key: cord-0695766-v1nakd1m
authors: Peter, Antonia Sophia; Roth, Edith; Schulz, Sebastian R.; Fraedrich, Kirsten; Steinmetz, Tobit; Damm, Dominik; Hauke, Manuela; Richel, Elie; Mueller‐Schmucker, Sandra; Habenicht, Katharina; Eberlein, Valentina; Issmail, Leila; Uhlig, Nadja; Dolles, Simon; Grüner, Eva; Peterhoff, David; Ciesek, Sandra; Hoffmann, Markus; Pöhlmann, Stefan; McKay, Paul F.; Shattock, Robin J.; Wölfel, Roman; Socher, Eileen; Wagner, Ralf; Eichler, Jutta; Sticht, Heinrich; Schuh, Wolfgang; Neipel, Frank; Ensser, Armin; Mielenz, Dirk; Tenbusch, Matthias; Winkler, Thomas H.; Grunwald, Thomas; Überla, Klaus; Jäck, Hans‐Martin
title: A pair of non‐competing neutralizing human monoclonal antibodies protecting from disease in a SARS‐CoV‐2 infection model
date: 2021-08-06
journal: Eur J Immunol
DOI: 10.1002/eji.202149374
sha: e6a09944ff3aebf598b782cb2e8b8eff475f7dd2
doc_id: 695766
cord_uid: v1nakd1m

TRIANNI mice carry an entire set of human immunoglobulin V region gene segments and are a powerful tool to rapidly isolate human monoclonal antibodies. After immunizing these mice with DNA encoding the spike protein of SARS‐CoV‐2 and boosting with spike protein, we identified 29 hybridoma antibodies that reacted with the SARS‐CoV‐2 spike protein. Nine antibodies neutralize SARS‐CoV‐2 infection at IC50 values in the subnanomolar range. ELISA‐binding studies and DNA sequence analyses revealed one cluster of three clonally related neutralizing antibodies that target the receptor‐binding domain and compete with the cellular receptor hACE2. A second cluster of six clonally related neutralizing antibodies bind to the N‐terminal domain of the spike protein without competing with the binding of hACE2 or cluster 1 antibodies. SARS‐CoV‐2 mutants selected for resistance to an antibody from one cluster are still neutralized by an antibody from the other cluster. Antibodies from both clusters markedly reduced viral spread in mice transgenic for human ACE2 and protected the animals from SARS‐CoV‐2‐induced weight loss. The two clusters of potent non‐competing SARS‐CoV‐2 neutralizing antibodies provide potential candidates for therapy and prophylaxis of COVID‐19. The study further supports transgenic animals with a human immunoglobulin gene repertoire as a powerful platform in pandemic preparedness initiatives. This article is protected by copyright. All rights reserved

Since December 2019 [1] , SARS-CoV-2 has rapidly spread throughout the world, leading to more than 133,971,287 confirmed cases of COVID- 19 and 2.902 .493 million deaths [2] . Several vaccines have been developed and licensed in worldwide efforts at an unprecedented speed [3, 4] . In countries or age groups with high vaccine coverage, case counts have decreased substantially [5, 6] . For all licensed vaccines, the vaccine antigen is the ectodomain of the spike protein. Its receptorbinding domain (RBD) interacting with the cellular receptor human Angiotensin-converting enzyme 2 (hACE2) and its N-terminal domain (NTD) were identified as primary targets of the neutralizing activity of convalescent sera and monoclonal antibodies [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] . Clinical development of some of these monoclonal antibodies is ongoing, already providing evidence of their utility in therapeutic trials [20] [21] [22] . Some have since been licensed for the treatment of COVID-19 (reviewed in [23] ).

After crossing species barriers, zoonotic viruses adapt to new species. Early after the new entry, adaptation to efficient transmission is most likely the most substantial selective pressure, explaining the rapid emergence of SARS-CoV-2 variants, such as those carrying the D614G mutation [24] or more complex mutational signatures in the spike protein [25, 26] . Once a larger percentage of a population has overcome a first infection with an emerging virus, immune escape variants of this virus will have a selective advantage. Evidence that immune escape may already contribute to the emergence of new SARS-CoV-2 variants has been reported for the Beta (B.1.351), the Gamma (P.1) and the Delta variant [27] [28] [29] [30] . Since the antibody response to current mRNA vaccines mimics the antibody response to natural infection [31] , the vaccines' efficacy against these variants may be This article is protected by copyright. All rights reserved. 3

impaired [27, [31] [32] [33] . Similarly, the neutralizing potency of monoclonal antibodies may be reduced or completely lost [15, 34, 35] .

Concerning the loss of vaccine efficacy against emerging variants, it has been argued that mRNA and viral vector vaccines can be rapidly adapted to include the spike proteins of the variants of concern (VOC). While this should work for individuals with no prior exposure to SARS-CoV-2 or COVID-19 vaccines, the outcome is less predictable in the presence of pre-existing memory B cells specific for the founder spike protein. Therefore, more targeted vaccine approaches employing structure-based designs of spike vaccines against emerging VOCs and a reliable animal models for validation and optimization are required. Transgenic mice with a complete human antibody repertoire could be such a model.

