Isolation of Strongly Neutralized Monoclonal Antibodies Using Nanoparticles Displaying SARS-CoV-2S Glycoprotein

The emergence of a pandemic of coronavirus disease 2019 (COVID-19) caused by Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) has had a negative impact on global health and the economy. Isolation of the monoclonal antibody (mAb), which helped provide therapeutic benefit or prophylactic protection against the SARS-CoV-2 mutant of concern, played a major role in suppressing the pandemic. These therapeutic mAbs target the receptor binding domain (RBD) of the SARS-CoV-2 spike (S) protein. The role of RBD is very important because it binds to the human angiotensin converting enzyme 2 (ACE2) receptor in the lungs and promotes viral invasion and infection.

study: Low dose in vivo protection and neutralization across SARS-CoV-2 mutants with a combination of monoclonal antibodies.. Image Credit: Kateryna Kon / Shutterstock

Second class of Neutralizing antibody Target the N-terminal domain (NTD) of the Spike protein, which is a domain located around the S-trimer. All NTD-directed antibodies are known to target a single antigenic supersite within this subdomain. However, despite these advances, some variant virus strains (VOCs) of concern can avoid neutralization with mAb therapy. This highlights the need for a wide range of treatments.

New research published in Nature immunology We aimed to identify some potent neutralizing RBD- and NTD-oriented mAbs that capture SARS-CoV-2 specific B cells using nanoparticles displaying S glycoproteins. Enhanced virus protection with a combination of NTD and RBD mAb In vivo, Prevented the virus from escaping In vitro, VOC is widely covered.

About research

This study included healthy and mild participants Symptoms of COVID-19.. Plasma and peripheral blood monocytes were collected from infected patients 7 weeks after a positive SARS-CoV-2 PCR test. This was followed by two different assays. Multiplex antibody binding assay and SARS-CoV-2 pseudovirus neutralizing assay.

After SARS-CoV-2 positive B cells were classified into plates, antibody sequencing and production, recombinant protein production, and Fab production were performed. In the SARS-CoV-2 plaque reduction neutralization test, Vero E6 cells were used to detect antibody-viral interactions. In addition, antibody Fc effector function was measured using recombinant and peplomer proteins.

A biolayer interference measurement binding assay was used to characterize the epitope. Epitope mapping was also done using an alanine scan. We performed X-ray crystallography and structural analysis to get ideas for the structure of mAbs. finally, In vivo Conservation studies were performed using transgenic mice.

Investigation result

Screening Convalescent plasma Samples collected from 56 SARS-CoV-2 infected human donors with mild to moderate symptoms were sent to NTD and RBD by one donor (donor 3) as a potent neutralization and prefusion-stabilized S trimmer. It was shown to have high antibody binding.

The first classification strategy included a combination of SARS-CoV-2 probes containing RBD, S trimmer, S1 and S1 subunits. The second sorting strategy replaced the S trimer with multivalent spike ferritin nanoparticles (SpFN). It mimicked SARS-CoV-2 and displayed eight S trimers. The results of the two sort strategies show that most of the N-oriented mAbs have been separated from the SpFN sort. In contrast, RBD neutralized mAbs were obtained from both strategies.

Overall, 213 antibody heavy and light chain pairs were harvested and sequenced from both sorting strategies.Antibodies were generated In vivo Human IgG1 was used on Expi293F cells and screened for neutralization and binding. It was found that most of the mAbs bind to S2, followed by RBD and NTD.

In addition, a comparison of the neutralizing potency of these mAbs using pSV and the genuine SARS-CoV-2 virus neutralization assay showed that NTD mAbs neutralized 75% in the pSV assay and in SARS-CoV-2 virus. It was shown that the sum assay showed 100%. In contrast, RBD mAb showed 100% neutralization in both assays.

After that, the ability of NTD and RBDmAb to promote Fc effector function was investigated. NTD mAbs were superior to RBD mAbs in mediating the opsonization of cells expressing S proteins on the surface. In addition, NTD-neutralized mAbs showed higher antibody-dependent complementation (ADCD) activity than RBD-neutralized mAbs.

A biolayer interferometry (BLI) competitive binding assay was used to characterize an epitope of mAbs that targets NTDs. All neutralizing antibodies were clustered into one group (NTD A). In contrast, all non-neutralizing antibodies were clustered into two groups (NTD B and C) with different abilities to bind to S trimmers. Shotgun mutagenesis was found to affect the neutralization potency and binding affinity of NTDmAb by mutations in the N3 and / or N5 loop.

Similarly, the epitope of RBDmAb was also characterized. RBD neutralized mAbs were divided into RBD A, B, and C groups. For RBD A, the CDR H2 and H3 loops play a major role in the neutralizing potential, CDRH1-3 and CDRL1 and L2 for RBDB, and CDRH2-3 and CDRL1-3 for RBDC.

In addition, the prophylactic protection provided by NTD and RBD neutralized mAbs was determined using a mouse model. The results showed that 5 micrograms of NTD mAb was sufficient to suppress viral replication in the lung, and RBD mAb required a dose of 1 mg / body weight.Also, the Fc effector In vivo NTD protection was much more pronounced than RBDmAb.

However, the combination of NTD and RBD mAb has several advantages over virus evasion. Several combinations of both mAbs were used to determine their neutralizing activity. Neutralization of the combined mAbs was found to be comparable to a single dose of mAbs. It was also found that the mAbs of NTD and RBD retain their overall activity when mixed.In addition, using the mouse model In vivo Neutralization of combined mAbs. It was found that all mAb-treated groups showed lower viral titers compared to the control group. However, all groups treated with the mAb combination showed undetectable virus in the lungs except in two mice. The minimum protective dose of combined mAbs was shown to be 50 micrograms, while partial protection was observed at 12.5 micrograms. In addition, the combined mAb has also succeeded in preventing virus escape.

The advent of VOCs threatened the strategies used by neutralizing mAbs. Alpha, beta, gamma, delta, and the two mutants of interest had specific mutations that interfered with the neutralizing activity of NTD and RBDmAb. Most of the NTD-neutralized mAbs lost their activity against alpha, beta, and delta variants while maintaining their activity against gamma variants. Most RBD-neutralized mAbs lost activity against beta and gamma variants and retained activity against alpha and delta variants.

However, one mAb, the Walter Reed Army Institute of Research (WRAIR) -2125, retained activity against all VOCs, either alone or in combination with NTDs or RBD mAbs. WRAIR-2125 was found to target the minimal epitope required for ACE2 involvement in RBD and was centered on residue F486. The interaction with VOC-mutated residues was minimal, allowing effective binding to VOCs.

Conclusion

Therefore, we conclude that the combination of NTD and RBD mAb, although less protected in vivo, was very effective in preventing viral escape and provided a wider range for various circulating VOCs. I can. Therefore, the combination of mAbs can combat current and future variants of SARS-CoV-2, especially in people with immunodeficiency and those who do not respond to conventional vaccination.