key: cord-0987333-dtl4olyy
authors: Amanat, Fatima; Strohmeier, Shirin; Lee, Wen-Hsin; Bangaru, Sandhya; Ward, Andrew B.; Coughlan, Lynda; Krammer, Florian
title: Murine monoclonal antibodies against RBD of SARS-CoV-2 neutralize authentic wild type SARS-CoV-2 as well as B.1.1.7 and B.1.351 viruses and protect in vivo in a mouse model in a neutralization dependent manner
date: 2021-04-06
journal: bioRxiv
DOI: 10.1101/2021.04.05.438547
sha: b7e559cade82c3a7aa2e92b085c9b5027116f33b
doc_id: 987333
cord_uid: dtl4olyy

After first emerging in December 2019 in China, severe acute respiratory syndrome 2 (SARS-CoV-2) has since caused a pandemic leading to millions of infections and deaths worldwide. Vaccines have been developed and authorized but supply of these vaccines is currently limited. With new variants of the virus now emerging and spreading globally, it is essential to develop therapeutics that are broadly protective and bind conserved epitopes in the receptor binding domain (RBD) or the whole spike of SARS-CoV-2. In this study, we have generated mouse monoclonal antibodies (mAbs) against different epitopes on the RBD and assessed binding and neutralization against authentic SARS-CoV-2. We have demonstrated that antibodies with neutralizing activity, but not non-neutralizing antibodies, lower viral titers in the lungs when administered in a prophylactic setting in vivo in a mouse challenge model. In addition, most of the mAbs cross-neutralize the B.1.351 as well as the B.1.1.7 variants in vitro. Importance Crossneutralization of SARS-CoV-2 variants by RBD-targeting antibodies is still not well understood and very little is known about the potential protective effect of non-neutralizing antibodies in vivo. Using a panel of mouse monoclonal antibodies, we investigate both of these aspects.

CoV-2 correlate with protection and vaccination has been shown to be highly efficacious and 50 effective as well. However, it is still crucial to develop therapeutics that can be used to treat 51 individuals that are infected with SARS-CoV-2, particularly those at high risk for severe disease. 52 While mAbs have been developed and approved for use, there remains a significant concern 53 from the virus acquiring mutations that would lead to escape rendering the mAbs and vaccines concern as most neutralizing antibodies target the RBD and block entry. Another region heavily 58 mutated in the new circulating variant viruses is the N-terminal domain (NTD) which is also a 59 target of neutralizing antibodies (14) . Hence, the efficacy of vaccines and therapeutics could be 60 compromised as more and more mutations in the NTD and RBD occur and persist in nature (15, 61 16).

In this study, we isolated and characterized fourteen mouse mAbs against the RBD of SARS-63 CoV-2 and assessed their binding to recombinant RBD and spike protein as well as tested their 64 ability to neutralize live virus. In addition, we tested if non-neutralizing mAbs can lower viral 65 loads in a mouse challenge model. Due to the new variants of concern which have been detected, 66 we also tested if mAbs can bind mutant RBDs that contain single amino acid changes as well as 67 multiple mutations found in B. 

Generation of monoclonal antibodies. After two vaccinations of BALB/c mice with 71 recombinant RBD protein supplemented with poly I:C, murine hybridoma technology was used 72 to generate hybridoma cell lines that secreted RBD-specific monoclonal antibodies (17) (18) (19) .

Fourteen unique hybridoma lines were isolated and picked that produced IgGs (Table 1) . Twelve 74 monoclonal antibodies belonged to the IgG1 isotype while two monoclonal antibodies were from 75 the IgG2a subclass.

All antibodies bind the RBD of SARS-CoV-2 and six mAbs can neutralize live virus. Once 77 all antibodies were purified from hybridoma supernatant, a standard enzyme-linked 78 immunosorbent assay (ELISA) was performed to assess binding of the monoclonal antibodies to 79 the RBD of SARS-CoV-2 ( Figure 1A) , full spike of SARS-CoV-2 ( Figure 1B) , and RBD of 80 SARS-CoV-1 ( Figure 1C ). All antibodies bound well to SARS-CoV-2 RBD and most had very 81 low minimal binding concentrations (MBC). Of note, the MBC values for KL-S-1B5 and KL-S-82 2A1 (0.1 ug/ml) against SARS-CoV-2 RBD were higher than the rest of the antibodies, 83 indicating lower affinity. Next, antibodies were tested in an ELISA against the full spike protein 84 of SARS-CoV-2 ( Figure 1B) . It is interesting to note that while most mAbs bound well with low Antibodies can lower viral titers in vivo in a mouse challenge model. To further study the 97 biological functionality of these mAbs, all mAbs were tested in vivo. Hence, an animal model 98 was utilized to test if antibodies are able to block viral entry and thus lower titers in the lung.