Here we report the rapid isolation of fully human SARS-CoV-2 neutralizing monoclonal antibodies from immunized TRIANNI mice harboring the complete human antibody repertoire (patent US 2013/0219535 A1; Fig 1A) [36, 37] . The neutralizing antibodies target the RBD or the NTD of the spike protein, providing a pair of non-competing human monoclonal antibodies for clinical development. Further characterization of the antibodies´ human VH gene usage and divergence from germline sequences support the concept of mouse models with a human antibody repertoire in pandemic preparedness efforts and suggests the use of these models in structure-based optimization of vaccine antigens against emerging SARS-CoV-2 variants.

Based on the published nucleotide sequence of SARS-CoV-2 [38] , the first immunogens available early during the current pandemic were expression plasmids encoding the spike protein, the primary target of neutralizing antibodies to coronaviruses [39, 40] . In addition to a DNA vaccine encoding the wildtype S protein (SARS-CoV-2-S DNA), we generated a DNA vaccine in which the coding region of the cytoplasmic domain of the SARS-CoV-2 spike protein was replaced by the cytoplasmic domain of the G protein of VSV (SARS-CoV-2-SG DNA). For SARS-CoV, it was previously shown that such a modification increases S protein expression at the cell surface and induces higher neutralizing antibody responses after immunization with exosomal vaccines [41] . The time interval between the DNA priming immunization and the booster immunization allowed for the production and purification of a recombinant S protein stabilized in the pre-fusion state (SARS-CoV-2-S protein) and an exosomal vaccine presenting a membrane-anchored form of the SARS-CoV-2-S protein (SARS-CoV-2-Exosome) (Fig. 1B) .

TRIANNI mice were selected for these immunization experiments (Fig. 1A ) because they produce antibodies with human Ig variable (V) region and murine constant (C) regions. One unique feature of the TRIANNI mice is that the intronic regions between the exons of the variable segments of Ig genes are of murine origin. This design aims to maintain proper regulation of expression and splicing of the V regions of the immunoglobulin exons as a prerequisite for proper affinity maturation. After selecting antibodies of interest, the human V region gene segments can be recombinantly fused to human C region gene segments allowing the expression of fully human antibodies for clinical applications.

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Two groups of TRIANNI mice were either immunized by intramuscular electroporation with the SARS-CoV-2-S DNA and/or the SARS-CoV-2-SG DNA (Fig. 1B) . Three weeks later, the SARS-CoV-2-S DNA primed mice were either boosted with the SARS-CoV-2-S DNA or the SARS-CoV-2-S protein adjuvanted with MPLA liposomes. The SARS-CoV-2-SG DNA primed mice were boosted with SARS-CoV-2-SG or SARS-CoV-2 exosomes at weeks 3 and 4, respectively (Fig. 1B) . Using a flow cytometric assay with cells transiently transfected with SARS-CoV-2-S DNA [41] , antibody responses were analyzed two weeks after the priming and the boosting immunization (Fig. 1C ). Antibodies to SARS-CoV-2-S were detectable in all four animals after the DNA priming immunization and further increased after the booster immunizations.

Sera of the immunized mice were also analyzed for competition with the binding of the cellular receptor hACE2 to the SARS-CoV-2-S protein (Fig. 1D ). Two weeks after the booster immunization, all four sera reduced hACE2 binding. Sera from the mice primed with SARS-CoV-2-S DNA and boosted with SARS-CoV-2-S protein or SARS-CoV-2-S DNA also neutralized wild type SARS-CoV-2 at a 1:100 dilution by 50% and 27%, respectively ( Fig 1E) .

Hybridoma screening for spike binding, ACE2 competing and neutralizing antibodies

The TRIANNI mouse (M2) with the best neutralizing activity was boosted with the adjuvanted SARS-CoV-2-S protein on week 6 and sacrificed five days later to generate hybridoma lines. A 1:100 dilution of the serum from this time point neutralized wildtype SARS-CoV-2 by 86%. Cells from the spleen and draining lymph nodes were fused with Sp2/0-Ag14 cells, and hybridoma cells were grown in 106 96-well plates seeded at a density of 1,000 cells per well. Between ten to 20 days after the fusion, antibodies secreted by the hybridoma cells were screened by flow cytometry for binding to the S protein. A total of 29 hybridomas could be identified that secreted antibodies binding to the S protein. These antibodies were designated TRES antibodies (TRIANNI-Erlangen anti-SARS-CoV-2-Spike). The screening results of ten representative hybridoma supernatants containing TRES antibodies are displayed in Fig. 2A (for a list of all binding antibodies and their characteristics, see Table S1 ). Intracellular staining revealed that most hybridomas expressed the IgG2c subtype.

