Live imaging of SARS-CoV-2 infection in mice reveals neutralizing antibodies require Fc function for optimal efficacy

SUMMARY

Neutralizing antibodies (NAbs) are effective in treating COVID-19 but the mechanism of immune protection is not fully understood. Here, we applied live bioluminescence imaging (BLI) to monitor the real-time effects of NAb treatment in prophylaxis and therapy of K18-hACE2 mice intranasally infected with SARS-CoV-2-nanoluciferase. We visualized sequential spread of virus from the nasal cavity to the lungs followed by systemic spread to various organs including the brain, culminating in death. Highly potent NAbs from a COVID-19 convalescent subject prevented, and also effectively resolved, established infection when administered within three days of infection. In addition to direct neutralization, in vivo efficacy required Fc effector functions of NAbs, with contributions from monocytes, neutrophils and natural killer cells, to dampen inflammatory responses and limit immunopathology. Thus, our study highlights the requirement of both Fab and Fc effector functions for an optimal in vivo efficacy afforded by NAbs against SARS-CoV-2.

Introduction

SARS-CoV-2-neutralizing monoclonal antibodies (NAbs) are an attractive countermeasure for both COVID-19 prevention and therapy (Schafer et al., 2021; Voss et al., 2020; Weinreich et al., 2021). To date, multiple NAbs against the spike (S) glycoprotein of SARS-CoV-2 have been identified from convalescent subjects. The majority of NAbs bind to the receptor binding domain (RBD) in the S1 subunit for inhibiting virus attachment to the human Angiotensin Converting Enzyme 2 (hACE2) receptor. NAbs against the N-terminal domain (NTD) of S1 as well as the S2 subunit have also been isolated (Anand et al., 2021b; Liu et al., 2020; Voss et al., 2020). NAbs have demonstrated varying levels of efficacy and protection in multiple animal models of SARS-CoV-2 (Alsoussi et al., 2020; Baum et al., 2020; Fagre et al., 2020; Hansen et al., 2020; Hassan et al., 2020; Li et al., 2020; Rogers et al., 2020; Shi et al., 2020b; Winkler et al., 2020; Zost et al., 2020a; Zost et al., 2020b). However, the in vitro neutralization potency of NAbs has not consistently correlated with in vivo protection (Bournazos et al., 2014; Schafer et al., 2021). While the antigen binding domain (Fab) of antibodies are critical for neutralization, the fragment crystallizable (Fc) domain can contribute significantly to their in vivo efficacy (Bournazos et al., 2019; Bournazos et al., 2014; DiLillo et al., 2014). Fc engagement of Fc gamma receptors (FcγRs) can elicit complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). The Fc region can recruit natural killer (NK) cells, monocytes, or neutrophils that can facilitate clearance of infected cells and shape the cytokine response produced by these cells for enhancing adaptive and cell-mediated immune responses (Lu et al., 2018). Fc effector functions can also be detrimental to the host, especially against respiratory diseases such as respiratory syncytial virus (RSV) and SARS-CoV-1 leading to antibody-dependent enhancement (ADE) and aggravated disease pathology (Bolles et al., 2011; Halstead and Katzelnick, 2020; Ruckwardt et al., 2019). Therefore, a careful investigation NAb mechanisms that elicit protective or pathological consequences is required before their clinical deployment.

Animal models evaluated to date (Johansen et al., 2020; Leist et al., 2020a; Leist et al., 2020b) have not fully recapitulated pathological features of human COVID-19. Transgenic mice expressing hACE2 under the cytokeratin 18 promoter (K18-hACE2 mice) however, have some distinct advantages. Primarily, they are permissive and highly susceptible to human-tropic SARS-CoV-2 virus strains and succumb to infection within a week (McCray et al., 2007; Shi et al., 2020a; Winkler et al., 2020). This allows for a rapid turnaround and a high bar for identifying effective prophylactic or therapeutic intervention strategies. In susceptible humans, SARS-CoV-2 infection disables innate immunity and elicits an imbalanced inflammatory cytokine response in the lungs leading to acute respiratory distress syndrome (ARDS) which is the major cause of death (Graham and Baric, 2020). Lethality in K18-hACE2 mice was initially associated with lung inflammation, cytokine storm and impaired respiratory function (Winkler et al., 2020). However, it has recently been recognized that the pathogenic endpoint in K18-hACE2 mice is a result of viral neuroinvasion and an ensuing neuronal disease (Carossino et al., 2021; Golden et al., 2020; Leist et al., 2020a). Infact, many patients display a myriad of neurological symptoms during and after recovery from SARS-CoV-2 infection (Ellul et al., 2020). Finally, mouse FcγRs display similar affinities to human antibodies (Dekkers et al., 2017). Therefore, K18-hACE2 mice serve as excellent models for evaluating and screening candidate human NAbs for their effects on SARS-CoV-2 replication and pathogenesis.

Bioluminescence imaging (BLI)-guided studies permit live visualization of pathogen spread to diverse anatomical site, the identification of relevant tissues of interest and a real-time readout of treatment regimens accelerating the evaluation process. A BLI-driven platform has not been harnessed for studying infectious respiratory pathogens like SARS-CoV-2 that require level 3 biosafety containment. Here, we have established a BLI-driven approach to study SARS-CoV-2 infection with a well characterized replication competent virus carrying a nanoluciferase (nLuc) reporter in the place of the Orf7A gene (Xie et al., 2020a; Xie et al., 2020b). SARS-CoV-2-nLuc closely mimics the wildtype virus replication kinetics and stably maintains the nLuc reporter over five generations in vitro. In addition, Orf7a deletion was recently shown to induce comparable pathology to the wild-type virus (Silvas et al., 2021). Our in vivo imaging studies revealed that the virus spreads from the nasal cavity to lungs for establishing infection. This was followed by infection of cervical lymph nodes (cLNs), brain, and systemic dissemination. Once neuroinvasion occurred, the virus replicated rapidly in the brain leading to fulminant infection and death by 6-7 days post infection (dpi). A single prophylactic intraperitoneal (i.p.) administration of highly potent NAbs isolated from a convalescent COVID-19 subject completely prevented SARS-CoV-2 infection and mortality in K18-hACE2 mice. Protection was associated with widespread localization of administered NAbs and Fc-mediated effector functions with contributions from monocytes and NK cells as well as reduced induction of inflammatory cytokines. BLI also revealed a therapeutic window of 3 dpi for NAb for successfully halting progression of established infection in the lungs as well as to distal tissues. Thus, our BLI-driven study highlighted the requirement for both neutralizing and Fc effector functions of NAbs to elicit optimal virological and immunological outcome against SARS-CoV-2.

Results

BLI allows Visualization of SARS-CoV-2 Replication Dynamics and Pathogenesis

We tracked spread of SARS-CoV-2-nLuc using BLI after intranasal (i.n.) challenge in K18-hACE2 mice (Figure 1A). 1 x 10⁵ FFU of SARS-CoV-2 generated sufficient photon flux to allow non-invasive BLI. Importantly, luciferase signal was absent in C57BL/6J (B6) mice lacking hACE2 (Figure 1B). Temporal tracking of emitted light intensities revealed that the virus replicated in the nasal cavity in a biphasic manner (Figure 1C). Luminescent signal in the nose increased in the first two days of infection after which it diminished before increasing again between 5 to 6 dpi when systemic spread occurred. The first signs of infection in the lungs were observed at 1 dpi. The nLuc signal then steadily increased in the lungs until 3 dpi and plateaued thereafter. We detected nLuc signals in the cLNs and brain region (imaging in ventral position) at 4 dpi. There was a steep rise in nLuc activity in the brain from 4 to 6 dpi indicating neuroinvasion and robust virus replication (Figure 1B, C, Video S1). This was accompanied by widespread replication of the virus in the gut and genital tract with concomitant loss in body weight. By 6 dpi, the infected K18-hACE2 mice lost 20% of their initial body weight, became moribund and succumbed to the infection (Figure 1D, E). In contrast, as expected, B6 mice did not experience any weight loss and survived the virus challenge.

Figure 1. Visualization of SARS-CoV-2 Replication Dynamics in hACE2 Transgenic Mice

(A) Experimental strategy utilizing SARS-CoV-2 carrying nLuc reporter in ORF7 for non-invasive BLI of virus spread following intranasal (i.n.) challenge of B6 or K18-hACE2 mice.

(B) Representative images from temporal BLI of SARS-CoV-2-nLuc-infected mice in ventral (v) and dorsal (d) positions at the indicated dpi and after necropsy at 6 dpi.

(C) Temporal quantification of nLuc signal as flux (photons/sec) acquired non-invasively in the indicated tissues of each animal. The color bar above the x-axis (yellow to orange) represents computed signal intensities in K18-hACE2 mice that are significantly above those in B6 mice.

(D) Temporal changes in mouse body weight at the indicated dpi with initial body weight set to 100%.

(E) Kaplan-Meier survival curves of mice for experiment as in A statistically compared by log-rank (Mantel-Cox) test.

(F) Ex vivo imaging of indicated organs and quantification of nLuc signal as flux(photons/sec) at indicated dpi after necropsy

(G, H) Viral loads (FFUs/g or nLuc activity/g) from indicated tissue using Vero E6 cells as targets. Undetectable virus amounts were set to 1.

(I) Ratio of C_t values for SARS-CoV-2 nucleocapsid (N) and nLuc estimated by RT-PCR using RNA extracted from input virions (inoculum) and virions from sera of mice at 6 dpi.

(J, K) Cytokine mRNA levels in lung and brain tissues at 6 dpi after normalization to Gapdh in the same sample and that in uninfected mice.

Each curve in (C) and (D) and each data point in (F), (I), (J), and (K) represents an individual mouse. Scale bars in (B and (F) denote radiance (photons/sec/cm²/steradian). p values obtained by non-parametric Mann-Whitney test for pairwise comparison. ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001; ∗∗∗∗, p < 0.0001; ns, not significant; Mean values ± SD are depicted.

To visualize the extent of viral spread with enhanced sensitivity and resolution, we imaged individual organs after necropsy (Figure 1B, F). nLuc signal was absent in B6 mice while most organs analyzed from K18-hACE2 mice showed nLuc activity with maximum signal detected in the brain followed by the lung and nasal cavity (Figure 1F). These observations mirrored viral loads [Focus Forming Units (FFUs) and nLuc activity] in the brain, lung and nasal cavity (Figure 1G, H). Real-time PCR analyses to detect N gene mRNA as well as histological analyses of organs confirmed widespread infection (Figure S1A, B).

Reporter-expressing viruses often purge foreign genes, particularly in vivo, due to fitness and immune pressure (Falzarano et al., 2014; Ventura et al., 2019). To estimate the stability of nLuc reporter, we compared the copy numbers of SARS-CoV-2 nucleocapsid (N) to nLuc in the viral RNA by real-time PCR analyses of input virions and virions isolated from sera of mice at 6 dpi. The ratio of copy numbers between the two samples sets did not change significantly (Figure 1I) indicating that the reporter was stable throughout the experimental timeline. Thus, nLuc activity was an excellent surrogate to follow virus replication in vivo.

SARS-CoV-2 infection triggers an imbalanced immune response and a cytokine storm that contributes significantly to pathogenesis (Del Valle et al., 2020). We compared the mRNA levels of inflammatory cytokines IL6, CCL2, CXCL10 and IFNγ in the lungs and brains of mice after necropsy at 6 dpi. Indeed, most cytokines mRNAs were significantly upregulated in both organs of infected K18-hACE2 mice compared to B6 (Figure 1J, K). Overall, cytokine mRNAs were higher in the brain compared to lungs with CXCL10 mRNA copy numbers reaching ∼1000 fold higher in K18-hACE2 than in B6 mice corroborating extensive infection (Figure 1J, K).

We next used BLI to illuminate areas of infected regions within lungs, brain, and testis for directed histology and electron tomographic studies (Figure 2). Higher resolution imaging revealed that SARS-CoV-2 virions were associated to large extent with capillary endothelial cell and/or alveolar type-1 cells in the lungs (Figure 2A-D; Video S2). In the brain, neuronal cells (MAP2⁺GFAP^-CD68^-CD11b^-) were positive for SARS-CoV-2 N and EM tomography revealed an array of SARS-CoV-2 virions associated within the dendrites (Figure 2E-J, Figure S1C, Video S3). In the testis, Sertoli cells stained positively for N (Figure 2K-N, Video S4). EM tomography also showed a large population of virions within pleomorphic membrane-bound compartments of Sertoli cells.

Figure 2. EM localization of SARS-CoV-2 Virions in Lung, Brain and Testis of Infected K18-hACE2 Mice.

(A) 2D overview of a lung region featuring red blood cells (rbc) within a pulmonary capillary, an alveolar Type 2 cell (AT2).

(B) Slice from a 3D tomogram of square region in A showing membrane-enclosed cytoplasmic compartments (arrowheads) containing presumptive SARS-CoV-2 virions in capillary endothelial cells.

(C) Presumptive virions from tomogram in B displayed at equatorial views. Presumptive virions were identified as described in the Methods and are directly comparable to those in SARS-CoV-2 infected Vero-E6 cells (panels O-Q).

(D) ImmunoEM tomography of presumptive SARS-CoV-2 virions from infected lung tissue, labeled with antiserum against Spike protein and gold (10 nm) conjugated 2° antibodies. Gold particles localized to the outer peripheries of the virions indicate specific labeling of SARS-CoV-2 Spikes.

(E) Tomographic of SARS-CoV-2 infected brain tissue. Presumptive SARS-CoV-2 virions (red arrowheads) are present within a neuron (pale green). A dendritic synaptic terminal to the left of the virus-containing neuron shows that presumptive SARS-CoV-2 virions are easily distinguished from typical synaptic neurotransmitter vesicles.

(F) 2D overview of brain tissue illustrating the complex spatial relationship among neurons and other brain cell types. Presumptive SARS-CoV-2 virions are present in two compartments (black squares) within a single neuron.

(G, H) Tomographic slices of black squares in F. Presumptive SARS-CoV-2 virions (red arrowheads) appear to be aligned within compartments that border the edges of a neural projection.

(I) Presumptive SARS-CoV-2 virions from tomograms in G and H.

(J) ImmunoEM tomography as in D of presumptive SARS-CoV-2 virions from infected brain tissue.

