Fragment crystallizable-altered COVID-19 antibodies efficacious across two animal models

A recent article posted on the bioRxiv* preprint server has identified numerous effective antibodies against the coronavirus 2 (SARS-CoV-2) of severe acute respiratory syndrome (SARS-CoV-2) from recovering SARS-CoV-2 patients.

Study: Fc-modified SARS-CoV-2 neutralizing antibodies with therapeutic effects in two animal models.  Image Credit: NIAIDStudy: Fc-modified SARS-CoV-2 neutralizing antibodies with therapeutic effects in two animal models. Image Credit: NIAID

Background

The SARS-CoV-2 pandemic continues through the sequentially emerging viral variants. Several vaccines against coronavirus disease 2019 (COVID-19) have been developed that target the original SARS-CoV-2 Wuhan strains. Coincidentally, they were effective against the subsequently elicited viral strains.

The number of SARS-CoV-2 infections has decreased in certain countries, possibly due to the effectiveness of the vaccination. Nevertheless, the global COVID-19 pandemic is not yet under control.

Antiviral therapy is effective during the SARS-CoV-2 replication phase, which occurs in the early phases of infection. Similarly, the use of therapeutic neutralizing antibodies against COVID-19 has been significantly effective. Unfortunately, few useful antibodies exist to fight developing SARS-CoV-2 strains.

About the study

In the current study, the scientists developed multiple monoclonal antibodies from SARS-CoV-2 recovering patients. Since the onset of the SARS-CoV-2 epidemic in Japan in March 2020, the authors have collected peripheral blood samples from recovering COVID-19 patients, which have been used to generate neutralizing antibodies.

The researchers obtained blood samples from discharged SARS-CoV-2 patients from Keio University Hospital. The cell-based SARS-CoV-2 spike (S)-angiotensin-converting enzyme 2 (ACE2) inhibition assay was used to evaluate the neutralizing capacity of sera. The team selected 12 patients who showed prominent neutralizing concentrations for antibody generation.

The authors profiled patient-strain antibodies using 1) S-ACE2 inhibition assessment and 2) the association between the binding ability of these antibodies to S-expressing cells and their potential to inhibit the attachment of ACE2 to S-expressing cells. to hinder. To analyze these antibodies more thoroughly, they also examined their neutralizing potential using a cell fusion experiment. The researchers performed an endpoint microneutralization screening to verify that the chosen antibodies could neutralize the authentic SARS-CoV-2.

To further identify potential antibodies, the scientists evaluated affinity for SARS-CoV-2 receptor binding domain (RBD) antigen and analyzed epitope overlap. They chose five antibodies and used a pseudovirus harboring the S protein of the original SARS-CoV-2 Wuhan sequence and four significant variants to perform a neutralization experiment before the emergence of variants of concern (VOCs). After the emergence of VOCs, they tested the ability of the antibodies to neutralize the original WK-521 virus and its variants, including Beta, Alpha, Gamma, Kappa, Delta and Omicron BA.2 and BA.1.

The team conducted a cryoelectron microscopy (cryo-EM) study to gain a structural understanding of antibodies and the SARS-CoV-2 S protein. The currently discovered antibodies used in the in vivo study possessed the N297A mutation in the crystallizable (Fc) region of the immunoglobulin G1 (IgG1) fragment to prevent antibody-dependent enhancement (ADE). In addition, the N297A mutation reduces the attachment to the Fc receptor. The researchers then examined the effects of these antibodies in two animal models (a cynomolgus macaque model and a hamster model) to assess the impact of these antibodies in in vivo settings.