The binding antibodies were further characterized in an hACE2 competition assay. Three (TRES6, 224, 567) of the 29 binding antibodies were able to outcompete hACE2 binding very efficiently ( Fig.  2B ), indicating that these antibodies might bind the RBD and possibly neutralize SARS-CoV-2.

Subsequently, all supernatants were tested in a virus neutralization assay with SARS-CoV-2 MUC-IMB-1, an early isolate from the Webasto cluster in Bavaria carrying the D614G mutation in the spike protein [42, 43] . Supernatants from the hybridomas competing with ACE2-binding (TRES antibodies 224, 567 and 6) strongly neutralized SARS-CoV-2 ( Fig. 2C ) and, indeed, an RBD-specific ELISA confirmed the RBD-binding of these three antibodies (Fig.S1 A) . Three additional hybridoma supernatants could be identified to inhibit virus infection by at least 40% (TRES49, 328, 1293). They do not bind to the RBD, recombinant S1 proteins or the S2 protein ( Fig.S1 A-C). TRES49, 328, 1293 bind to a stabilized S protein trimer and recombinant NTD (Fig. S1D, E) . A similar binding profile was also observed for the TRES antibodies 618, 219 and 1209. Thus, these six antibodies differ in their binding profile from TRES6, TRES224 and TRES567. We also identified one hybridoma supernatant (TRES1) that strongly binds to S2 (Fig.S1 B) .

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Based on the screening results, hybridomas were subcloned, and antibodies from the supernatants of hybridoma subclones of the TRES antibodies 6, 224, 567, 49, 219, 328, 618, 1209 , 1293 and 1 were purified and further characterized. Because the TRES antibodies 6, 224, and 567 compete with hACE2 for binding to RBD ( Fig. 2A) , the 50% effective concentration (EC50) was determined in an hACE2 competition ELISA (Fig. 3A ). Surface plasmon resonance (SPR) analyses further revealed K D Values of purified RBD and NTD binding antibodies against the S protein stabilized in the prefusion state ranging from 9.5 nM to 1.6 nM (Table S1 ).

The neutralizing activity of purified antibodies was determined against a second wild type SARS-CoV-2 isolate, CoV-2-ER1 ( Fig. 3B ) which also carries the D614G mutation dominating the SARS-CoV-2 populations in Europe [44, 45] . Antibodies that compete with hACE2 (TRES6, 224, and 567) had IC50s ranging from 64-102 ng/ml. The difference in the IC50s between TRES6 and 567 reflects experimental variation of the assay and the quantification of antibody concentrations as both antibodies share the same amino acid sequence (see below). The IC50s of antibodies not interfering with hACE2-binding (TRES49, 328, 618 and 1293) ranged between 5 and 34 ng/ml. Although the initial screening of hybridoma supernatants had not detected neutralization by TRES618, 219, 1209, the antibodies had an IC50 of 18 ng/ml, 5 ng/ml and 11 ng/ml, respectively. The S2-binding antibody TRES1 did not display neutralizing activity (Fig. 3B ). When the antibodies were assessed in a cell-cell fusion assay using Vero-E6 cells and HEK-293T cells expressing the S protein, the RBD-binding antibodies seemed to inhibit cell-cell fusion more efficiently than the neutralizing antibodies that do not bind to RBD (Fig.S2 ). In general, cell-cell fusion inhibition required higher antibody concentrations, and total inhibition of cell-cell fusion was, therefore, not achieved.

Sequencing of productive immunoglobulin variable regions expressed by hybridomas encoding TRES antibodies revealed a broad usage of V regions of H and L chains with a preference for L chains (Table S1 ). Compared to the corresponding germline sequences, TRES VH and VL nucleotide sequences showed similarities as low as 94.44% and 97.85%, respectively, indicating the presence of somatic mutations and suggesting affinity maturation. Consistent with their similar binding profiles ( Fig.S1 ), TRES6, 224, and 567 H and L chain exons contain the same V H DJ H and V L J L recombination joining sequences and are therefore designated cluster 1 antibodies. All three antibodies express the VH3-33*01 or VH3-33*06 and KV1D-17*01 gene segments (Table S1 ). An alignment of amino acid sequences of VH and VL of cluster 1 antibodies with the inferred germline-encoded antibody revealed an identical L chain with 5 non-silent somatic mutations in the antigen bindings loop (complementary determining regions, CDR) and one in the framework (FR) (Fig.S3 ) TRES6 and 567 utilize the same VDJ H chain exon with four somatic mutations in the framework. TRES224 contains two additional mutations in the CDR1 and CDR2 and an unusual change of a tyrosine conserved in 98% of all human VH segments. A second cluster comprises six TRES antibodies (Table S1 ; TRES49, 219, 328, 618, 1209 and 1293) that share the V H DJ H and V L J L recombination joining sequences.

Various somatic mutations are also present in the CDRs and the FR of their VH and VL segments (Fig.S3 ). The non-neutralizing antibodies of a third cluster bind to S1, but not to RBD, and are composed of an H chain with a human VH and an L chain with an unusual mouse VλX region (Table  S1 ) [46] , which could be explained by the fact that the mouse VλX region is still present in TRIANNI mice.

