizing activity of MAb362 sIgA (IC50 = 9.54 µg/ml), while MAb362 IgG failed to neutralize at the highest tested concentration. The study strongly suggests that the IgA isotype may play a critical role in SARS-CoV-2 neutralization and that IgA antibodies may be a promising avenue for the development of mucosal vaccines.

The findings of this study highlight the potential of IgA antibodies as a therapeutic agent against SARS-CoV-2. The significantly enhanced neutralization potency of the IgA isotype compared to IgG, along with its ability to block ACE2 binding, suggests that IgA may offer superior protection against SARS-CoV-2 infection. The cross-reactivity of MAb362 against both SARS-CoV and SARS-CoV-2, targeting a conserved epitope, implies a broader protective effect against potential future coronavirus outbreaks. The superior neutralizing activity of IgA likely arises from its multimeric structure and increased flexibility compared to IgG. The data strongly suggest that IgA antibodies may play a unique role in providing protective mucosal immunity, a crucial feature in respiratory viral infections. The successful neutralization of authentic SARS-CoV-2 virus by MAb362 sIgA further underscores its therapeutic potential. Further research is warranted to explore the clinical implications of IgA-based therapies or vaccines for COVID-19 prevention and treatment.

This study demonstrates the potent neutralizing activity of a cross-reactive human IgA monoclonal antibody, MAb362, against SARS-CoV-2. The significantly higher neutralization potency of the IgA isotype compared to IgG highlights the potential of IgA-based therapies or vaccines. Future studies should focus on evaluating the in vivo efficacy and safety of MAb362 and explore the potential for developing IgA-based vaccines to induce robust mucosal immunity against SARS-CoV-2.

The study primarily focused on in vitro experiments and the results need to be validated in in vivo studies to confirm efficacy and safety. The sample size in some assays could be considered relatively small; larger sample sizes might provide more robust statistical analyses. Further studies are needed to investigate the long-term effects and potential adverse events associated with IgA-based therapies and vaccines.