(K) (Upper) BLI of testis from a SARS-CoV-2 infected mouse to identify infected regions for IF and EM analyses. (Lower) IF image of an infected testis region stained with antibodies to SARS-CoV-2 Nucleocapsid (red)

(L) 2D overview of testis corresponding to region of high intensity (red) in the upper panel of K, showing Sertoli cells surrounded by developing sperm (left) and one primary spermatocyte (1°S, upper right). Presumptive SARS-CoV-2 virions are localized to membrane-bound compartments in Sertoli cells (black squares).

(M, N) Slices from two 3D tomograms of squares in L. Presumptive SARS-CoV-2 virions (arrowheads) are present within membrane-enclosed cytoplasmic compartments. These compartments contain additional structures amongst the discernable SARS-CoV-2 virions (insets).

(O) EM localization of virions in SARS-CoV-2 infected Vero-E6 cells, processed for EM as above tissue samples. Virions were characterized (see Methods) and compared to presumptive virions in the tissue samples to confidently verify their identities. 2D overview of infected Vero-E6 cell in a 150 nm section.

(P) Tomogram of rectangle in O showing >100 presumptive SARS-CoV-2 virions contained within cytoplasmic exit compartments.

(Q) Virions from the tomogram in P showing common features of dense RNC puncta, discernable surface spikes, vary in size (∼60-120 nm) and shape. Virions are directly comparable to those shown for the tissue samples in C and I.

Highly Potent SARS-CoV-2 NAbs CV3-1 and CV3-25 from a Convalescent Donor

We recently reported and characterized plasma from a COVID-19 convalescent subject (S006) with potent neutralizing activity and high levels of SARS-CoV-1 cross-reactive Abs (Lu et al., 2020). We probed the B cell receptor (BCR) repertoire from this donor to isolate broad and potent NAbs. Using a recombinant SARS-CoV-2 S ectodomain (S2P) as a bait to identify antigen-specific B cells, we collected and screened a library of S-targeted BCR clones and identified two most potent NAb candidates: CV3-1 and CV3-25. We first characterized their epitope specificity using ELISA, cell-surface staining, virus capture assay and surface plasmon resonance (SPR) (Ding et al., 2020; Prevost et al., 2020). Both NAbs recognized SARS-CoV-2 S efficiently with a low-nanomolar affinity, as a stabilized ectodomain (S-6P) or when displayed on cells and virions (Figure 3A-E). While CV3-1 bound the SARS-CoV-2 RBD, CV3-25 targeted the S2 subunit (Figure 3A, D-E) and cross-reacted with SARS-CoV-1 S on cells or virions, but not with S from other human Coronaviruses (Figure 3B-C). In agreement with the previous smFRET data for S006 plasma, CV3-1 stabilized S in the RBD-up (∼0.1 FRET) conformation (Figure 3F-H), as seen with hACE2 and most RBD-directed NAbs (Lu et al., 2020). Interestingly, CV3-25-bound S showed a partial shift towards downstream conformations (∼0.1 and ∼0.3 FRET), suggesting a distinct inhibitory mechanism from CV3-1 (Figure 3F-H).

Figure 3. In vitro Characterization of CV3-1 and CV3-25 NAbs.

(A) NAb binding to SARS-CoV-2 Spike ectodomain (S-6P) or RBD estimated by ELISA. Relative light unit (RLU) were normalized to the cross-reactive SARS-CoV-1 NAb CR3022. NAb binding to SARS-CoV-2 S2 N-His protein on cell-surface of transfected 293T cells analyzed by flow cytometry. Median fluorescence intensities (MFIs) for anti-Spike NAbs were normalized to the signal obtained with an anti-His tag mAb.

(B) Flow cytometric detection of S-expressing 293T cells from the different human CoVs: SARS-CoV-2, SARS-CoV-1, OC43, HKU1, MERS-CoV, NL63 and 229E. MFI from 293T cells transfected with empty vector were used for normalization.

(C) Pseudoviruses bearing SARS-CoV-2 or SARS-CoV-1 S were tested for capture by anti-Spike NAbs. The cross-reactive CR3022 mAb was used for normalization.

(D-E) NAb binding affinity and kinetics to SARS-CoV-2 S using Surface Plasmon Resonance (SPR). SARS-CoV-2 S-6P or S2 ectodomain was immobilized as the ligand on the chip and CV3-1 or CV3-25 Fab was used as analytes at concentrations in a range from 1.56 to 100 nM for both Fabs to S-6P and 3.125nM to 200nM for CV3-25 to S2 (2-fold serial dilution, see Methods for details). Alternatively, CV3-1 IgG was immobilized on the chip and SARS-CoV-2 RBD was used as the analyte with concentrations ranging from 1.56 to 50 nM (2-fold serial dilution). Kinetic constants were determined using a 1:1 Langmuir model in BIA evaluation software (experimental readings shown in blue and fitted curves shown in black).

(F-H) FRET histograms of ligand-free S on S-MEN coronavirus-like particles (VLPs) or in presence of 50 µg/mL of CV3-1 (G) or CV3-25 (H). VLPs were incubated for 1 h at 37°C before smFRET imaging. N_m is the number of individual FRET traces compiled into a conformation-population FRET histogram (gray lines) and fitted into a 4-state Gaussian distribution (solid black) centered at 0.1-FRET (dashed cyan), 0.3-FRET (dashed red), 0.5-FRET (dashed green), and 0.8-FRET (dashed magenta).

(I) Neutralizing activity of CV3-1 and CV3-25 alone or in combination (1:1 ratio) on SARS-CoV-2 S bearing pseudoviruses using 293T-ACE2 cells.

(J) Microneutralization activity of anti-Spike NAbs on live SARS-CoV-2 virus using Vero E6 cells.

(K) Inhibition of cell-to-cell fusion between 293T cells expressing HIV-1 Tat and SARS-CoV-2 S and TZM-bl-ACE2 cells by NAbs.

Half maximal inhibitory antibody concentration (IC₅₀) values in I-K were determined by normalized non-linear regression analyses.

(L) MFI of CEM.NKr cells expressing SARS-CoV-2 Spike (CEM.NKr-Spike) stained with indicated amounts of NAbs and normalized to parental CEM.NKr.

(M) % ADCC in the presence of titrated amounts of NAbs using 1:1 ratio of parental CEM.NKr cells and CEM.NKr-Spike cells as targets when PBMCs from uninfected donors were used as effector cells

(N) % ADCP in the presence of titrated amounts of NAbs using CEM.NKr-Spike cells as targets and THP-1 cells as phagocytic cells.

We next measured the ability of CV3-1 and CV3-25 to neutralize and mediate Fc-dependent antibody functions. While both NAbs blocked infection by SARS-CoV-2 pseudovirus or live virus and interfered with S-driven cell-to-cell fusion, CV3-1 was ∼10 times more potent than CV3-25 (Figure 3I-L). To evaluate Fc-mediated effector functions of the NAbs, we used assays that quantify the ADCC and ADCP activities against S-expressing cells. CV3-1 and CV3-25 efficiently bound and eliminated S-expressing cells by stimulating cytotoxic and phagocytic responses in immune effector cells (Figure 3L-N). Overall, both NAbs displayed significant neutralization and Fc-dependent antibody functions, although CV3-1 was found to be more effective for most of the assessed functions. The combination of the two NAbs (1:1 ratio) was found to be phenotypically similar to the responses seen with CV3-1 alone (Figure 3I-N).

Prophylactic Treatment with NAbs Protects K18-hACE2 Mice from SARS-CoV-2 Infection

We first monitored the biodistribution of Alexa Fluor (AF) conjugated CV3-1 and CV3-25 in various tissues 24 h after i.p. delivery in mice by fluorescence imaging, histology and ELISA. All three approaches revealed widespread distribution of the NAbs to multiple organs and target tissues including the nasal cavity, lung and the brain (Figure S2, S3A-D). Next, we tested a prophylactic regimen where each NAb was delivered i.p. alone (12.5 mg/kg body weight) or in 1:1 combination (6.25 mg each NAb/kg body weight) 24 h before i.n. challenge with SARS-CoV-2 nLuc (Figure 4A). Temporal monitoring by whole-body BLI revealed that all three prophylactic regimens substantially reduced SARS-CoV-2 infection in the lungs and subsequent spread (Figure 4B-D). Remarkably, pretreatment with CV3-1 alone or in combination with CV3-25 (cocktail 1:1) produced near complete protection from SARS-CoV-2 infection with no signals detected in multiple organs after non-invasive imaging or after terminal necropsy at 22 dpi (Figure 4B-D, G, H). Moreover, all test cohorts survived with no discernible weight loss, nLuc activity or viral loads in all organs tested signifying complete control of virus infection (Figure 4E-I). Lung and subsequent neuroinvasion did occur, albeit at reduced intensity, in CV3-25-pretreated animals which was corroborated by imaging of individual organs after necropsy (Figure 4B, G, H). CV3-25 delayed mortality by ∼2 days in 4 out of the 6 animals and viral loads in the nasal cavity, lungs, and brain at the time of necropsy (8 dpi) were similar to that in the control cohorts treated with isotype-matched antibodies at 6 dpi (Figure 4F, I).

Figure 4. Prophylactic Treatment with CV3-1 Protects Mice from Lethal SARS-CoV-2 Infection.

(A) Experimental design for testing in vivo efficacy of NAbs CV3-1 and CV3-25 administered alone (12.5 mg/kg body weight) or as a 1:1 cocktail (6.25 mg/kg body weight each) 1 day prior to challenging K18-hACE2 mice (i.n.) with SARS-CoV-2-nLuc followed by non-invasive BLI every 2 days. Human IgG1-treated (12.5 mg Ig/kg) mice were use as the isotype control (Iso)

(B) Representative images from BLI of SARS-CoV-2-nLuc-infected mice in ventral (v) and dorsal (d) positions at the indicated dpi and after necropsy at indicated days for experiment as in A.

(C-D) Temporal quantification of nLuc signal as flux (photons/sec) computed non-invasively in indicated areas of each animal.

(E) Temporal changes in mouse body weight at the indicated dpi with initial body weight set to 100%.

(F) Kaplan-Meier survival curves of mice statistically compared by log-rank (Mantel-Cox) test for experiment as in A.

(G, H) Ex-vivo imaging of organs and quantification of nLuc signal as flux (photons/sec) at the indicated dpi after necropsy.

(I) Viral loads (nLuc activity/g) from indicated organs using Vero E6 cells as targets. Undetectable virus amounts were set to 1.

(J, K) Cytokine mRNA levels in lung and brain tissues after necropsy normalized to Gapdh in the same sample and that in uninfected mice.

Viral loads (I) and inflammatory cytokine profile (J, K) were determined after necropsy for mice that succumbed to infection and in mice surviving at 22 dpi.

Scale bars in (B) and (G) denote radiance (photons/sec/cm²/steradian). Each curve in (C)-(E) and each data point in (H)-(K) represents an individual mouse. Grouped data in (C)-(K) were analyzed by 2-way ANOVA followed by Dunnett’s or Tukey’s multiple comparison tests. Statistical significance for group comparisons to isotype control are shown in black and for those to CV3-25 are shown in red ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001; ∗∗∗∗, p < 0.0001; Mean values ± SD are depicted.

CV3-1 or NAb cocktail pre-treatment also prevented the inflammatory cytokine induction seen in control and CV3-25 pre-treated cohorts (Figure 4J, K). In contrast, heightened levels of cytokine mRNA were detected in mice that had succumbed to infection and the control cohort (Figure 4J, K). Mice that survived in the CV3-25 pretreated cohorts, regained body weight and at 22 dpi, had no detectable virus in organs and exhibited base-line inflammatory cytokine induction (Figure 4E-K). Overall, our data indicated that CV3-1 alone, or in combination with CV3-25, inhibited establishment of virus infection and fully protected K18-hACE2 mice. Histology of brain tissue revealed that NAbs CV3-1 and CV3-25 persisted even at 6 dpi (Figure S3E). Consistent with virus infection in the brain, CV3-25 localized heavily to the surface of infected neurons at 6 dpi, in addition to endothelial cells, which was predominantly observed before infection. In contrast, CV3-1 localization remained unaltered due to the absence of viral neuroinvasion in this cohort (Figure S3E). In addition, in vivo dose response studies revealed that as little as 0.75 mg CV3-1/kg body weight protected 50% of the treated cohort from lethal SARS-CoV-2 infection (Figure S4). These data indicated that CV3-1 is highly potent at halting SARS-CoV-2 infection and contributed to protection observed in the cohort treated with the cocktail.

CV3-1 Therapy Recues Mice from Lethal SARS-CoV-2 Infection

CV3-1 NAb alone completely protected K18-hACE2 mice against SARS-CoV-2-induced mortality. We therefore explored if CV3-1 could also cure infected mice. Mice infected with SARS-CoV-2-nLuc were administered CV3-1 at 1, 3, and 4 dpi after confirming SARS-CoV-2 infection was established in the lungs of all mice (Figure 5A). Temporal imaging and quantification of nLuc signal revealed that CV3-1, when administered at 1 and 3 dpi, controlled virus spread and successfully prevented neuroinvasion (Figure 5B-D, G, H). This was corroborated by no weight loss and/or recuperation of body weight, undetectable viral loads as well as near-baseline levels of inflammatory cytokines in tissues (Figure 5E-K). CV3-1 therapy at 4 dpi, however, could neither control virus spread nor neuroinvasion resulting in death of 75% of the mice in this cohort (Figure 5B-F) with loss in body weight, high levels of inflammatory cytokines and tissue viral loads, similar to that in the control cohort (Figure 5E-K). Thus, the therapeutic window of maximal efficacy for CV3-1 treatment extends for up to 3 days from the initiation of SARS-CoV-2 infection to successfully prevent lethality.

Figure 5. CV3-1 Therapy Protects Mice from Lethal SARS-CoV-2 Infection.

(A) Experimental design to test in vivo efficacy of CV3-1 administered i.p. (12.5 mg/kg body weight) at indicated times after i.n. challenge of K18-hACE2 mice with SARS-CoV-2 nLuc followed by non-invasive BLI every 2 days. Human IgG1 treated (12.5 mg/kg body weight) mice were the control cohort.