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Gene segments encoding the human V exons of the H and L chains of all neutralizing TRES antibodies were fused with the human 1 H and the L chain constant regions, respectively. Recombinant antibodies entirely composed of human Ig regions were produced in HEK-293F cells and purified by affinity chromatography. As expected, all recombinant TRES antibodies bound to the S protein in the flow cytometric assay (Fig. 4A ).

To explore a potential overlap of epitopes between TRES antibodies of cluster 1 and cluster 2, cells expressing the SARS-CoV-2-S protein were incubated with TRES224hu or TRES618hu in the presence of increasing concentrations of TRES224, 567, 49, 328, 618 and 1293 hybridoma antibodies containing mouse C regions (Fig. 4B , C).

No competition between the cluster 1 and cluster 2 antibodies could be observed. In contrast and as expected, binding of the recombinant TRES224 with human C regions (TRES224hu) was efficiently blocked by TRES567 and 224 ( Fig. 4B ), indicating that these two neutralizing antibodies from cluster 1 recognize the same epitope. Accordingly, the binding of TRES618hu was blocked by TRES49, 328, 618 and 1293, verifying that cluster 2 antibodies bind the same epitope. Most importantly, however, the humanized TRES antibodies also neutralized SARS-CoV-2 with similar IC50s as the parental hybridoma TRES antibodies ( Fig. 4D ), confirming that the identified antibody sequences confer neutralization.

Neutralization assays and spike protein binding assays ( Fig.S4 A, B, C) were also performed for two emerging SARS-CoV-2 variants of concern and three antibodies from each cluster. The To further explore the possibility for de novo emergence of resistant variants to cluster 1 and cluster 2 antibodies, the CoV-2 ER-1 isolate carrying the D614G mutation was passaged in the presence of increasing concentrations of TRES6 and TRES328 antibodies. After 5 passages, neutralization assays were performed. While the passaged virus became resistant to antibody neutralization when the antibody was present during passaging, the virus remained sensitive to the TRES antibody when the antibodies were not included during passaging (Fig.S4 E) . Whole-genome sequencing of the passaged SARS-CoV-2 viruses revealed the emergence of a TRES6 antibody escape variant with an I68R mutation in the NTD and a T478K mutation in the RBD (Fig.S4 F, displayed in pink). In contrast, the TRES328 escape variant harbored an L241-Y248 deletion with a phenylalanine insertion in the NTD (Fig.S4 F, displayed in blue). Additional point mutations and deletions were observed in proximity to the S1-S2 cleavage sites. These mutations probably reflect adaptation to the replication in the Vero-E6 host cells [47] since these sites were also deleted in the virus variant (P5) passaged in the absence of a neutralizing antibody (Fig.S4 F, displayed in white).

The experimental characterization has shown that TRES328 binds to the NTD. Modeling of the respective complex suggests that TRES328 contacts the sequence stretch 241-248 that is deleted in This article is protected by copyright. All rights reserved. 7 the escape variant ( Fig. S5 A) . In the wildtype virus, NTD residues R246 and Y248 play an essential role for antibody binding by forming specific polar interactions with Y27 and E31 of the CDRH1 (Fig.S5 B) . Replacement of residues 241-248 by a single phenylalanine in the escape variant significantly reduces the length of the N5-loop and causes a loss of the stabilizing interactions mediated by R246/Y248 (Fig.S5 C) . In addition, the deletion places D246 (corresponding to D253 of the wildtype) in the vicinity of E31 (Fig. S5 C) , thereby further destabilizing the interaction due to electrostatic repulsion between the two negatively charged residues. Interestingly, is as described above, TRES328 did not neutralize the Alpha (B.1.1.7) variant, although this variant carries no mutation in the putative TRES328 binding site. This loss of binding might however be due to a deletion of the amino acids 69-70 in the Alpha (B.1.1.7) NTD, which might lead to a change in the allosteric conformation of the S1 epitope by pulling the loop of amino acids 69-76 inward. [30, 33, 48] This conformational change in the NTD might result in a concealed TRES328 binding site and a subsequent loss of neutralization.

TRES6 exhibits only limited sequence similarity to other Spike-binding antibodies of known 3Dstructure, thus impeding molecular modeling of the RBD-TRES6 complex. However, the observation that a T478K mutation results in a loss of TRES6 binding indicates that T478 is part of the binding epitope. Since this epitope is also mutated in the recently isolated Delta (B.1.617.2) variant [49] , this variant of concern is very likely able to escape TRES6-mediated neutralization. Inspection of other known antibody structures reveals that a subset of RBD-binding antibodies also recognizes T478. One example for this group of antibodies is COVOX-253 ( Fig. S5 D, E), which recognizes T478 by Y33 of CDRL1 and D108 of CDRH3. These two amino acids are also present in TRES6 at the respective sequence positions suggesting that TRES6 might use similar structural principles for RBD recognition. However, a more detailed analysis will require an experimental structure determination of the TRES6-RBD complex.