Since mouse ACE2 does not facilitate entry of SARS-CoV-2, an adenovirus that expresses the 100 human ACE2 gene was used to first transduce mice (20, 21). Five days later, monoclonal 101 antibodies were administered at 10 mg/kg two hours prior to infection with SARS-CoV-2 and 102 lungs were collected on day 3 and day 5 post infection to assess viral titers via a plaque assay.

Only neutralizing mAbs were able to confer a protective benefit and lowered viral titers in the 104 lungs (Figure 2A Neutralizing antibodies eliminate viral presence in the lungs and little differences were 115 found between the groups in terms of lung pathology. In addition to assessment of viral titers 116 in the lungs in a prophylactic setting, we also wanted to test if mAbs can protect from 117 inflammation and/or tissue damage in the lungs or lead to enhanced disease which has been 118 noted for SARS-CoV-1 (22). Lungs were harvested on day 4 post vaccination from all the 119 antibody groups (n=2) and subjected to pathological analysis (Histowiz) such as hematoxylin and 120 eosin (H&E) staining as well as immunohistochemistry (IHC) using an antibody specific for the 121 nucleoprotein of SARS-CoV-2. A 5-point grading scheme that took into account six different 122 parameters ("perivascular inflammation", "bronchial/bronchiolar epithelial 123 degeneration/necrosis", "bronchial/bronchiolar inflammation", "intraluminal debris", "alveolar 124 inflammation" and "congestion/edema") was utilized to score lung sections. Interestingly, mice 125 from all groups treated with antibodies displayed some pulmonary histopathological lesions of 126 interstitial pneumonia (Figure 3) . This could be a result of the high dose (10 5 PFU per mouse) of antibodies bind on the RBD, structural analysis was performed, and negative stain three while non-neutralizing antibodies had no effect. However, there is an important caveat that needs 213 to be discussed for this experiment. All non-neutralizing antibodies that we isolated were of the 214 IgG1 subtype, which in mice, is known to have low affinity for activating FcRs. This is in 215 contrast to murine IgG2a and IgG2b which have high affinity for these FcRs. Therefore, we can BioTek Synergy H1. All data was analyzed using GraphPad Prism 7. An anti-histidine antibody 293 was used for ELISAs as positive control (Takara, catalog # 631212). Vero.E6 cells were seeded in a 96-well cell culture plate and used the next day. Antibody 300 dilutions were prepared starting at 30 ug/ml and 3-fold subsequent dilutions were prepared. The 301 protocol has been described earlier (21, 29, 35, 36) . Cells were stained for the nucleoprotein and trimers were expressed and purified as described previously (39).

Negative stain EM sample preparation and data collection. Spike protein was complexed 340 with purified Fab at three times molar excess per trimer and incubated for thirty minutes at room 341 temperature. Complexes were diluted to 0.02mg/ml in TBS and 3µl applied to a 400mesh Cu 342 grid, blotted with filter paper, and stained with 2% uranyl formate for 30 seconds. Images were 343 collected on a Tecnai Spirit microscope operating at 120 kV with a FEI Eagle CCD (4k) camera.

Particles were picked using DogPicker and 3D classification was done using Relion 3.0 (40, 41). to RBDs that contain single or multiple mutations found in new variants. The line at 100% 535 indicates binding to wild type and binding to each mutant RBD is graphed as percent binding 536 compared to wild type. A negative control mAb, anti-influenza H10, was run against all the 537 RBDs to ensure that there is no unspecific binding. A positive control, anti-histidine antibody, 538 was used to ensure that the RBD proteins that have a hexa-histidine tag are coated properly. 

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