(B) Representative images from temporal BLI of SARS-CoV-2-nLuc-infected mice in ventral (v) and dorsal (d) positions at the indicated dpi and after necropsy.

(C-D) Temporal quantification of nLuc signal acquired non-invasively as flux (photons/sec) in indicated regions of each mice.

(E) Temporal changes in mouse body weight at indicated dpi with initial body weight set to 100%.

(F) Kaplan-Meier survival curves of mice statistically compared by log-rank (Mantel-Cox) test for experiment as in A.

(G, H) Ex vivo imaging of indicated organs and quantification of nLuc signal as flux(photons/sec) at indicated dpi after necropsy.

(I) Viral loads estimated as nLuc activity/g of indicated organs using Vero E6 cells as targets.

Non-detectable virus amounts were set to 1.

(J, K) Cytokine mRNA levels in lung and brain tissues after necropsy. The data was normalized to Gapdh in the same sample and that in uninfected mice.

Viral loads (I) and inflammatory cytokine profile (J, K) were determined after necropsy at times indicated in G. Each curve in (C)-(E) and each data point in (H)-(K) represents an individual mouse. CV3-1 treatment times are indicated in (C)-(E). Grouped data in (C)-(K) were analyzed by 2-way ANOVA followed by Dunnett’s or Tukey’s multiple comparison tests. Statistical significance for group comparisons to isotype control are shown in black and for those under CV3-1 therapies to 4 dpi-treated cohorts are shown in red. ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001;

∗∗∗∗, p < 0.0001; Mean values ± SD are depicted.

CV3-1 and CV3-25 Require Antibody Effector Functions For in vivo efficacy

Highly potent antibodies can effectively neutralize free viruses and may also mediate Fc-recruitment of immune cells to eliminate infected cells. We therefore explored a role for Fc-mediated effector functions in protection in vivo. We generated Leucine to Alanine (L234A/L235A, LALA) mutant versions of both NAbs to impair interaction with Fc receptors (Saunders, 2019). Our in vitro assays confirmed that, while ADCC and ADCP activities were compromised, LALA mutations had no impact on S binding and neutralizing capacities of both NAbs (Figure S5). Biodistribution analyses of AF647-conjugated CV3-1 and CV3-25 LALA NAbs, 24h after i.p. administration indicated penetration into most tissues (Figure S5).

We next tested the impact of LALA mutations on the prophylactic efficacy of CV3-1 and CV3-25 (Figure S6A). Longitudinal non-invasive BLI and terminal imaging analyses after necropsy, body weight changes, survival and viral load estimations revealed that LALA mutations had indeed compromised the protective efficacy of both antibodies (Figure S6A-I). SARS-CoV-2 replicated better, invaded the brain and induce body weight loss in cohorts treated with LALA NAbs compared to the corresponding wild-type NAbs (Figure S6D-E). Histology at 6 dpi revealed that both LALA NAbs had penetrated the brain tissue during the course of infection and bound the surface of infected neurons (Figure S5G, H). While CV3-1-treated animals had no detectable viral loads at 6 dpi, CV3-1 LALA pretreated mice had higher tissue viral loads indicating compromised protective efficacy (Figure S6I). Similarly, while tissue viral loads in CV3-25 treated mice were reduced by a log, those in CV3-25 LALA treated mice were comparable to that in control cohorts. Moreover, the delayed mortality and 25% protective efficacy offered by CV3-25 was abrogated and the ability of CV3-1 to provide 100% protection from SARS-CoV-2-induced mortality was reduced to 62.5% with the corresponding LALA mutants (Figure S6F). Additionally, there was an overall increase in the signature inflammatory cytokine profile in mice pre-treated with LALA NAbs (Figure S6J, K).

The requirement for Fc effector function during CV3-1 prophylaxis was surprising as we did not detected infection in CV3-1 treated mice both by non-invasive and post-necropsy tissue imaging at 6 dpi (Figure 4). However, examination of tissues at 3 dpi did reveal weak nLuc signals in the nasal cavity and lungs despite absence of signal by non-invasive imaging (Figure S5J-M). PCR analyses also confirmed the presence SARS-CoV-2 N RNA in these tissues at 3 dpi (Figure S5N). The data indicated that some of the incoming virions did not encounter CV3-1 and managed to establish infection during prophylaxis and hence Fc effector functions were required to eliminate them.

Our data thus implied that immune cell components would be critical during CV3-1 therapy (Figure 6A). Indeed, while CV3-1 treatment at 3 dpi controlled infection, cohorts treated with CV3-1 LALA displayed rapidly spreading lung infection and fully succumbed by 6 dpi after an accelerated loss in body weight (Figure 6B-F). High viral loads and cytokine levels in nose, lung, and brain also reflected the failure of the LALA NAbs to treat pre-established viral infection (Figure 6G). Notably, while the lung viral loads in CV3-1 LALA NAb-treated cohort were similar to that in the control, inflammatory cytokine mRNA levels in lungs, CXCL10 in particular, were significantly higher suggesting a crucial requirement for Fc-engagement in curbing a cytokine-storm like phenotype (Figure 6H-I).

Figure 6. Fc-mediated Antibody Effector Functions Contribute to the In Vivo Efficacy of CV3-1

(A) Experimental design to test therapeutic efficacy of NAb CV3-1 and its corresponding Leucine to Alanine (LALA) mutant administered ip (12.5 mg/kg body weight) 3 dpi to K18-hACE2 mice challenged with SARS-CoV-2 nLuc followed by non-invasive BLI every 2 days. Human IgG1-treated (12.5 mg/kg body weight) mice were used as the control cohort.

(B) Representative images from temporal BLI of SARS-CoV-2-nLuc-infected mice in ventral (v) and dorsal (d) positions at the indicated dpi and after necropsy. Scale bars denote radiance (photons/sec/cm²/steradian).

(C-D) Temporal quantification of nLuc signal acquired non-invasively as flux (photons/sec) in indicated regions.

(E) Temporal changes in mouse body weight at indicated dpi with initial body weight set to 100%.

(F) Kaplan-Meier survival curves of mice statistically compared by log-rank (Mantel-Cox) test. for experiment as in A.

(G) Viral loads (nLuc activity/g) in indicated organs using Vero E6 cells as targets. Undetectable virus amounts were set to 1.

(H, I) Cytokine mRNA levels in lung and brain tissues after necropsy. The values were normalized to Gapdh in the same sample and that in uninfected mice.

Viral loads (G) and inflammatory cytokine profile (H, I) was determined after necropsy at times indicated in B. Each curve in (C)-(E) and each data point in (G)-(I) represents an individual mouse. CV3-1 treatment times are indicated in (C)-(E). Grouped data in (C)-(I) were analyzed by 2-way ANOVA followed by Dunnett’s or Tukey’s multiple comparison tests. Statistical significance for group comparisons to isotype control are shown in black and between CV3-1 and CV3-1 LALA treated cohorts are shown in red. ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001; ∗∗∗∗, p < 0.0001; Mean values ± SD are depicted.

Monocytes and NK cells Contribute to Antibody-mediated Effector Functions In Vivo

To identify the immune cell types engaged by NAbs, we initiated NK cell depletion (α-NK1.1) prior to CV3-1 prophylaxis. Flow cytometric analyses confirmed NK cell depletion in α-NK1.1 treated cohorts. (Figure S7J, K). Our BLI-guided analyses showed appearance of weak nLuc signals in lungs of infected mice prophylactically treated with CV3-1 as well as αNK1.1 mAbs compared to those in the control cohort treated with CV3-1 and with an isotype control (Figure S7A-D). In addition, two of the mice that underwent NK cell depletion in the CV3-1 pretreated group experienced a temporary but significant decrease in body weight before recovering (Figure S7E). Nevertheless, all the mice receiving CV3-1 prophylaxis survived despite NK cell depletion (Figure S7F). We did observe a marginal increase in viral loads in target organs upon NK depletion in mice under CV3-1 prophylaxis (Figure S7G). In addition, the ability of CV3-1 to suppress inflammatory cytokines was significantly compromised upon NK cell-depletion (Figure S7H, I-K). Thus, while NK cells do contribute to in vivo efficacy of CV3-1, their requirement was not significant enough to compromise protection offered by CV3-1 prophylaxis.

To investigate NK cell-requirement during CV3-1 therapy, we depleted them in SARS-CoV-2-nLuc infected mice where CV3-1 treatment was initiated at 3 dpi (Figure 7A). Our BLI-centric multiparametric analyses revealed that NK cell depletion partially compromised the efficacy of CV3-1 therapy with 25% of the mice succumbing to SARS-CoV-2 infection compared to CV3-1-treated controls where all the mice survived (Figure 7B-F). We next investigated if Ly6G⁺ neutrophils and Ly6C^hi CD11b⁺ classical monocytes accounted for additional Fc effector activities by using the anti-Ly6G and anti-CCR2 depleting mAbs in mice under CV3-1 therapy (Figure 7A, B, S7L-O) (Mack et al., 2001). Neutrophil or monocyte depletion under CV3-1 therapy led to 75% and 80% of the mice failed to control SARS-CoV-2 spread and loss in body weight resulting in death although neuroinvasion was overall weaker than isotype control cohorts (Figure 7B-E, G). Neutrophil or monocyte depletion in the setting of CV3-1 therapy led to increased viral burden and enhanced expression of CCL2, CXCL10, and Il6 mRNA in target tissues compared to the cohort under CV3-1 therapy (Figure 7H-J). These data indicated that neutrophils, monocytes and NK cells contributed to the antibody-dependent cure of mice from lethal SARS-CoV-2 infection and are critical for the success of SARS-CoV-2 NAb-directed therapies.

Figure 7. Monocytes, Neutrophils and Natural Killer Cells Contribute to Antibody Effector Functions In Vivo.

(A) Experimental design to test the contribution of NK cells, neutrophils (CD11b⁺Ly6G⁺) and monocytes (CCR2⁺Ly6^hi CD11b⁺) in K18-hACE2 mice therapeutically treated with CV3-1 NAb (i.p.,12.5 mg/kg body weight) at 3 dpi after challenge with SARS-CoV-2-nLuc. αNK1.1 mAb (i.p., 20 mg/kg body weight), αLy6G mAb (i.p., 20 mg/kg body weight) and αCCR2 mAb (i.p., 2.5 mg/kg body weight) were used to deplete NK cells, neutrophils and monocytes respectively every 48h starting at 1 dpi. Corresponding human (for CV3-1) and rat (for αNK1.1 and αLy6G mAb or αCCR2) monoclonal antibodies served as non-specific isotype controls (Iso). The mice were followed by non-invasive BLI every 2 days from the start of infection.

(B) Representative images from temporal BLI of SARS-CoV-2-nLuc-infected mice in ventral (v) and dorsal (d) positions at the indicated dpi and after necropsy.

(C-D) Temporal quantification of nLuc signal acquired non-invasively as flux (photons/sec) in indicated regions.

(E) Temporal changes in mouse body weight at indicated dpi with initial body weight set to 100%.

(F) Kaplan-Meier survival curves of mice statistically compared by log-rank (Mantel-Cox) test for experiment as in A.

(H) Viral loads (nLuc activity/g) of indicated organs using Vero E6 cells as targets. Undetectable virus amounts were set to 1.

(I, J) Cytokine mRNA levels in lung and brain tissues after necropsy and normalization to GAPDH in the same sample and that in uninfected mice.

Viral loads (H) and inflammatory cytokine profile (I, J) was determined after necropsy at times indicated in B. Each curve in C-E and each data point in H-J represents an individual mouse.

Grouped data in (C)-(I) were analyzed by 2-way ANOVA followed by Dunnett’s or Tukey’s multiple comparison tests. Statistical significance: group comparisons to isotype control are shown in black; group comparisons to Iso+CV3-1 within the NK and neutrophil depleted cohorts are shown in purple; group comparisons to Iso+CV3-1 within the monocyte-depleted cohorts are shown in red. ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001; ∗∗∗∗, p < 0.0001; Mean values ± SD are depicted.

Together, our data demonstrate the utility of a BLI-guided platform for temporo-spatial visualization of SARS-CoV-2 replication, pathogenesis and the mechanisms contributing to an effective outcome with NAb-based interventions in vivo in the K18-ACE2 mouse model.

Discussion

NAb therapies are being explored to augment current vaccination strategies against SARS-CoV-2 to expand the protection afforded towards newly arising virus variants. However, prior evidence of antibody-dependent enhancement of pathology caused by respiratory viruses like RSV and SARS-CoV-1 warrants careful investigation of antibody effects in vivo before clinical implementation (Iwasaki and Yang, 2020; Klasse and Moore, 2020). We have established a whole-body imaging approach to follow the dynamics and pathogenesis of SARS-CoV-2 infection in mice to facilitate preclinical studies for identifying effective therapeutic measures against COVID-19. Temporal tracking of K18-hACE2 mice revealed that SARS-CoV-2 first replicates in the nasal cavity, reaches the lungs at 1 dpi where the infection expands till 3 dpi before spreading systemically to other organs including the brain by 4 dpi. BLI also helped illuminate how the highly potent human NAbs CV3-1 (targets Spike RBD) and CV3-25 (binds S2 domain) differed in their ability to protect or treat SARS-Cov-2 infection in the highly susceptible K18-hACE2 mouse model. Imaging analyses revealed widespread NAb distribution within the animals, including in the nasal cavity and lung where the virus initially establishes infection, and persistence for at least a week after administration, features that were critical for efficacy in this acute model for SARS-Cov-2. BLI also revealed a therapeutic window of 3 dpi for CV3-1 NAb to successfully halt progression of established infection in the lungs and distal tissues. Given that the previously reported SARS-CoV-2 NAbs have a therapeutic window of 1 dpi (Alsoussi et al., 2020; Hassan et al., 2020; Schafer et al., 2021; Winkler et al., 2021). CV3-1 displays one of the most potent in vivo efficacy profile with a widened therapeutic window till 3 dpi. Most protective human NAbs for SARS-Cov-2 tested in animal models and also in humans, target RBD (Baum et al., 2020; Chen et al., 2021; Rogers et al., 2020; Schafer et al., 2021; Tortorici et al., 2020; Weinreich et al., 2021), some NTD targeting NAbs also display potent antiviral activity in vivo (Li et al., 2021; Noy-Porat et al., 2021; Voss et al., 2020). We show that the S2-directed CV3-25 NAb also conferred protection, albeit not as potent as CV3-1. This of significance as newly emerging variants display fewer mutations in the S2 subunit compared to the S1 subunit. Indeed, CV3-25 has been found to efficiently neutralize the B.1.351 variant, while neutralization capacities of anti-NTD and anti-RBD NAbs were greatly diminished (Stamatatos et al., 2021).