To assess whether the antibodies generated in this study can confer protection from disease, we evaluated the efficacy of one antibody from each cluster in a stringent hACE2 transgenic mouse [50] viral challenge model under post-exposure prophylactic settings. Three groups of mice (n=12/group) were infected intranasally with 300 FFUs of SARS-CoV-2 (MUC-IMB-1). One day later, mice were injected intravenously with 5.25 mg/kg body weight of the cluster 1 antibody TRES6, the cluster 2 antibody TRES328, or the isotype-matched control antibody TRES480.

Six animals from each group were sacrificed on day 4 or 10 or according to humane endpoints, and viral loads were determined in the lung, BAL, brain, liver and spleen. Both antibodies reduced the amount of viral RNA in the lung and BAL samples approximately 30-to 200-fold after four days (Fig.  5A ) or approximately 100-o 450-fold after 10 days (Fig. 5B ). Viral load in the other organs was reduced close to the level of detection. Interestingly, the titer of infectious virus in the BAL samples from TRES6-and TRES328-treated animals was below the detection level and, therefore, at least 1000-fold lower than in mice receiving the control antibody (Fig. 5C ). Compared to the isotype control, TRES480, treatment of mice with TRES6 and TRES328 prevented the loss of body weight induced by the viral infection (Fig. 5) and reduced clinical symptoms assessed by a clinical score (Fig.  5E) . Importantly, none of the TRES antibody-treated mice reached clinical endpoints requiring euthanasia, while 4 of 6 SARS-CoV-2-infected mice receiving the isotype control antibody TRES480 had to be euthanized (Fig. 5F ).

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By immunizing TRIANNI mice with a DNA prime protein boost regimen, 29 monoclonal antibodies were obtained that bind to the S protein of SARS-CoV-2 expressed on the cell surface of transfected HEK-293T cells. The use of immunization and screening approaches based on the expression of the wildtype S protein avoids the need for prior knowledge of domains targeted by neutralizing antibodies and allows to obtain a panel of antibodies binding to different epitopes. Indeed, different binding patterns were observed, including antibodies that i) bind to the RBD and compete with ACE2 binding, ii) bind to the S1 subunit without binding to RBD, iii) only bind to the ectodomain of the S protein stabilized in a trimeric form and the NTD and iv) bind to the S2 subunit. In those mice that we first challenged with SARS-CoV-2 and subsequently treated with the monoclonal antibodies; we could also assess any potential disease-enhancing properties. This issue has been suggested as a potential outcome of infections occurring in the presence of vaccine-induced antiviral antibodies not providing sterilizing immunity [51] . However, no evidence for such an enhancing effect could be observed. A combination therapy with an antibody from cluster 1 and cluster 2 seems especially attractive because this will extend the neutralization breadth and enhance the genetic barrier for the emergence of antibody-resistant SARS-CoV-2 variants. This is further supported by the observation that the Alpha (B.1.1.7) and the Beta (B.1.351) variants maintain neutralization sensitivity to at least one of the two clusters of antibodies. In addition, virus escape mutants from one antibody of each cluster are still sensitive to a representative TRES antibody from the other cluster.

Most of the neutralizing antibodies in convalescent COVID-19 patients are close to the germline sequence [52] . However, it was recently shown that antibodies derived from convalescent COVID-19 patients, 3-6-month post symptom onset, show significant somatic hypermutation [53, 54] . It was also shown, that this affinity maturation further enhances the neutralization capacity of RBD-binding antibodies [31] . Accordingly, the TRES antibodies had mutations in the VH and VL regions, including the CDRs (Fig.S3) , suggesting that affinity maturation can also occur in TRIANNI mice after a DNA prime protein boost immunization regimen. Although mice encoding an entire [11] or partial [55] human immunoglobulin repertoire have been used previously to generate monoclonal antibodies targeting the spike protein of SARS-CoV-2, direct evidence for affinity maturation, in the context of SARS-CoV-2, has not yet been provided. Using a three-week immunization regimen consisting of a single intradermal priming immunization with DNA encoding the S protein and booster immunizations with an RBD-Fc fusion protein in VelocImmune TM mice, Hansen et al. showed induction of RBD-specific antibody responses and also reported the isolation of neutralizing RBDbinding antibodies from B cells that were sorted based on RBD binding [11] . Wang et al. performed sequential immunizations of H2L2 TM (Harbour Antibodies) mice with spike ectodomains of different human coronaviruses and derived a monoclonal antibody that cross-neutralized wildtype SARS-CoV-2 moderately with an IC50 of 570 ng/ml [55] .

Although extensive affinity maturation does not seem to be necessary for the formation of potent neutralizing antibodies against SARS-CoV-2 [52] , this may be relevant for elicitation of neutralizing antibodies against other pathogens as exemplified by HIV-1 [56] [57] [58] . As observed for four of seven other potent neutralizing antibodies targeting the NTD and recovered from convalescent patients [7] , our TRIANNI mouse-derived NTD antibodies also use the VH1-24 gene segment. Therefore, transgenic animal models supporting affinity maturation of antibodies with human variable regions This article is protected by copyright. All rights reserved. 9

should be an essential part of future pandemic preparedness efforts and be explored in structureguided vaccine antigen design against SARS-CoV-2.