Cryo-EM structure of neutralizing antibodies (A) The structures of RBD and Ab159, Ab188, Ab326, Ab354, Ab445 and Ab496 are shown.  Only the variable domains of antibodies are modeled and drawn as a cartoon tube (individual color) on the RBD surface (grey), and the epitope of each antibody is stained the same as each antibody.  The red region in the central RBD is the binding residue of ACE2 (7A94) (Benton et al., 2020), showing the relationship between the binding sites of the antibodies, which are roughly divided into three groups.  The positions of the major amino acids are indicated by black arrows.  (B) Spike residues 400-506 are shown.  The epitopes revealed by cryo-EM are colored red and the residues affected by the mutation described in Figure 3A are shown in squares.Cryo-EM structure of neutralizing antibodies (A) The structures of RBD and Ab159, Ab188, Ab326, Ab354, Ab445 and Ab496 are shown. Only the variable domains of antibodies are modeled and drawn as a cartoon tube (individual color) on the RBD surface (grey), and the epitope of each antibody is stained the same as each antibody. The red region in the central RBD is the binding residue of ACE2 (7A94) (Benton et al., 2020), showing the relationship between the binding sites of the antibodies, which are roughly divided into three groups. The positions of the major amino acids are indicated by black arrows. (B) Spike residues 400-506 are shown.

Results

The researchers discovered 494 antibodies from COVID-19-recovered individuals, most of which showed an identical SARS-CoV-2 neutralizing ability as clinically used antibodies in the neutralization assessment. Initially, antigen-specific memory B cells and antigen-non-specific plasma cells were used to make antibodies. Nevertheless, the first harbored superior antibodies, emphasizing the importance of selecting B cells by antigen. The data from the authentic viral neutralization assay at the endpoint confirmed the findings of the cell-based S-ACE2 inhibition and cell fusion assays that screen neutralizing antibodies.

Cryo-EM and cell-based mutant S-ACE2 inhibition experiments identified the epitopes on the S protein, as antibodies were selected by competing with ACE2, with antibody attachment to S classified as class 1/2. N297 insertion on IgG1-Fc was one of the hallmarks of the antibodies discovered in this study. This mutation nearly eradicated adhesion to Fc receptors. Indeed, it stopped the Fc-facilitated uptake of the virus to Raji cells.

The antibodies selected were comparable to or better than imdevimab, a therapeutic agent for COVID-19, in neutralization tests against the Wuhan strain and VOCs using authentic viruses and pseudoviruses. Concerning the in vivo activity of these antibodies, they showed potential for therapeutic use in macaque and hamster models. At doses of approximately 5 to 7 mg/kg, the current antibodies demonstrated therapeutic efficacy in hamsters and macaques without increasing viral uptake via ADE.

conclusions

Overall, in the current study, the authors generated many antibodies from the B cells of recovering COVID-19 patients infected with the SARS-CoV-2 D614G mutant or Wuhan strain. In addition, they identified numerous neutralizing antibodies with potent neutralization properties of the SARS-CoV-2 variant.

These Fc-modified neutralizing antibodies from SARS-CoV-2 recovered individuals had neutralizing properties comparable to clinical COVID-19 antibodies. The effectiveness of these antibodies was illustrated by infection studies with macaque and hamster models in vivo and authentic viral and pseudovirus neutralization assays in vitro† These findings showed that the currently discovered antibodies had sufficient antiviral activity to serve as treatment options for COVID-19.

*Important announcement

bioRxiv publishes preliminary scientific reports that have not been peer-reviewed and therefore should not be considered conclusive, that should guide clinical practice/health-related behavior or be treated as established information.

Reference magazine:

  • Fc-modified SARS-CoV-2 neutralizing antibodies with therapeutic effects in two animal models; Masaru Takeshita, Hidehiro Fukuyama, Katsuhiko Kamada, Takehisa Matsumoto, Chieko Makino-Okamura, Tomomi Uchikubo-Kamo, Yuri Tomabechi, Kazuharu Hanada, Saya Moriyama, Yoshimasa Takahashi, Hirohito Ishigaki, Misako Nakad Uchikubo-Kamo, Hidehiro Masaki Ma Imai, , Kiyoko Iwatsuki-Horimoto, Mutsumi Ito, Seiya Yamayoshi, Yoshihiro Kawaoka, Mikako Shirouzu, Makoto Ishii, Hideyuki Saya, Yasushi Kondo, Yasushi Kondo, Yuko Kaneko Ko, Katsuya Suzuki,. bioRxiv preprint 2022. DOI: https://doi.org/10.1101/2022.06.21.496751, https://www.biorxiv.org/content/10.1101/2022.06.21.496751v1

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