Our data also establishes that neutralizing capacity alone is not enough to garner clinical protection by NAbs. LALA variants of CV3-1 revealed a crucial role for Fc-mediated interactions in augmenting in vivo protection for prophylaxis as well as therapy. The requirement for Fc effector functions during CV3-1 prophylaxis, needed for eliminate infected cells originating from virions that eluded neutralization, is noteworthy and contrasting to a recent report where they were required only during NAb therapy (Winkler et al., 2021). Introducing LALA mutation in CV3-1 completely compromised its ability to therapeutically cure and was in agreement with previous observations (Winkler et al., 2021). Surprisingly, we noticed a more severe loss in body weight in mice that were therapeutically administered CV3-1 LALA variants and significantly higher inflammatory responses (CCL2, CXCL10, IFNγ) in lungs than isotype-treated control animals. These data suggest that the Fc region plays an additional protective role by limiting immunopathology through dampening of inflammatory responses. A previously reported NAb engaged only monocytes for in vivo activity (Winkler et al., 2021). However, our studies revealed that CV3-1 engaged Fc-interacting neutrophils, monocytes and NK cells for its in vivo efficacy. Thus, in addition to its potent neutralizing activity, a superior engagement of innate immune components contributed to the high in vivo potency of CV3-1

Experiments using low doses of NAbs indicated that CV3-1 did not enhance infection at concentrations that protected only 50% of animals in the group. Thus, our data add to the growing body of evidence that suggest the absence of an antibody-dependent enhancement (ADE) mechanism with a protective rather than a pathogenic role for Fc effects during SARS-CoV-2 infection (Schafer et al., 2021; Winkler et al., 2021). However, while mouse FcγRs bind human antibodies with similar affinities as mouse antibodies, their expression pattern on various mouse immune cells differs from those in humans. Therefore, additional investigations in other models are required to confirm definitive absence of ADE during human SARS-CoV-2 infection (Gorman et al., 2021). Moreover, elucidation of the major FcγR(s) (FcγRI, FcγRIII and/or FcγRIV) engaged by NAbs will help design ultrapotent SARS-CoV-2 Nab therapies (Smith et al., 2012).

In summary, our study demonstrates the utility of the BLI-guided approach to study SARS-CoV-2 pathogenesis and identify effective antiviral therapies for rapid translation to clinical use in humans.

Supplemental information

7 Supplementary figures and 4 Videos

Author contributions

PDU, IU, JP, PK, AF, & WM: conceptualization, experimental design, interpretation, and manuscript preparation and writing; IU: animal experiments, BLI, antibody biodistribution, viral load analyses & data processing; PDU: histological analyses, FACS, data processing, figure generation & initial draft; LS & ATM: isolated NAbs; JP, ML, SPA, GBB, MB, SD, RG, CF, YC, AT, GG, CB, HM, GAD, JDD, DEK, JR, MP, WM and AF: generation & in vitro characterization of SARS-CoV-2 S NAbs; MSL & PJB: EM tomography; IU, HS: cytokine, N gene mRNA PCR; MM: anti-CCR2 antibody; CBW: aliquot of the reporter virus. PK, WM, AF, PJB: funding for the work.

Disclaimer

The views expressed in this presentation are those of the authors and do not reflect the official policy or position of the Uniformed Services University, US Army, the Department of Defense, or the US Government.

Declaration of Interests

The authors declare no competing interests.

Supplementary Figure Legends

Figure S1. Widespread SAR-CoV-2 infection in K18-hACE2 mice. Related to Figure 1. (A) A plot showing real-time PCR analyses to detect SARS-CoV-2 nucleocapsid (N) gene mRNA in indicated organs of B6 and K18-hACE2 mice at 6 dpi intranasally challenged with 1 x 10⁵ FFU of SARS-CoV-2-nLuc. The data were normalized to N RNA seen in uninfected mice and GAPDH mRNA levels. p values obtained by non-parametric Mann-Whitney test for pairwise comparison. ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001; ∗∗∗∗, p < 0.0001; ns, not significant; Mean values ± SD are depicted.

(B) Images of cryosections from indicated tissues of SARS-CoV-2-nLuc infected K18-hACE2 mouse harvested at 6 dpi. Actin (green), nucleocapsid (red) and hACE2 (magenta) were detected using phalloidin and respective antibodies. Notably, hACE2 appeared as puncta on the surface of infected neurons and lung tissue compared to other organs where the signal was more uniform and stained a region of the cell surface. Scale bar: 50 µm

(C) Images of cryosections from brain tissues of SARS-CoV-2-nLuc infected K18-hACE2 mouse harvested at 6 dpi to characterize infected cells. Glial cells (top panel) were identified using antibodies to markers CD68 (magenta) and CD11b (green). Neurons (lower panel) were identified using antibodies to MAP2 (green) and mature astrocytes were identified using antibodies to GFAP (magenta). SARS-CoV-2 infected cells were identified using antibodies to nucleocapsid (red). Nucleocapsid positive cells were predominantly positive for MAP2. Scale bar: 20 µm

Figure S2. Widespread biodistribution of CV3-25 and CV3-1 NAbs in mice 24 h after intraperitoneal delivery. Related to Figure 3. (A) C57BL/6J mice were either mock treated (PBS) or intraperitoneally administered 12.5 mg/kg body weight of CV3-25 monoclonal antibody conjugated to Alexa Fluor 594 (CV3-25 AF594). 24 h later, indicated tissues were imaged using the fluorescence module in IVIS spectrum to detect AF594. The plot shows the quantified radiance detected in indicated tissues after normalization with corresponding organs from control mouse.

(B) Images of cryosections from indicated tissues from CV3-25 AF594-treated mouse as in A. Actin and CV3-25 were detected using phalloidin-AF488 and AF647 conjugated anti-human IgG respectively. Scale bar: 20 µm

(C) C57BL/6J mice were either mock treated (PBS) or intraperitoneally administered 12.5 mg/kg of CV3-1 monoclonal antibody conjugated to Alexa Fluor 647 (CV3-25 AF647). 24 h later, indicated tissues were imaged using the fluorescence module in IVIS spectrum to detect AF647. The plot shows the quantified radiance detected in indicated tissues after normalization with corresponding organs from control mouse.

(B) Images of cryosections from indicated tissues from CV3-1 AF647-treated mouse as in A. Actin was detected using phalloidin-Alexa Fluor 488. CV3-1 AF647 was detected in the red channel using Alexa Fluor 568 conjugated anti-human IgG. Scale bar: 20 µm

Figure S3. Assessment of CV3-1 and CV3-25 NAbs biodistribution in mice using ELISA and immunohistology. Related to Figure 4. (A-D) Estimation of CV3-25 and CV3-1 NAbs biodistribution in mice using ELISA. Measurement of anti-Spike NAbs levels in organs was performed using quantitative ELISA. (A-B) Recombinant SARS-CoV-2 RBD and (C-D) S-6P proteins were used to quantify CV3-1 and CV3-25 antibody levels, respectively. Linear standard curves using known concentrations of CV3-1 or CV3-25 NAbs were established for inferring the antibody concentration in organ homogenates. Serial dilutions of homogenized mice organs were prepared in PBS and incubate on antigen-coated plates. The presence of anti-Spike NAbs was revealed using HRP-conjugated anti-human IgG secondary Abs. The signal obtained with BSA (negative control) was subtracted for each organ. Relative light unit (RLU) values were transformed into a NAb concentrations based on the standard curve and the dilution factor. Subsequently, these concentration values were multiplied with the homogenization volume and divided by the total organ weight.

(E) Persistence and redistribution of neutralizing NAbs in SARS-CoV-2 infected mice. Images of brain tissue from K18-hACE2 mice infected with SARS-CoV-2-nLuc at 6 dpi that were prophylactically treated with CV3-1 or CV3-25 (12.5 mg/kg body weight), 24 h before infection. Actin (green) was labelled using phalloidin, CV3-1 and CV3-25 (magenta) were detected using anti-hIgG conjugated to Alexa Fluor 647 and infected cells (red) were identified using antibodies to SARS-CoV-2 N. CV3-1 localizes to the endothelial walls of blood vessels and CV3-25 redistributes to decorate infected neurons in addition to endothelium (seen in UI mice; Figure S2). Scale bar: 50 µm

Figure S4. Efficacious dose for CV3-1 NAb during prophylaxis. Related to Figure 4. (A) A scheme showing experimental design for testing the dose of CV3-1 NAb to achieve protection for lethal SARS-CoV-2-nLuc infection. Indicated concentration of CV3-1 NAb was delivered (i.p.) 1 day before challenging K18-hACE2 mice with 1 x 10⁵ FFU of SARS-CoV-2 nLuc. Human IgG1-treated (12.5 mg/kg) mice were used as control (isotype treated). Mice were followed by non-invasive BLI every 2 days from the start of infection using IVIS Spectrum after retroorbital administration of furimazine (nLuc substrate).

(B) SARS-CoV-2 replication and dissemination in K18-hACE2 transgenic mice (n = 4-6 per group) for experiment as in A, were monitored via BLI in ventral (v) and dorsal (d) positions at the indicated days post infection every 2 days. Images from two mice under CV3-1 prophylaxis (0.7 mg/kg) are shown where one mouse succumbed at 6 dpi and the other survived despite weak but observable neuroinvasion. Images from one representative experiment are shown for the rest. (C-D) A plot showing temporal quantification of nLuc signal acquired non-invasively and displayed as photon flux (photons/sec) in whole body or brain region of SARS-CoV2-nLuc infected K18-hACE2 mice for an experiment as in A. Each curve represents luminescent signal computed for individual mouse. Scale bars denote radiance in photons per second per square centimeter per steradian (p/sec/cm2/sr).

(E) A plot showing temporal body weight changes in indicated groups of K18-hACE2 mice at indicated days post infection for an experiment shown in A. Each curve represents one animal. The body weight at the start of the experiment was set to 100 %.

(F) Kaplan-Meier survival curves of mice statistically compared by log-rank (Mantel-Cox) test for experiment as in A.

(G) Plot showing viral loads as nLuc activity per gram of indicated organs using Vero E6 cells as targets. Undetectable virus amounts were set to 1 for display on log plots.

Grouped data in (C)-(E) and G were analyzed by 2-way ANOVA followed by Dunnett’s or Tukey’s multiple comparison tests. Statistical significance: group comparisons to isotype control are shown in black; group comparisons to CV3-1 (0.7 mg/kg) are shown in red. ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001; ∗∗∗∗, p < 0.0001; Mean values ± SD are depicted.

Figure S5. LALA mutations Diminish Antibody Effector Functions of CV3-1 and CV3-25 without Compromising Neutralizing Activity. Related to Figure 6. (A) Cell-surface staining of CEM.NKr cells stably expressing full-length SARS-CoV-2 Spike (CEM.NKr-Spike) using CV3-1 and CV3-25 mAbs or their LALA mutant counterpart. The graph shown represent the mean fluorescence intensities (MFI) obtained. with titrated concentrations of anti-Spike NAbs. MFI values obtained with parental CEM.NKr were subtracted.

(B) Pseudoviral particles encoding for the luciferase reporter gene and bearing the SARS-CoV-2 S glycoproteins were used to infect 293T-ACE2 cells. Neutralizing activity was measured by incubating pseudoviruses with titrated concentrations of anti-Spike NAbs at 37°C for 1 h prior to infection of 293T-ACE2 cells. Neutralization half maximal inhibitory antibody concentration (IC₅₀) values were determined using a normalized non-linear regression using GraphPad Prism software.

(C) Using a FACS-based ADCC assay, CEM.NKr parental cells were mixed at a 1:1 ratio with CEM.NKr-Spike cells and were used as target cells. PBMCs from uninfected donors were used as effector cells. The graph shown represent the percentages of ADCC obtained in the presence of titrated concentrations of anti-Spike NAbs.

(D) Using a FACS-based ADCP assay, CEM.NKr-Spike cells were used as target cells and THP-1 monocytic cell line was used as effector cells. The graph shown represent the percentages of effector cells that had phagocytosed target cells obtained in the presence of titrated concentrations of anti-Spike NAbs.

Statistical significance was tested using a non-parametric Mann-Whitney test for pairwise comparison between WT and LALA NAbs (*, p < 0.05; **, p < 0.01; ns, not significant)

(E-F) Biodistribution of CV3-25 and CV3-1 LALA mutants in mice 24 h after i.p. delivery. B6 mice were either isotype treated (control) or intraperitoneally administered of Alexa Fluor 647 conjugated LALA mutants of CV3-25 or CV3-1 NAb mutants (12.5 mg/kg body weight). 24 h later, indicated tissues were imaged using the fluorescence module in IVIS spectrum to detect Alexa Flour 647. The plot shows the quantified radiance detected in indicated tissues after normalization with corresponding organs from control mouse.

(G, H) Images of cryosections from brain tissues of K18-hACE2 mice pretreated with LALA mutants of CV3-25 or CV3-1 (i.p., 12.5 μg/g body weight) at 6 dpi. Actin was detected using phalloidin-Alexa Fluor 488. CV3-25 and CV3-1 (magenta) were detected using Alexa Fluor 647 conjugated anti-human IgG respectively. Infected cells were detected using antibodies to SARS-CoV-2 nucleocapsid (red). Images show penetration of both CV3-25 and CV3-1 mAbs into the brain and localization to the surface of infected neurons. Scale bar: 20 µm

(I) SARS-CoV-2 can establish infection in nasal cavity and lungs during CV3-1 prophylaxis. A scheme showing experimental design to test establishment of virus infection in K18-hACE2 mice pretreated with CV3-1 NAb (i.p.,12.5 mg/kg body weight), 1 day before challenging with 1 x 10⁵ FFU of SARS-CoV-2 nLuc. Mice treated similarly with Isotype matched hIgG1 were used as controls. The mice were followed by non-invasive BLI at 0 and 3 dpi using IVIS Spectrum after retroorbital administration of furimazine (nLuc substrate).