Besides generating potent neutralizing antibodies that also confer protection in the hACE2 mouse model, a key criterion for applying TRIANNI mice in future pandemic responses is the time needed to obtain such antibodies. The immunization schedule we used took seven weeks until hybridomas were generated. Growing the hybridomas, their screening and subcloning took approximately six weeks. Sequence analyses from these hybridomas and gene synthesis for the generation of fully human antibodies took another six weeks. Accelerating the development time would certainly be desirable. Whether shortening the immunization schedule still results in potent neutralizing antibodies is difficult to predict and may depend on the degree of affinity maturation needed to generate such antibodies. Since, as described above, an extensive antibody affinity maturation is needed for the elicitation of neutralizing antibodies against some pathogens, as for example HIV, a shortened immunization schedule in this case might not be advantageous [56] [57] [58] . The general time needed for the isolation of antibodies against such pathogens, however, could be accelerated by staining antigen-specific memory B cells or germinal center-selected surface IgG-positive plasma blasts with fluorescence-labeled antigen and sorting them by flow cytometry instead of the generation of hybridomas (Fig. S6 ). Paired Ig VH and VL sequences from single cells could then be amplified by PCR and directly cloned into expression plasmids described for memory B cells from convalescent COVID-19 patients [10, 59] . Transiently expressed paired recombinant antibodies could then be screened for binding and neutralization, shortening the development time by several weeks. 

Two 9-12-week-old TRIANNI mice were primed with pCG1-CoV-2019-S ( [60] designated SARS-CoV2-S DNA) encoding wild type SARS-COV-2 spike protein (position 21580 -25400 from GenBank NC_045512). Two additional TRIANNI mice were primed by SARS-CoV-2-SG DNA encoding a chimeric protein in which the intracytoplasmic domain of SARS-CoV-2-S was replaced by the intracytoplasmic domain of VSV-G as previously described for SARS-CoV [41] . The DNA vaccines were delivered by intramuscular electroporation as previously described [61] . Briefly, a total of 30 µg DNA diluted in 60 µl PBS were injected in both hind legs under constant isoflurane (CP-Pharma, Burgdorf, Germany) anesthesia. Immediately after the intramuscular injection, electrical impulses were applied to the injection site with a TriGrid electrode array (provided by Drew Hannaman, Ichor Medical Systems, Inc., San Diego, USA). Mice were boosted intramuscularly either with i) 5 µg of the S protein of SARS-CoV-2 stabilized in a pre-fusion conformation (designated SARS-CoV-2-S protein) and adjuvanted with 25 µg Monophosphoryl Lipid A (MPLA) liposomes (Polymun Scientific GmbH, Klosterneuburg, Austria) into the hind leg, ii) by electroporation of the DNA vaccines used for priming, or iii) with exosomes purified from HEK-293T cells transiently transfected with SARS-CoV-2 DNA as described previously [41] . Details for the immunization schedule are summarized in Figure 1B . Mice were bled by punctuation of the retro-orbital sinus using non-heparinized capillaries (Hirschmann Laborgeräte, Eberstadt, Germany). Serum was collected, inactivated for 30 min at 60°C and kept at -20°C for longterm storage.

One of the immunized mice received a second booster immunization with SARS-CoV-2-S protein at week 5. Five days later, spleen and inguinal lymph nodes were harvested and fused with the azaguanine-resistant SP2/0 Ag14 hybridoma line (ATCC#CRL-1581) using the PEG (Sigma Aldrich, Taufkirchen, Germany) method [62] . Briefly, 2x10 8 spleen cells and inguinal lymph node cells (about 1x10 8 B cells) were mixed with 10 8 Sp2/0 cells and washed 3 times with RPMI1640 medium (Gibco, Thermo Fisher Scientific, Waltham, USA) without FCS. 2 ml PEG was added dropwise within one minute to the suspended cell pellet. The mix of fused cells was resuspended in R10+ medium (RPMI 1640 with 10% FCS, 0.05 mM mercaptoethanol, 2 mM L-glutamine, 1 mM sodium pyruvate, and 1% penicillin/streptomycin; (all from Gibco, Thermo Fisher Scientific, Waltham, USA); enriched with HAT (Gibco, Thermo Fisher Scientific, Waltham, USA) and OPI Media supplement (Sigma Aldrich, Taufkirchen, Germany). Cells were seeded into 96-well plates with approximately 1x10 4 cells per well. 10-20 days post-fusion, hybridoma clones were tested for spike-binding antibodies. Positive clones were expanded in R10+ supplemented with HT (Sigma Aldrich, Taufkirchen, Germany) and subcloned by the limiting dilution method.