(J) SARS-CoV-2 replication and dissemination in indicated groups of K18-hACE2 transgenic mice (n = 5-3 per group) for experiment as in I, were monitored via BLI at the indicated times. The mice were euthanized at 3 dpi and imaged again after necropsy. Images from one representative experiment are shown.

(K) A plot showing temporal quantification of nLuc signal acquired non-invasively and displayed as photon flux (photons/sec) in whole body of SARS-CoV2-nLuc infected K18-hACE2 mice for an experiment as in I. Each line represents luminescent signal computed for individual mouse.

(L, M) Ex vivo imaging of indicated organs after necropsy at 3 dpi and quantification of nLuc signal displayed as photon flux (photons/sec) in K18-ACE2 mice for experiment as in I.

(N) A plot showing real-time PCR analyses to detect SARS-CoV-2 nucleocapsid (N) gene mRNA in indicated organs of K18-hACE2 mice for an experiment as in I. The data were normalized to N gene mRNA seen in uninfected mice and Gapdh mRNA levels. Scale bars denote radiance (photons/sec/cm²/steradian). p values obtained by non-parametric Mann-Whitney test for pairwise comparison with isotype-treated controls; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; individual data points along with mean values ± SD are depicted.

Figure S6. Fc-mediated Antibody Effector Functions Contribute to In Vivo Efficacy of CV3-1 and CV3-25 During Prophylaxis. Related to Figure 6. (A) A scheme showing experimental design for testing in vivo efficacy of neutralizing antibody CV3-1, CV3-25 and their corresponding Leucine to Alanine (LALA) mutants (12.5 mg/kg body weight) delivered intraperitoneally (i.p.) 1 day before challenging K18-hACE2 mice with 1 x 10⁵ FFU of SARS-CoV-2 nLuc. Human IgG1-treated (12.5 mg/kg body weight) mice were used as control (Iso). Mice were followed by non-invasive BLI every 2 days from the start of infection using IVIS Spectrum after retroorbital administration of furimazine (nLuc substrate).

(B) SARS-CoV-2 replication and dissemination in K18-hACE2 transgenic mice (n = 4-8 per group) for experiment as in A, were monitored via BLI in ventral (v) and dorsal (d) positions at the indicated days post infection every 2 days. The mice were euthanized on indicated days and imaged again after necropsy. Images from one representative experiment are shown.

(C-D) A plot showing temporal quantification of nLuc signal acquired non-invasively and displayed as Flux (photons/sec) in whole body or brain region of SARS-CoV2-nLuc infected K18-hACE2 mice for an experiment as in A. Each curve represents luminescent signal computed for individual mouse.

(E) A plot showing temporal body weight changes of K18-hACE2 mice at indicated days post infection for an experiment shown in A. Each curve represents one animal. The body weight at the start of the experiment was set to 100%.

(F) Kaplan-Meier survival curves of mice statistically compared by log-rank (Mantel-Cox) test for experiment as in A.

(G, H) Ex vivo imaging of indicated organs after necropsy at indicated dpi and quantification of nLuc signal displayed as Flux (photons/sec) in K18-ACE2 mice for experiment as in A.

(I) Plot showing viral loads as nLuc activity per gram of indicated organs using Vero E6 cells as targets. Nluc activity was determined 24 h after infection. Undetectable virus amounts were set to 1 for display on log plots.

(J, K) A plot showing mRNA levels of indicated cytokines from lung and brain tissues of K18-hACE2 mice at the time of euthanasia as shown in F. The mRNA amounts were normalized to the levels seen in uninfected mice and the house keeping gene Gapdh.

Scale bars in (B) and (G) denote radiance (photons/sec/cm2/steradian). Grouped data in (C)-(E) and (H)-(K) were analyzed by 2-way ANOVA followed by Dunnett’s or Tukey’s multiple comparison tests. Statistical significance: group comparisons to isotype control are shown in black; group comparisons between CV3-25 LALA and CV3-25 treated cohorts are shown in red; group comparison between CV3-1 LALA and CV3-1 treated cohorts are shown in purple. ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001; ∗∗∗∗, p < 0.0001; Mean values ± SD are depicted.

Figure S7. NK Cells Contribute Marginally to In Vivo Efficacy During CV3-1 Prophylaxis. Related to Figure 7. (A) A scheme showing experimental design for testing the contribution of NK cells in K18-hACE2 mice pretreated with CV3-1 NAb (i.p.,12.5 mg/kg body weight), 1 day before challenging with 1 x 10⁵ FFU of SARS-CoV-2 nLuc. αNK1.1 mAb (i.p., 20 mg/kg body weight) was used to deplete NK cells at indicated time points. Corresponding human (for CV3-1) and rat (for αNK1.1) antibodies served as non-specific isotype controls. The mice were followed by non-invasive BLI every 2 days from the start of infection using IVIS Spectrum after retroorbital administration of furimazine (nLuc substrate).

(B) SARS-CoV-2 replication and dissemination in indicated groups of K18-hACE2 transgenic mice (n = 5 per group) for experiment as in A, were monitored via BLI at ventral (v) and dorsal (d) positions at the indicated days post infection every 2 days. The mice were euthanized at indicated times and imaged again after necropsy. Images from one representative experiment are shown. (C-D) A plot showing temporal quantification of nLuc signal acquired non-invasively and displayed as photon flux (photons/sec) in whole body or brain region of SARS-CoV2-nLuc infected K18-hACE2 mice for an experiment as in A. Each curve represents luminescent signal computed for individual mouse. Scale bars denote radiance (photons/sec/cm²/steradian).

(E) A plot showing temporal body weight changes in designated groups of K18-hACE2 mice at indicated days post infection for an experiment shown in A. Each curve represents one animal. The body weight at the start of the experiment was set to 100%.

(F) Kaplan-Meier survival curves of mice statistically compared by log-rank (Mantel-Cox) test for experiment as in A.

(G) Plot showing viral loads as nLuc activity per gram of indicated organs using Vero E6 cells as targets. Nluc activity was determined 24 h after infection. Undetectable virus amounts were set to 1 for display on log plots.

(H, I) A plot showing mRNA levels of indicated cytokines from lung and brain tissues of K18-hACE2 mice at the time of euthanasia as shown in F. The mRNA amounts were normalized to the levels seen in uninfected mice and the house keeping gene GAPDH.

(J, K) Representative FACS plots showing the gating strategy to identify NK cells (CD3^-NK1.1⁺) and quantification to ascertain their depletion in PBMCs of indicated groups of mice.

(L, M) Representative FACS plots showing the gating strategy to identify neutrophils cells (CD45⁺ CD11b⁺Ly6G⁺) and quantification to ascertain their depletion in PBMCs of indicated groups of mice.

(N, O) Representative FACS plots showing the gating strategy to identify Ly6C^hi monocytes and quantification to ascertain their depletion in PBMCs of indicated groups of mice.

Grouped data in (C)-(E) and (G)-(I) were analyzed by 2-way ANOVA followed by Dunnett’s or Tukey’s multiple comparison tests. Statistical significance: group comparisons to isotype control are shown in black; group comparisons to Iso+CV3-1 treated cohort are in red. Pairwise comparisons in (K), (M) and (O) were analyzed using non-parametric Mann-Whitney test. ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001; ∗∗∗∗, p < 0.0001; Mean values ± SD are depicted.

Supplementary Multimedia Files

Video S1. Longitudinal Non-invasive BLI of SARS-CoV-2-nLuc infection and dissemination in K18-hACE2 mice, Related to Figure 1. SARS-CoV-2-nLuc challenged mice were imaged daily in dorsal (d) and ventral (v) positions for 6 days using IVIS Spectrum to monitor virus spread in the whole body as well as neuroinvasion.

Video S2. Tomographic Reconstruction of SARS-CoV-2 Infected Lung Tissue, Related to Figure S2, panels B-D. Virus particles are found within membrane-enclosed exit compartments of two adjacent pulmonary capillary endothelial cells. The movie traverses the reconstructed volume to illustrate the compartments (red arrowheads) then increases in magnification to detail the virions within the compartments.

Video S3. Tomographic Reconstruction of SARS-CoV-2 Infected Brain Tissue, Related to Figure S2, panel F. Virus particles are found within neurons, often appearing in linear groups within compartments bordering the edges of neuronal projections. The movie details the distinction between presumptive SARS-CoV-2 virions and typical synaptic neurotransmitter vesicles found in an adjacent synaptic terminal.

Video S4. Tomographic Reconstruction of SARS-CoV-2 Infected Testis Tissue, Related to Figure S2, panel M. Virus particles are found within membrane-enclosed compartments of Sertoli cells. Additional material and structures coexist with the virions in these compartments, suggesting they may be defined as lysosomes. Presumptive SARS-CoV-2 virions can be discerned from the other structures.

RESOURCE AVAILABILITY

Lead Contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Pradeep Uchil (), Priti Kumar (), Andrés Finzi () and Walther Mothes().

Materials Availability

All other unique reagents generated in this study are available from the corresponding authors with a completed Materials Transfer Agreement.

Data and Code Availability

The data that support the findings of this study are available from the corresponding authors upon reasonable request.

EXPERIMENTAL MODEL AND SUBJECT DETAILS

Cell and Viruses

Vero E6 (CRL-1586, American Type Culture Collection (ATCC), were cultured at 37°C in RPMI supplemented with 10% fetal bovine serum (FBS), 10 mM HEPES pH 7.3, 1 mM sodium pyruvate, 1× non-essential amino acids, and 100 U/ml of penicillin–streptomycin. The 2019n-CoV/USA_WA1/2019 isolate of SARS-CoV-2 expressing nanoluciferase was obtained from Craig B Wilen, Yale University and generously provided by K. Plante and Pei-Yong Shi, World Reference Center for Emerging Viruses and Arboviruses, University of Texas Medical Branch) (Xie et al., 2020a; Xie et al., 2020b). The SARS-CoV-2 USA-WA1/2020 virus strain used for microneutralization assay was obtained through BEI Resources. Virus was propagated in Vero-E6 by infecting them in T150 cm² flasks at a MOI of 0.1. The culture supernatants were collected after 72 h when cytopathic effects were clearly visible. The cell debris was removed by centrifugation and filtered through 0.45-micron filter to generate virus stocks. Viruses were concentrated by adding one volume of cold (4 °C) 4x PEG-it Virus Precipitation Solution (40 % (w/v) PEG-8000 and 1.2 M NaCl; System Biosciences) to three volumes of virus-containing supernatant. The solution was mixed by inverting the tubes several times and then incubated at 4 °C overnight. The precipitated virus was harvested by centrifugation at 1,500 × g for 60 minutes at 4 °C. The concentrated virus was then resuspended in PBS then aliquoted for storage at −80°C. All work with infectious SARS-CoV-2 was performed in Institutional Biosafety Committee approved BSL3 and A-BSL3 facilities at Yale University School of Medicine or the University of Western Ontario using appropriate positive pressure air respirators and protective equipment. CEM.NKr, CEM.NKr-Spike, THP-1 and peripheral blood mononuclear cells (PBMCs) were maintained at 37°C under 5% CO₂ in RPMI media, supplemented with 10% FBS and 100 U/mL penicillin/ streptomycin. 293T (or HEK293T), 293T-ACE2, CF2Th, TZM-bl and TZM-bl-ACE2 cells were maintained at 37°C under 5% CO₂ in DMEM media, supplemented with 5% FBS and 100 U/mL penicillin/ streptomycin. CEM.NKr (NIH AIDS Reagent Program) is a T lymphocytic cell line resistant to NK cell-mediated lysis. CEM.NKr-Spike stably expressing SARS-CoV-2 Spike were used as target cells in ADCC and ADCP assays (Anand et al., 2021a). THP-1 monocytic cell line (ATCC) was used as effector cells in the ADCP assay. PBMCs were obtained from healthy donor through leukapheresis and were used as effector cells in ADCC assay. 293T cells (obtained from ATCC) were derived from 293 cells, into which the simian virus 40 T-antigen was inserted. 293T-ACE2 cells stably expressing human ACE2 is derived from 293T cells (Prevost et al., 2020). Cf2Th cells (obtained from ATCC) are SARS-CoV-2-resistant canine thymocytes and were used in the virus capture assay. TZM-bl (NIH AIDS Reagent Program) were derived from HeLa cells and were engineered to contain the Tat-responsive firefly luciferase reporter gene. For the generation of TZM-bl cells stably expressing human ACE2, transgenic lentiviruses were produced in 293T using a third-generation lentiviral vector system. Briefly, 293T cells were co-transfected with two packaging plasmids (pLP1 and pLP2), an envelope plasmid (pSVCMV-IN-VSV-G) and a lentiviral transfer plasmid coding for human ACE2 (pLenti-C-mGFP-P2A-Puro-ACE2) (OriGene). Forty-eight hours post-transfection, supernatant containing lentiviral particles was used to infect TZM-bl cells in presence of 5 µg/mL of polybrene. Stably transduced cells were enriched upon puromycin selection. TZM-bl-ACE2 cells were then cultured in medium supplemented with 2 mg/mL of puromycin (Millipore Sigma).

Ethics statement

PBMCs from healthy individuals as a source of effector cells in our ADCC assay were obtained under CRCHUM institutional review board (protocol #19.381). Research adhered to the standards indicated by the Declaration of Helsinki. All participants were adults and provided informed written consent prior to enrollment in accordance with Institutional Review Board approval.

Antibodies

The human antibodies (CV3-1 and CV3-25) used in the work were isolated from blood of male convalescent donor S006 (male) recovered 41 days after symptoms onset using fluorescent recombinant stabilized Spike ectodomains (S2P) as probes to identify antigen-specific B cells as previously described (Lu et al., 2020; Seydoux et al., 2020). Site-directed mutagenesis was performed on plasmids expressing CV3-1 and CV3-25 antibody heavy chains in order to introduce the LALA mutations (L234A/L235A) using to the QuikChange II XL site-directed mutagenesis protocol (Stratagene).

Mouse Experiments

All experiments were approved by the Institutional Animal Care and Use Committees (IACUC) of and Institutional Biosafety Committee of Yale University (IBSCYU). All the animals were housed under specific pathogen-free conditions in the facilities provided and supported by Yale Animal Resources Center (YARC). All IVIS imaging, blood draw and virus inoculation experiments were done under anesthesia using regulated flow of isoflurane:oxygen mix to minimize pain and discomfort to the animals.

C57BL/6 (B6), hACE2 transgenic B6 mice (heterozygous) were obtained from Jackson Laboratory. 6–8-week-old male and female mice were used for all the experiments. The heterozygous mice were crossed and genotyped to select heterozygous mice for experiments by using the primer sets recommended by Jackson Laboratory.

METHOD DETAILS

SARS-CoV-2 infection and treatment conditions

For all in vivo experiments, the 6 to 8 weeks male and female mice were intranasally challenged with 1 x 10⁵ FFU in 25-30 µl volume under anesthesia (0.5 - 5 % isoflurane delivered using precision Dräger vaporizer with oxygen flow rate of 1 L/min). For NAb treatment using prophylaxis regimen, mice were treated with 250 µg (12.5 mg/kg body weight) of indicated antibodies (CV3-1 or CV3-25) or in combination (1:1; 6.25 mg/kg body weight of each) via intraperitoneal injection (i.p.) 24 h prior to infection. For mAb treatment under therapeutic regimen, mice were treated at 1, 3 and 4 dpi intraperitoneally with CV3-1(12.5 mg/kg body weight). Body weight was measured and recorded daily. The starting body weight was set to 100 %. For survival experiments, mice were monitored every 6-12 h starting six days after virus administration. Lethargic and moribund mice or mice that had lost more than 20% of their body weight were sacrificed and considered to have succumbed to infection for Kaplan-Meier survival plots.

Bioluminescence Imaging (BLI) of SARS-CoV-2 infection

All standard operating procedures and protocols for IVIS imaging of SARS-CoV-2 infected animals under ABSL-3 conditions were approved by IACUC, IBSCYU and YARC. All the imaging was carried out using IVIS Spectrum® (PerkinElmer) in XIC-3 animal isolation chamber (PerkinElmer) that provided biological isolation of anesthetized mice or individual organs during the imaging procedure. All mice were anesthetized via isoflurane inhalation (3 - 5 % isoflurane, oxygen flow rate of 1.5 L/min) prior and during BLI using the XGI-8 Gas Anesthesia System. Prior to imaging, 100 µL of nanoluciferase substrate, furimazine (NanoGlo^TM, Promega, Madison, WI) diluted 1:40 in endotoxin-free PBS was retroorbitally administered to mice under anesthesia. The mice were then placed into XIC-3 animal isolation chamber (PerkinElmer) pre-saturated with isothesia and oxygen mix. The mice were imaged in both dorsal and ventral position at indicated days post infection. The animals were then imaged again after euthanasia and necropsy by spreading additional 200 µL of substrate on to exposed intact organs. Infected areas of interest identified by carrying out whole-body imaging after necropsy were isolated, washed in PBS to remove residual blood and placed onto a clear plastic plate. Additional droplets of furimazine in PBS (1:40) were added to organs and soaked in substrate for 1-2 min before BLI.

Images were acquired and analyzed with the manufacturer’s Living Image v4.7.3 in vivo software package. Image acquisition exposures were set to auto, with imaging parameter preferences set in order of exposure time, binning, and f/stop, respectively. Images were acquired with luminescent f/stop of 2, photographic f/stop of 8. Binning was set to medium. Comparative images were compiled and batch-processed using the image browser with collective luminescent scales. Photon flux was measured as luminescent radiance (p/sec/cm2/sr). During luminescent threshold selection for image display, luminescent signals were regarded as background when minimum threshold levels resulted in displayed radiance above non-tissue-containing or known uninfected regions. To determine the pattern of virus spread, the image sequences were acquired every day following administration of SARS-CoV-2 (i.n). Image sequences were assembled and converted to videos using Image J.

Biodistribution of therapeutic neutralizing antibodies using IVIS

Mice were intraperitoneally (i.p) administered with 250 µg of unconjugated (12.5 mg/kg body weight), Alexa Fluor 647 or Alexa Fluor 594-labeled antibodies to non-infected or SARS-CoV-2 infected hACE2 mice. 24 h later all organs (nose, trachea, lung, cervical lymph nodes, brain, liver, spleen, kidney, gut, testis and seminal vesicles) were isolated after necropsy and images were acquired with an IVIS Spectrum® (PerkinElmer) and fluorescence radiance intensities were analyzed with the manufacturer’s Living Image v4.7.3 in vivo software package. Organs were cut into half and weighed. One half was fixed in 4 % PFA and processed for cryoimmunohistology. The other half was resuspended in serum-free RPMI and homogenized in a bead beater for determination of antibody levels using quantitative ELISA.

Measurement of therapeutic antibody levels in organs by quantitative ELISA

Recombinant SARS-CoV-2 RBD and S-6P proteins were used to quantify CV3-1 and CV3-25 antibody levels, respectively, in mice organs. SARS-CoV-2 proteins (2.5 μg/ml), or bovine serum albumin (BSA) (2.5 μg/ml) as a negative control, were prepared in PBS and were adsorbed to plates (MaxiSorp; Nunc) overnight at 4 °C. Coated wells were subsequently blocked with blocking buffer (Tris-buffered saline [TBS], 0.1% Tween20, 2% BSA) for 1 hour at room temperature. Wells were then washed four times with washing buffer (TBS 0.1% Tween20). Titrated concentrations of CV3-1 or CV3-25 or serial dilutions of mice organ homogenates were prepared in a diluted solution of blocking buffer (0.1 % BSA) and incubated in wells for 90 minutes at room temperature. Plates were washed four times with washing buffer followed by incubation with HRP-conjugated anti-IgG secondary Abs (Invitrogen) (diluted in a diluted solution of blocking buffer [0.4% BSA]) for 1 hour at room temperature, followed by four washes. HRP enzyme activity was determined after the addition of a 1:1 mix of Western Lightning oxidizing and luminol reagents (Perkin Elmer Life Sciences). Light emission was measured with a LB941 TriStar luminometer (Berthold Technologies). Signal obtained with BSA was subtracted for each organ. Titrated concentrations of CV3-1 or CV3-25 were used to establish a standard curve of known antibody concentrations and the linear portion of the curve was used to infer the antibody concentration in tested organ homogenates.

Cryo-immunohistology of organs

Organs were isolated after necropsy and fixed in 1X PBS containing freshly prepared 4% PFA for 12 h at 4 °C. They were then washed with PBS, cryoprotected with 10, 20 and 30% ascending sucrose series, snap-frozen in Tissue-Tek® O.C.T.^TM compound and stored at −80 °C. The nasal cavity was snap-frozen in 8% gelatin prepared in 1X PBS and stored at −80 °C. 10 - 30 µm thick frozen sections were permeabilized with Triton X-100 and treated with Fc receptor blocker (Innovex Biosciences) before staining with indicated conjugated primary, secondary antibodies or Phalloidin in PBS containing 2 % BSA containing 10 % fetal bovine serum. Stained sections were treated with TrueVIEW Autofluorescence Quenching Kit (Vector Laboratories) and mounted in VECTASHIELD® Vibrance™ Antifade Mounting Medium. Images were acquired using Nikon W1 spinning disk confocal microscope equipped with 405, 488, 561 and 647 nm laser lines. The images were processed using Nikon Elements AR version 4.5 software (Nikon Instruments Inc, Americas) and figures assembled with Photoshop CC and Illustrator CC (Adobe Systems, San Jose, CA, USA).

Focus forming assay

Titers of virus stocks was determined by standard plaque assay. Briefly, the 4 x 10⁵ Vero-E6 cells were seeded on 12-well plate. 24 h later, the cells were infected with 200 µL of serially diluted virus stock. After 1 hour, the cells were overlayed with 1ml of pre-warmed 0.6% Avicel (RC-581 FMC BioPolymer) made in complete RPMI medium. Plaques were resolved at 48 h post infection by fixing in 10 % paraformaldehyde for 15 min followed by staining for 1 hour with 0.2 % crystal violet made in 20 % ethanol. Plates were rinsed in water to visualize plaques.

Measurement of viral burden

Indicated organs (nasal cavity, brain, lungs from infected or uninfected mice were collected, weighed, and homogenized in 1 mL of serum free RPMI media containing penicillin-streptomycin and homogenized in 2 mL tube containing 1.5 mm Zirconium beads with BeadBug 6 homogenizer (Benchmark Scientific, TEquipment Inc). Virus titers were measured using three highly correlative methods. Frist, the total RNA was extracted from homogenized tissues using RNeasy plus Mini kit (Qiagen Cat # 74136), reverse transcribed with iScript advanced cDNA kit (Bio-Rad Cat #1725036) followed by a SYBR Green Real-time PCR assay for determining copies of SARS-CoV-2 N gene RNA using primers SARS-CoV-2 N F: 5’-ATGCTGCAATCGTGCTACAA-3’ and SARS-CoV-2 N R: 5’-GACTGCCGCCTCTGCTC-3’.

Second, serially diluted clarified tissue homogenates were used to infect Vero-E6 cell culture monolayer. The titers per gram of tissue were quantified using standard plaque forming assay described above. Third, we used nanoluciferase activity as a shorter surrogate for plaque assay. Infected cells were washed with PBS and then lysed using 1X Passive lysis buffer. The lysates transferred into a 96-well solid white plate (Costar Inc) and nanoluciferase activity was measured using Tristar multiwell Luminometer (Berthold Technology, Bad Wildbad, Germany) for 2.5 seconds by adding 20 µl of Nano-Glo® substrate in nanoluc assay buffer (Promega Inc, WI, USA). Uninfected monolayer of Vero cells treated identically served as controls to determine basal luciferase activity to obtain normalized relative light units. The data were processed and plotted using GraphPad Prism 8 v8.4.3.

Analyses of signature inflammatory cytokines mRNA

Brain and lung samples were collected from mice at the time of necropsy. Approximately, 20 mg of tissue was suspended in 500 µL of RLT lysis buffer, and RNA was extracted using RNeasy plus Mini kit (Qiagen Cat # 74136), reverse transcribed with iScript advanced cDNA kit (Bio-Rad Cat #1725036). To determine levels of signature inflammatory cytokines, multiplex qPCR was conducted using iQ Multiplex Powermix (Bio Rad Cat # 1725848) and PrimePCR Probe Assay mouse primers FAM-GAPDH, HEX-IL6, TEX615-CCL2, Cy5-CXCL10, and Cy5.5-IFNgamma. The reaction plate was analyzed using CFX96 touch real time PCR detection system. Scan mode was set to all channels. The PCR conditions were 95 °C 2 min, 40 cycles of 95 °C for 10 s and 60 °C for 45 s, followed by a melting curve analysis to ensure that each primer pair resulted in amplification of a single PCR product. mRNA levels of Il6, Ccl2, Cxcl10 and Ifng in the cDNA samples of infected mice were normalized to Gapdh with the formula ΔC_t(target gene)=C_t(target gene)-C_t(Gapdh). The fold increase was determined using 2^-ΔΔCt method comparing treated mice to uninfected controls.

Antibody depletion of immune cell subsets

For evaluating the effect of NK cell depletion during CV3-1 prophylaxis, anti-NK1.1 (clone PK136; 12.5 mg/kg body weight) or an isotype control mAb (BioXCell; clone C1.18.4; 12.5 mg/kg body weight) was administered to mice by i.p. injections every 2 days starting at 48 h before SARS-CoV-2-nLuc challenge till 8 dpi. The mice were bled after two days of antibody depletion, necropsy or at 10 dpi (surviving mice) for analyses. To evaluate the effect of NK cell and neutrophil depletion during CV3-1 therapy, anti-NK1.1 (clone PK136; 12.5 mg/kg body weight) or anti-Ly6G (clone: 1A8; 12.5 mg/kg body weight) was administered to mice by i.p injection every two days starting at 1 dpi respectively. Rat IgG2a mAb (BioXCell; clone C1.18.4; 12.5 mg/kg body weight) was used as isotype control. The mice were sacrificed and bled at 10 dpi for analyses. For evaluating the effect of monocyte depletion on CV3-1 therapy, anti-CCR2 (clone MC-21; 2.5 mg/kg body weight) (Mack et al., 2001) or an isotype control mAb (BioXCell; clone LTF-2; 2.5 mg/kg body weight) was administered to mice by i.p injection every two days starting at 1 dpi. The mice were sacrificed and bled 2-3 days after antibody administration or at 10 dpi to ascertain depletion of desired population.

Flow Cytometric Analyses

For analysis of immune cell depletion, peripheral blood was collected before infection and on day of harvest. Erythrocytes were lysed with RBC lysis buffer (BioLegend Inc), PBMCs fixed with 4 % PFA and quenched with PBS containing 0.1M glycine. PFA-fixed cells PBMCs were resuspended and blocked in Cell Staining buffer (BioLegend Inc.) containing Fc blocking antibody against CD16/CD32 (BioLegend Inc) before staining with antibodies. NK cells were identified as CD3-NK1.1+ cells using PE/Cy7 anti-mouse CD3(17A2) and APC anti-mouse NK-1.1 (PK136). Neutrophils were identified as CD45+CD11b+Ly6G+ cells using APC Rat anti-mouse CD45 (30-F11), PE anti-mouse CD11b (M1/70) APC/Cy7 and anti-mouse Ly-6G (1A8). Ly6C^hi monocytes were identified as CD45⁺CD11b⁺Ly6C^hi cells using APC Rat anti-mouse CD45 (30-F11), PE anti-mouse CD11b (M1/70) and APC/Cy7 anti-mouse Ly-6C (HK1.4). Data were acquired on an Accuri C6 (BD Biosciences) and were analyzed with Accuri C6 software. FlowJo software (Treestar) was used to generate FACS plot shown in Figure S7. 100,000 – 200,000 viable cells were acquired for each sample.