To detect antibody binding to SARS-CoV-2-S protein, HEK-293T cells were co-transfected with SARS-CoV-2-S DNA and a GFP reporter plasmid (e.g., pEGFP-C1) using the PEI method as described previously [63] . 10 5 thawed or freshly transfected cells were incubated first in wells of a 96-well plate with 100µl undiluted hybridoma supernatant or 100 µl mouse serum (1:200 dilutions in R10+ medium), bound antibodies were detected with a Cy5-conjugated goat anti-pan-mouse IgG antibody (Southern Biotechnology, Birmingham, USA, #SBA-1030-15). Cells were washed in FACS buffer and analyzed with an Attune Nxt, CytoFlex or a Gallios flow cytometer (Thermo Fisher Scientific, Waltham, USA or Beckman Coulter, Brea, USA, respectively) and evaluated with Flow Logic TM (IInivai Technologies Mentone, Australia). Flow cytometric analysis adhered to 'Guidelines for the use of flow cytometry and cell sorting in immunological studies' [64] .

Hybridoma cells were stained intracellularly for the assessment of the monoclonality of the colonies and the determination of the IgG subtype. Briefly, 10 5 cells were fixed for 20 min in 2% paraformaldehyde (Morphisto, Frankfurt am Main, Germany) diluted in PBS, washed twice with FACS buffer, resuspended in permeabilization buffer (0.5% Saponin Sigma Aldrich, Taufkirchen, Germany, in FACS buffer) containing fluorochrome-conjugated murine H and L isotype-specific antibodies (anti-mouse IgG1-APC # 550874 and anti-mouse IgG3-bio # 020620 from BD, Franklin Lakes, USA, anti-mouse IgG2b-PE SBA-1090-09 and anti-mouse IgG2c-bio SBA-1079-08 from Southern Biotech, Birmingham, USA, anti-mouse Ig light chain lambda APC # 407306 and anti-mouse IgG light chain kappa PE both from Biolegend, San Diego, USA) and incubated for 1 hour at 4°C. Cells were again washed in FACS buffer and analyzed with an Attune Nxt, CytoFlex or a Gallios flow and evaluated with Flow Logic TM .

Neutralizing activities of hybridoma supernatants, antibodies and sera were assessed in a microneutralization assay based on detecting the number of virus-producing cells via immunofluorescence. For this, 1x10 4 Vero-E6 cells were seeded per well of a 96-flat bottom plate one day before the infection. Purified antibodies, sera or hybridoma supernatants were first diluted and pre-incubated for one hour with 1,88x10E 4 infectious units of SARS-CoV-2 stocks per well in a final volume of 100 µl. All dilutions of hybridoma supernatants or purified antibodies were prepared in a cell OptiPRO TM culture medium. After removing the cell culture medium, seeded Vero-E6 cells were incubated for one hour with 100 µl per well of the pre-incubated virus-antibody mix. Afterward, the supernatant was discarded, the cells washed once with 100 µl PBS and 100 µl fresh cell culture medium added. The cells were incubated for 20-24 hours, washed with PBS, fixed with 4% paraformaldehyde for 20min, and subsequently stained. They were permeabilized for 15 min with 0.5% TritonX in PBS and blocked with 5% skimmed milk diluted in PBS for 1 hour. Subsequently, the plates treated with murine antibodies were stained with protein G-purified sera from a convalescent patient diluted 1:100 in PBS containing 2% skimmed milk. Plates that were treated with human antibodies were stained with an antibody mix of murine origin directed against SARS-CoV-2 at a concentration of 250ng/ml. After 1 hour, the cells were washed, and a goat anti-human IgG FITC (Jackson ImmunoResearch, West Grove, USA #109-096-088) antibody or a FITC-conjugated goat antimouse IgG serum (Jackson ImmunoResearch # 115-095-003) was applied. After 1 hour and a washing step, positive wells were identified by a CTL-ELISPOT reader (Immunospot; CTL Europe GmbH, Bonn, Germany). The signal was analyzed with the ImmunoSpot® fluoro-X™ suite (Cellular Technology Limited, Cleveland, USA). IC50 values were calculated by plotting the virus activity in percent against the antibody concentrations and using the normalized response vs. inhibitor equation (variable slope) or inhibitor vs. response-Variable slope (four parameters) of GraphPad Prism 7.02.

Quantitative antibody competition assay. For the assessment of the quantitative antibody competition, SARS-CoV-2-S DNA transfected HEK-293T cells were incubated with 100 µl of a humanized protein G purified TRES antibody at a concentration of 250 ng/ml and serial dilutions of TRES antibodies with a murine Fc region at final concentrations ranging from 2.5 µg/ml-0.01 ng/ml. The cells were incubated for 30min on ice, washed, and bound antibodies were detected with a mouse IgG2a Alexa647-conjugated antibody directed against human IgG-Fc (BioLegend, San Diego, USA #409320). After washing, the mean fluorescence intensities of transfected cells were determined with an Attune Nxt and the Flow Logic software TM .

Animal experiments were performed following the EU Directive 2010/63/EU for animal experiments and were approved by local authorities after review by an ethical commission (TVV 21/20) . Thirty-six female K18-hACE2 mice (Jackson Laboratory, Bar Harbor, USA) were infected intranasally under isoflurane anesthesia with 300 FFU of SARS-CoV-2 strain MUC-IMB-1 p.1 in a total volume of 50 µl. 24 hours after virus inoculation, twelve mice per group were injected intravenously either with 5.25 mg/kg body weight of TRES6 or TRES328 antibody or an isotype control antibody (TRES480) in a total volume of 100 µl. The animals were scored daily, and the survival and disease incidence were measured over a maximal 10-day period. One cohort (n=6) was euthanized at day 4, while the second cohort (n=6) was scored for humane endpoints and euthanized at day 10 post virus inoculation. After euthanasia, BALs (bronchoalveolar lavages) were collected, and lung, brain, liver, and spleen were taken. The organs were homogenized in 2 ml PBS using gentleMACS M tubes and gentleMACS Octo Dissociator (Miltenyi Biotec, Bergisch Gladbach, Germany). Afterward, the tubes were centrifuged at 2000 x g for 5 min at 4°C to discard cell debris. Viral RNA was isolated from 140 µl of homogenated supernatants or BAL using QIAamp Viral RNA Mini Kit (Qiagen, Aarhus, Denmark). The viral load in indicated organs and BAL was analyzed by RT-qPCR [65] . Reactions were performed using TaqMan® Fast Virus 1-Step Master Mix (Thermo Fisher Scientific, Waltham, USA) and 5 µl of isolated RNA as a template. Synthetic SARS-CoV-2 RNA (Twist Bioscience, San Francisco, USA) was used as a quantitative standard to obtain viral copy numbers. The viral load reduction was calculated in comparison to the isotype control. The clinical scoring system included the following items: weight loss and body posture (0-20 points), general conditions including the appearance of fur and eye closure (0-20 points), reduced activity and general behavior changes (0-20 points), and limb paralysis (0-20 points). Mice were euthanized when reaching a cumulative score of 20.

For the detection of infectious virus in BAL Vero-E6 cells were seeded at 2x10 4 cells/well in a 96-well plate in 200 µl of DMEM, 10 % FCS, 1x penicillin/streptomycin (Thermo Fisher Scientific, Waltham, USA) 24 h before infection with 20 -100 µl of BAL diluted in DMEM with 1x penicillin/streptomycin for 3 hours. After replacing the supernatant with overlay medium (DMEM with 1 % methylcellulose, 2 % FCS and 1x penicillin/streptomycin), cells were incubated for 27 hours. SARS-CoV-2 infected cells were visualized using SARS-CoV-2 S protein-specific immunochemistry staining with anti-SARS-CoV-2 spike glycoprotein S1 antibody (Abcam, Cambridge, Great Britain) as described previously [65] . Statistical evaluation of the data was performed by Kruskal-Wallis test (one-way ANOVA) and Dunn's Pairwise Multiple Comparison Procedures as post hoc test.

Detection of antibody binding, cloning and sequencing of Ig V exon sequences, generation of human IgG expression vectors, purification of the murine and human antibodies and the virus propagation were performed as described in the supplemental information. 

Transgenic mice with a complete human antibody repertoire (Trianni platform) were immunized with the spike protein (S protein) of SARS-CoV-2 and hybridoma clones were established. Two clusters of clonally related SARS-CoV-2-neutralizing monoclonal antibodies were isolated. Cluster 1 binds to the N-terminal domain (NTD) and Cluster 2 to the receptor-binding domain (RBD) of the CoV-2 spike protein. The antibodies also significantly reduce the viral spread in hACE2-transgenic mice.

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We would like to thank Isabell Schulz and Doris Jungnickl for their excellent technical assistance. We kindly thank Jasmin Fertey and Rosina Ehmann for providing high titer virus stocks of SARS-CoV-2 and Alexandra Rockstroh for the optimized SARS-CoV-2 detection protocol. The expression plasmid for the spike ectodomain was kindly provided by Jason McLellan, Austin, USA, the TriGrid electrode array for DNA electroporation by Drew Hannaman, Ichor Medical Systems, Inc., and S2 and S1 protein by Thomas Schumacher, Virion/Serion GmbH, Würzburg. The ELISPOT Analyzer was obtained with financial support from Foundation Dormeur, Vaduz. This work was supported by a grant (01KI2043) from the Bundesministerium für Bildung und Forschung (BMBF) and funds from the Bavarian State ministry for Science and the Arts to K.Ü., H-M J., and T.H.W. Further support was provided by B-FAST and COVIM, two BMBF-funded projects of the Netzwerk Universitätsmedizin (NaFoUniMedCovid19; FKZ: 01KX2021) and funds from the Deutsche Forschungsgemeinschaft (DFG) through the research training groups RTG 2504 and RTG1660 and the DFG-funded TRR130 (to HMJ and TW).The authors declare that they complied with all relevant ethical regulations. All relevant data supporting the findings of this study are available within the paper and its supplementary information files.