Sample Preparation for Electron Microscopy

Lung, brain and testis tissue samples from hACE2 transgenic mice challenged intranasally with SARS-CoV-2-nLuc (1 x 10⁵ FFU; 6 dpi) were imaged after necropsy using bioluminescence imaging (IVIS, Perkin Elmer), pruned to isolate regions with high nLuc activity and immediately pre-fixed with 3 % glutaraldehyde, 1 % paraformaldehyde, 5 % sucrose in 0.1 M sodium cacodylate trihydrate to render them safe for handling outside of BSL3 containment. Viral infections of cultured cells were conducted at the UVM BSL-3 facility using an approved Institutional Biosafety protocol. SARS-CoV-2 strain 2019-nCoV/USA_USA-WA1/2020 (WA1; generously provided by K. Plante, World Reference Center for Emerging Viruses and Arboviruses, University of Texas Medical Branch) and propagated in African green monkey kidney (Vero E6) cells. Vero E6 cells were maintained in complete Dulbecco’s Modified Eagle Medium (DMEM; Thermo Fisher, Cat. #11965–092) containing 10% fetal bovine serum (Gibco, Thermo-Fisher, Cat. #16140–071), 1% HEPES Buffer Solution (15630–130), and 1 % penicillin– streptomycin (Thermo Fisher, Cat. #15140–122). Cells were grown in a humidified incubator at 37 °C with 5 % CO₂. Vero E6 cells were seeded into six well dishes and infected with SARS-CoV-2 at a multiplicity of infection of 0.01 for 48 hours before fixing and preparing for electron microscopy. Cells were pre-fixed with 3% glutaraldehyde, 1% paraformaldehyde, 5 % sucrose in 0.1M sodium cacodylate trihydrate, removed from the plates and further prepared by high-pressure freezing and freeze-substitution as described below.

Tissues samples were further cut to ∼0.5 mm³ blocks and cultured cells were gently pelleted. Both samples were rinsed with fresh cacodylate buffer and placed into brass planchettes (Type A; Ted Pella, Inc., Redding, CA) prefilled with 10 % Ficoll in cacodylate buffer. The tissues were covered with the flat side of a Type-B brass planchette and rapidly frozen with an HPM-010 high-pressure freezing machine (Leica Microsystems, Vienna Austria). The frozen samples were transferred under liquid nitrogen to cryotubes (Nunc) containing a frozen solution of 2.5 % osmium tetroxide, 0.05 % uranyl acetate in acetone. Tubes were loaded into an AFS-2 freeze-substitution machine (Leica Microsystems) and processed at −90°C for 72 h, warmed over 12 h to −20°C, held at that temperature for 6 h, then warmed to 4°C for 2 h. The fixative was removed, and the samples rinsed 4 x with cold acetone, following which they were infiltrated with Epon-Araldite resin (Electron Microscopy Sciences, Port Washington PA) over 48 h. The spleen tissue was flat-embedded between two Teflon-coated glass microscope slides. Resin was polymerized at 60°C for 48 h.

Electron Microscopy and Dual-Axis Tomography

Flat-embedded tissue samples or portions of cell pellets were observed with a stereo dissecting microscope and appropriate regions were extracted with a microsurgical scalpel and glued to the tips of plastic sectioning stubs. Semi-thin (150-200 nm) serial sections were cut with a UC6 ultramicrotome (Leica Microsystems) using a diamond knife (Diatome, Ltd. Switzerland). Sections were placed on formvar-coated copper-rhodium slot grids (Electron Microscopy Sciences) and stained with 3 % uranyl acetate and lead citrate. Gold beads (10 nm) were placed on both surfaces of the grid to serve as fiducial markers for subsequent image alignment. Sections were placed in a dual-axis tomography holder (Model 2040, E.A. Fischione Instruments, Export PA) and imaged with a Tecnai T12-G2 transmission electron microscope operating at 120 KeV (ThermoFisher Scientific) equipped with a 2k x 2k CCD camera (XP1000; Gatan, Inc. Pleasanton CA). Tomographic tilt-series and large-area montaged overviews were acquired automatically using the SerialEM software package (Mastronarde, 2005, 2008; Mastronarde and Held, 2017). For tomography, samples were tilted +/− 62° and images collected at 1° intervals. The grid was then rotated 90° and a similar series taken about the orthogonal axis. Tomographic data was calculated, analyzed, and modeled using the IMOD software package (Mastronarde, 2005, 2008; Mastronarde and Held, 2017) on iMac Pro and MacPro computers (Apple, Inc., Cupertino, CA). Montaged projection overviews were used to illustrate spatial perspective, identify cell types and frequency within the tissue sections. High-resolution 3D electron tomography was used to confirm virus particles and characterize virus-containing compartments within infected cells.

Identification and Characterization of SARS-CoV-2 Virions in infected cells and tissues

Particles resembling virions were examined in 3D by tomography to determine their identity. Presumptive SARS-CoV-2 virions were identified from tomographic reconstructions of tissue samples by observing structures resembling virions described in cryo-electron tomography studies of purified SARS-CoV-2 and of SARS-CoV-2 in infected cells (Ke et al., 2020; Klein et al., 2020; Turonova et al., 2020; Yao et al., 2020). These were compared to identified virions within SARS-CoV-2–infected cultured Vero E6 cells that had been prepared for EM by the same methodology (Figure 2O-Q). We used the following criteria to positively identify SARS-CoV-2 virions in tissues: (i) Structures that were spherical in 3D with ∼60-120 nM diameters and were not continuous with other adjacent structures, (ii) Spherical structures with densities corresponding to a distinct membrane bilayer, internal puncta consistent with ribonucleoproteins (Yao et al., 2020), and densities corresponding to surface spikes on the external peripheries of the spheres. In further characterization of virions, we noted that the inner vesicles of multivesicular bodies (MVBs) have been mis-identified as SARS-CoV-2 by electron microscopy (Calomeni et al., 2020). We therefore compared measurements of MVB inner vesicles and presumptive coronavirus virions from what we identified as intracellular exit compartments within the same tomogram (data not shown) with our previous tomographic reconstructions of MVBs (He et al., 2008; Ladinsky et al., 2012). We distinguished virions inside of cytoplasmic exit compartments from the inner vesicles of MVBs based on differences in size (MVB inner virions are generally smaller in diameter than coronaviruses) and the presence of surface spikes and internal puncta (MVB inner vesicles do not present surface spikes or internal puncta).

Immunoelectron microscopy

SARS-CoV-2 infected tissues were extracted and immediately fixed with 4% paraformaldehyde, 5% sucrose in 0.1M cacodylate buffer. Tissues were cut into ∼0.5 mm3 pieces and infiltrated into 2.1M sucrose in 0.1M cacodylate buffer for 24 h. Individual tissue pieces were placed onto aluminum cryosectioning stubs (Ted Pella, Inc.) and rapidly frozen in liquid nitrogen. Thin (100 nm) cryosections were cut with a UC6/FC6 cryoultramicrotome (Leica Microsystems) using a cryo-diamond knife (Diatome, Ltd., Switzerland) at −110°C. Sections were picked up with a wire loop in a drop of 2.3M sucrose in 0.1M cacodylate buffer and transferred to Formvar-coated, carbon-coated, glow-discharged 100-mesh copper/rhodium grids (Electron Microscopy Sciences). Grids were incubated 1 hr with 10% calf serum in PBS to block nonspecific antibody binding, then incubated 2 hrs with anti-S antiserum (Cohen et al., 2021). Mosaic nanoparticles elicit cross-reactive immune responses to zoonotic coronaviruses in mice (Cohen et al., 2021). diluted 1:500 in PBS with 5% calf serum. Grids were rinsed (4x 10’) with PBS then labeled for 2 hrs with 10 nm gold conjugated goat anti-mouse secondary antibody (Ted Pella, Inc.). Grids were again rinsed (4x 10’) with PBS, then 3x with distilled water and negatively stained with 1% uranyl acetate in 1% methylcellulose (Sigma) for 20’. Grids were air-dried in wire loops and imaged as described for ET.

Protein expression and purification

FreeStyle 293F cells (Thermo Fisher Scientific) were grown in FreeStyle 293F medium (Thermo Fisher Scientific) to a density of 1×10⁶ cells/mL at 37°C with 8% CO₂ with regular agitation (150 rpm). Cells were transfected with a plasmid coding for recombinant stabilized SARS-CoV-2 ectodomain (S-6P; obtained from Dr. Jason S. McLellan) or SARS-CoV-2 RBD (Beaudoin-Bussieres et al., 2020) using ExpiFectamine 293 transfection reagent, as directed by the manufacturer (Thermo Fisher Scientific). One-week post-transfection, supernatants were clarified and filtered using a 0.22 µm filter (Thermo Fisher Scientific). The recombinant S-6P was purified by strep-tactin resin (IBA) following by size-exclusion chromatography on Superose 6 10/300 column (GE Healthcare) in 10 mM Tris pH 8.0 and 200 mM NaCl (SEC buffer). RBD was purified by Ni-NTA column (Invitrogen) and gel filtration on Hiload 16/600 Superdex 200pg using the same SEC buffer. Purified proteins were snap-frozen at liquid nitrogen and stored in aliquots at 80°C until further use. Protein purities were confirmed as one single-band on SDS-PAGE.

SARS-CoV-2 Spike ELISA (enzyme-linked immunosorbent assay)

The SARS-CoV-2 Spike ELISA assay used was recently described (Beaudoin-Bussieres et al., 2020; Prevost et al., 2020). Briefly, recombinant SARS-CoV-2 S-6P and RBD proteins (2.5 μg/ml), or bovine serum albumin (BSA) (2.5 μg/ml) as a negative control, were prepared in PBS and were adsorbed to plates (MaxiSorp; Nunc) overnight at 4 °C. Coated wells were subsequently blocked with blocking buffer (Tris-buffered saline [TBS] containing 0.1% Tween20 and 2% BSA) for 1 hour at room temperature. Wells were then washed four times with washing buffer (TBS containing 0.1% Tween20). CV3-1, CV3-25 and CR3022 mAbs (50 ng/ml) were prepared in a diluted solution of blocking buffer (0.1 % BSA) and incubated with the RBD-coated wells for 90 minutes at room temperature. Plates were washed four times with washing buffer followed by incubation with HRP-conjugated anti-IgG secondary Abs (Invitrogen) (diluted in a diluted solution of blocking buffer [0.4% BSA]) for 1 hour at room temperature, followed by four washes. HRP enzyme activity was determined after the addition of a 1:1 mix of Western Lightning oxidizing and luminol reagents (Perkin Elmer Life Sciences). Light emission was measured with a LB941 TriStar luminometer (Berthold Technologies). Signal obtained with BSA was subtracted for each plasma and was then normalized to the signal obtained with CR3022 mAb present in each plate.

Flow cytometry analysis of cell-surface Spike staining

Spike expressors of human coronaviruses SARS-CoV-2, SARS-CoV-1, MERS-CoV, OC43, NL63 and 229E were reported elsewhere (Hoffmann et al., 2020; Hoffmann et al., 2013; Hofmann et al., 2005; Park et al., 2016; Prevost et al., 2020). Expressors of HKU1 Spike and SARS-CoV-2 S2 N-His were purchased from Sino Biological. Using the standard calcium phosphate method, 10 μg of Spike expressor and 2 μg of a green fluorescent protein (GFP) expressor (pIRES2-eGFP) was transfected into 2 × 10⁶ 293T cells. At 48 hours post transfection, 293T cells were stained with CV3-1 and CV3-25 antibodies (5μg/mL), using cross-reactive anti-SARS-CoV-1 Spike CR3022 or mouse anti-His tag (Sigma-Aldrich) as positive controls. Alexa Fluor-647-conjugated goat anti-human IgG (H+L) Abs (Invitrogen) and goat anti-mouse IgG (H+L) Abs (Invitrogen) were used as secondary antibodies. The percentage of transfected cells (GFP+ cells) was determined by gating the living cell population based on the basis of viability dye staining (Aqua Vivid, Invitrogen). Samples were acquired on a LSRII cytometer (BD Biosciences) and data analysis was performed using FlowJo v10 (Tree Star).

Virus capture assay

The SARS-CoV-2 virus capture assay was previously reported (Ding et al., 2020). Briefly, pseudoviral particles were produced by transfecting 2×10⁶ HEK293T cells with pNL4.3 Luc R-E- (3.5 μg), plasmids encoding for SARS-CoV-2 Spike or SARS-CoV-1 Spike (3.5 μg) protein and VSV-G (pSVCMV-IN-VSV-G, 1 μg) using the standard calcium phosphate method. Forty-eight hours later, supernatant-containing virion was collected, and cell debris was removed through centrifugation (1,500 rpm for 10 min). To immobilize antibodies on ELISA plates, white MaxiSorp ELISA plates (Thermo Fisher Scientific) were incubated with 5 μg/ml of antibodies in 100 μl phosphate-buffered saline (PBS) overnight at 4°C. Unbound antibodies were removed by washing the plates twice with PBS. Plates were subsequently blocked with 3% bovine serum albumin (BSA) in PBS for 1 hour at room temperature. After two washes with PBS, 200 μl of virus-containing supernatant was added to the wells. After 4 to 6 hours incubation, supernatants were removed and the wells were washed with PBS 3 times. Virus capture by any given antibody was visualized by adding 1×10⁴ SARS-CoV-2-resistant Cf2Th cells per well in complete DMEM medium. Forty-eight hours post-infection, cells were lysed by the addition of 30 μL of passive lysis buffer (Promega) and three freeze-thaw cycles. An LB941 TriStar luminometer (Berthold Technologies) was used to measure the luciferase activity of each well after the addition of 100 μL of luciferin buffer (15 mM MgSO₄, 15 mM KH₂PO₄ [pH 7.8], 1 mM ATP, and 1 mM dithiothreitol) and 50 μL of 1 mM D-luciferin potassium salt (ThermoFisher Scientific).

Surface plasmon resonance (SPR)

All surface plasma resonance assays were performed on a Biacore 3000 (GE Healthcare) with a running buffer of 10 mM HEPES pH 7.5 and 150 mM NaCl, supplemented with 0.05% Tween 20 at 25°C. The binding affinity and kinetics to the SARS-CoV-2 spike (S) trimer (SARS-CoV-2 S HexaPro [S-6P]) (Hsieh et al., 2020) and SARS-CoV-2 S2 ectodomain (baculovirus produced his-tagged S2(686-1213) from BEI Resources (NR-53799) were evaluated using monovalent CV3-1 and CV3-25 Fab. Fabs were generated by standard papain digestion (Thermo Fisher) and purified by Protein A affinity chromatography and gel filtration. His-tagged SARS-CoV-2 S-6P or SARS-CoV-2 S2 ectodomain was immobilized onto a Ni-NTA sensor chip at a level of ∼1000 and ∼630 RU response units (RUs), respectively. Two-fold serial dilutions of CV3-1 or CV3-25 Fab were injected in a concentration range of 1.56-100 nM over the SARS-CoV-2 S-6P and CV3-25 Fab in a range of 3.125 to 200 to nM over the SARS-CoV-2 S2. After each cycle the Ni-NTA sensor chip was regenerated with a wash step of 0.1 M EDTA and reloaded with 0.1 M nickel sulfate followed by the immobilization of fresh antigens for the next cycle. The binding kinetics of SARS-CoV-2 RBD and CV3-1 were obtained in a format where CV3-1 IgG was immobilized onto a Protein A sensor chip (Cytiva) with ∼300 (RUs) and serial dilutions of SARS-CoV-2 RBD were injected with concentrations ranging from 1.56 to 50 nM. The protein A chip was regenerated with a wash step of 0.1 M glycine pH 2.0 and reloaded with IgG after each cycle.

All sensograms were corrected by subtraction of the corresponding blank channel and the kinetic constant determined using a 1:1 Langmuir model with the BIA evaluation software (GE Healthcare). Goodness of fit of the curve was evaluated by the Chi² value with a value below 3 considered acceptable

Pseudovirus neutralization assay

Target cells were infected with single-round luciferase-expressing lentiviral particles. Briefly, 293T cells were transfected by the calcium phosphate method with the pNL4.3 R-E-Luc plasmid (NIH AIDS Reagent Program) and a plasmid encoding for SARS-CoV-2 Spike at a ratio of 5:4. Two days post-transfection, cell supernatants were harvested and stored at –80°C until use. 293T-ACE2 (Prevost et al., 2020) target cells were seeded at a density of 1 × 10⁴ cells/well in 96-well luminometer-compatible tissue culture plates (Perkin Elmer) 24 h before infection. Recombinant viruses in a final volume of 100 μL were incubated with the indicated semi-log diluted antibody concentrations for 1 h at 37°C and were then added to the target cells followed by incubation for 48 h at 37°C; cells were lysed by the addition of 30 μL of passive lysis buffer (Promega) followed by one freeze-thaw cycle. An LB941 TriStar luminometer (Berthold Technologies) was used to measure the luciferase activity of each well after the addition of 100 μL of luciferin buffer (15 mM MgSO₄, 15 mM KH₂PO₄ [pH 7.8], 1 mM ATP, and 1 mM dithiothreitol) and 50 μL of 1 mM d-luciferin potassium salt. The neutralization half-maximal inhibitory dilution (IC₅₀) represents the plasma dilution to inhibit 50 % of the infection of 293T-ACE2 cells by recombinant viruses bearing the SARS-CoV-2 S glycoproteins.

Microneutralization assay

A microneutralization assay for SARS-CoV-2 serology was performed as previously described (Amanat et al., 2020). Experiments were conducted with the SARS-CoV-2 USA-WA1/2020 virus strain (obtained from BEI resources). One day prior to infection, 2×10⁴ Vero E6 cells were seeded per well of a 96 well flat bottom plate and incubated overnight at 37°C under 5% CO₂ to permit cell adherence. Titrated antibody concentrations were performed in a separate 96 well culture plate using MEM supplemented with penicillin (100 U/mL), streptomycin (100 mg/mL), HEPES, L-Glutamine (0.3 mg/mL), 0.12% sodium bicarbonate, 2% FBS (all from Thermo Fisher Scientific) and 0.24% BSA (EMD Millipore Corporation). In a Biosafety Level 3 laboratory (ImPaKT Facility, Western University), 10³ TCID₅₀/mL of SARS-CoV-2 USA-WA1/2020 live virus was prepared in MEM + 2% FBS and combined with an equivalent volume of respective antibody dilutions for one hour at room temperature. After this incubation, all media was removed from the 96 well plate seeded with Vero E6 cells and virus:antibody mixtures were added to each respective well at a volume corresponding to 600 TCID₅₀ per well and incubated for one hour further at 37°C. Both virus only and media only (MEM + 2% FBS) conditions were included in this assay. All virus:plasma supernatants were removed from wells without disrupting the Vero E6 monolayer. Each antibody concentration (100 µL) was added to its respective Vero E6-seeded well in addition to an equivalent volume of MEM + 2% FBS and was then incubated for 48 hours. Media was then discarded and replaced with 10% formaldehyde for 24 hours to cross-link Vero E6 monolayer. Formaldehyde was removed from wells and subsequently washed with PBS. Cell monolayers were permeabilized for 15 minutes at room temperature with PBS + 0.1% Triton X-100, washed with PBS and then incubated for one hour at room temperature with PBS + 3% non-fat milk. An anti-mouse SARS-CoV-2 nucleocapsid protein (Clone 1C7, Bioss Antibodies) primary antibody solution was prepared at 1 mg/mL in PBS + 1% non-fat milk and added to all wells for one hour at room temperature. Following extensive washing with PBS, an anti-mouse IgG HRP secondary antibody solution was formulated in PBS + 1% non-fat milk. One-hour post-incubation, wells were washed with PBS, SIGMAFAST OPD developing solution (Millipore Sigma) was prepared as per manufacturer’s instructions and added to each well for 12 minutes. Dilute HCl (3.0 M) was added to quench the reaction and the optical density at 490 nm of the culture plates was immediately measured using a Synergy LX multi-mode reader and Gen5 microplate reader and imager software (BioTek).

Cell-to-cell fusion assay

To assess cell-to-cell fusion, 2 × 10⁶ 293T cells were co-transfected with plasmid expressing HIV-1 Tat (1µg) and a plasmid expressing SARS-CoV-2 Spike (4 µg) using the calcium phosphate method. Two days after transfection, Spike-expressing 293T (effector cells) were detached with PBS-EDTA 1mM and incubated for 1 hour with indicated amounts of CV3-1 and/or CV3-25 NAbs at 37°C and 5% CO₂. Subsequently, effector cells (1 × 10⁴) were added to TZM-bl-ACE2 target cells that were seeded at a density of 1 × 10⁴ cells/well in 96-well luminometer-compatible tissue culture plates 24 h before the assay. Cells were co-incubated for 6 h at 37°C and 5% CO₂, after which they were lysed by the addition of 40 μl of passive lysis buffer (Promega) and one freeze-thaw cycles. An LB 941 TriStar luminometer (Berthold Technologies) was used to measure the luciferase activity of each well after the addition of 100 μl of luciferin buffer (15 mM MgSO₄, 15 mM KH₂PO₄ [pH 7.8], 1 mM ATP, and 1 mM dithiothreitol) and 50 μL of 1 mM d-luciferin potassium salt (ThermoFisher Scientific).

Antibody dependent cellular cytotoxicity (ADCC) assay

For evaluation of anti-SARS-CoV-2 ADCC activity, parental CEM.NKr CCR5+ cells were mixed at a 1:1 ratio with CEM.NKr-Spike cells. These cells were stained for viability (AquaVivid; Thermo Fisher Scientific) and a cellular dye (cell proliferation dye eFluor670; Thermo Fisher Scientific) and subsequently used as target cells. Overnight rested PBMCs were stained with another cellular marker (cell proliferation dye eFluor450; Thermo Fisher Scientific) and used as effector cells. Stained effector and target cells were mixed at a 10:1 ratio in 96-well V-bottom plates. Titrated concentrations of CV3-1 and CV3-25 mAbs were added to the appropriate wells. The plates were subsequently centrifuged for 1 min at 300xg, and incubated at 37°C, 5% CO2 for 5 hours before being fixed in a 2% PBS-formaldehyde solution.

ADCC activity was calculated using the formula: [(% of GFP+ cells in Targets plus Effectors)-(% of GFP+ cells in Targets plus Effectors plus antibody)]/(% of GFP+ cells in Targets) x 100 by gating on transduced live target cells. All samples were acquired on an LSRII cytometer (BD Biosciences) and data analysis performed using FlowJo v10 (Tree Star).

Antibody dependent cellular phagocytosis (ADCP) assay

The ADCP assay was performed using CEM.NKr-Spike cells as target cells that were fluorescently labelled with a cellular dye (cell proliferation dye eFluor450). THP-1 cells were used as effector cells and were stained with another cellular dye (cell proliferation dye eFluor670). Stained target and effector cells were mixed at a 5:1 ratio in 96-well U-bottom plates. Titrated concentrations of CV3-1 and CV3-25 mAbs were added to the appropriate wells. After an overnight incubation at 37 °C and 5% CO₂, cells were fixed with a 2% PBS-formaldehyde solution. Antibody-mediated phagocytosis was determined by flow cytometry, gating on THP-1 cells that were double-positive for efluor450 and efluor670 cellular dyes. All samples were acquired on an LSRII cytometer (BD Biosciences) and data analysis performed using FlowJo v10 (Tree Star).

smFRET imaging of S on SARS-CoV-2 VLPs (S-MEN particles)

S-MEN coronavirus-like particles carrying SARS-CoV-2 spikes were prepared similarly as previously described (Lu et al., 2020). The peptides tags-carrying spike plasmid (pCMV-S Q3-1 A4-1: Q3 - GQQQLG; A4 - DSLDMLEM) was used to make S-MEN coronavirus-like particles. Plasmids encoding wildtype pCMV-S, dual-tagged pCMV-S Q3-1 A4-1, pLVX-M, pLVX-E, and pLVX-N were transfected into 293T cells at a ratio of 20:1:21:21:21. Using this very diluted ratio of tagged-S vs. wildtype S, the vast majority of S-MEN particles carry wildtype spikes. For the rest of the virus particles containing tagged S, more than 95 % S trimers will have one dual-tagged protomer and two wildtype protomers within a trimer. Using this strategy, we generated S-MEN particles with an average of one dual-tagged S protomer for conjugating FRET-paired fluorophores among predominantly wildtype S trimers presented on VLP surface. S-MEN particles were harvested 40 h post-transfection, filtered with a 0.45 µm pore size filter, and partially purified using ultra-centrifugation at 25,000 rpm for 2 h through a 15 % sucrose cushion made in PBS. S-MEN particles were then re-suspended in 50 mM pH 7.5 HEPES buffer, labeled with Cy3B(3S) and Cy5 derivative (LD650-CoA) and purified through an optiprep gradient as previously described (Lu et al., 2019; Lu et al., 2020; Munro et al., 2014)

smFRET images of S-MEN particles was acquired on a home-built prism-based total internal reflection fluorescence (TIRF) microscope, as described previously (Lu et al., 2020). smFRET data analysis was performed using MATLAB (MathWorks)-based customized SPARTAN software package (Juette et al., 2016). The conformational effects of 50 ug/ml CV3-1 and CV3-25 antibodies on SARS-CoV-2 spike were tested by pre-incubating fluorescently labeled viruses for 60 mins at 37 °C before imaging in the continued presence of the antibodies. During smFRET imaging, fluorescently-labeled S-MEN particles were monitored for 80 seconds, where fluorescence from Cy3B(3S) and LD650-CoA labeled on S-MEN particles was recorded simultaneously at 25 frames per second for 80 seconds. Donor (Cy3B(3S)) and acceptor (LD650-CoA) fluorescence intensity traces were extracted after subtracting background signals and correcting cross-talks. The energy transfer efficiency (FRET) traces were generated from fluorescence intensity traces, according to FRET= I_A/(γI_D+I_A), where I_D and I_A are the fluorescence intensities of donor and acceptor, respectively, γ is the correlation coefficient compromising the discrepancy in quantum yields and detection efficiencies of two fluorophores. FRET is sensitive to changes in distances between the donor and the acceptor over time, ultimately translating into the conformational profiles and dynamics of S on S-MEN particles. S-MEN particles that contain incomplete FRET-paired fluorophores or more than one FRETing pairs of donor and acceptor on a single virus particle were automatically filtered from virus pools for further analysis. FRET traces of fluorescently-labeled S-MEN particles which meet the criteria of sufficient signal-to-noise ratio and anti-correlated fluctuations in donor and acceptor fluorescence intensity are indicative of live molecules. These FRET traces, indicated the number of traces in Figure 3, were then compiled into FRET histograms in Figure 3. Each FRET histogram was fitted into the sum of four Gaussian distributions in Matlab, where each Gaussian distribution represents one conformation and the area under each Gaussian curve estimates the occupancy.

Quantification and Statistical Analysis

Data were analyzed and plotted using GraphPad Prism software (La Jolla, CA, USA). Statistical significance for pairwise comparisons were derived by applying non-parametric Mann-Whitney test (two-tailed). To obtain statistical significance for survival curves, grouped data were compared by log-rank (Mantel-Cox) test. To obtain statistical significance for grouped data we employed 2-way ANOVA followed by Dunnett’s or Tukey’s multiple comparison tests.

p values lower than 0.05 were considered statistically significant. P values were indicated as ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001; ∗∗∗∗, p < 0.0001.

Schematics

Schematics for showing experimental design in figures were created with BioRender.com.

Acknowledgements

This work was supported by NIH grants P50AI150464 to WM and PJB; R33AI122384 and RO1AI145164 to PK; George Mason University Fast Grants to MSL and PJB; P20GM125498 (awarded to UVM Translational Global Infectious Disease Research Center) to EAB; le Ministère de l’Économie et de l’Innovation du Québec, Programme de soutien aux organismes de recherche et d’innovation, Foundation du CHUM, Canadian Institutes of Health Research (CIHR) foundation grant #352417 & Rapid Research Funding Opportunity #FRN440388 to J.D.D. and G.A.D, Canada Research Chair on Retroviral Entry no. RCHS0235 950-232424 to AF; Canada’s COVID-19 Immunity Task Force (CITF) & Canada Foundation for Innovation (CFI) #41027 to AF and DEK & #36287 to JDD. and GAD; FRQS Merit Research Scholarship to DEK; CIHR fellowships to JP, SPA and GBB, MITACS Accélération postdoctoral fellowship to RG; Fred Hutch COVID-19 Research Fund to LS and ATM.

Footnotes

• $ Lead Author