key: cord-0889208-0u14wypb authors: Brady, Molly; McQuaid, Conor; Solorzano, Alexander; Johnson, Angelique; Combs, Abigail; Venkatraman, Chethana; Rahman, Akib; Leyva, Hannah; Kwok, Wing-Chi Edmund; Wood, Ronald W.; Deane, Rashid title: Spike protein multiorgan tropism suppressed by antibodies targeting SARS-CoV-2 date: 2021-11-22 journal: Commun Biol DOI: 10.1038/s42003-021-02856-x sha: fe67539fef9aa1b1e5519a62d80d9dcb085925ab doc_id: 889208 cord_uid: 0u14wypb While there is SARS-CoV-2 multiorgan tropism in severely infected COVID-19 patients, it’s unclear if this occurs in healthy young individuals. In addition, for antibodies that target the spike protein (SP), it’s unclear if these reduce SARS-CoV-2/SP multiorgan tropism equally. We used fluorescently labeled SP-NIRF to study viral behavior, using an in vivo dynamic imaging system and ex in vivo tissue analysis, in young mice. We found a SP body-wide biodistribution followed by a slow regional elimination, except for the liver, which showed an accumulation. SP uptake was highest for the lungs, and this was followed by kidney, heart and liver, but, unlike the choroid plexus, it was not detected in the brain parenchyma or CSF. Thus, the brain vascular barriers were effective in restricting the entry of SP into brain parenchyma in young healthy mice. While both anti-ACE2 and anti-SP antibodies suppressed SP biodistribution and organ uptake, anti-SP antibody was more effective. By extension, our data support the efficacy of these antibodies on SARS-CoV-2 multiorgan tropism, which could determine COVID-19 organ-specific outcomes. The peak SP-NIRF signals were highest for the neck and upper abdomen, which was 1.3-to 1.9-fold greater than that for the thorax, lower abdomen, and paw, but there was no significant difference between these regions (Fig. 1d) . Although the area under the curve (AUC), the overall regional SP-NIRF exposure, was also similar for the upper abdomen and neck, these were greater by 1.30-to 1.8-fold compared to the thorax, lower abdomen, and paw. However, there was no significant differences between the AUC for the thorax, lower abdomen, and paw (Fig. 1e) . The elimination rate constants were no significant for the neck, thorax, and lower abdomen, which had elimination phases ( Supplementary Fig. 1c ). For the upper abdomen, after its distribution, the rate of SP-NIRF accumulation was determined from the rising phase of the intensity-time profile, using linear regression analysis ( Supplementary Fig. 2d ). SP-NIRF signal was detected in the bladder but this was variable, perhaps, due to changes in urination, as this was not controlled ( Supplementary Fig. 1e ). SP-NIRF plasma and CSF levels were not detectable at 60 min (Fig. 1f) , whereas the ROI signals were higher, suggesting that there was retention within these regions. In summary, these data show that SP-NIRF was widely distributed within the body of young mice, and taken up in many organs, especially in the upper abdominal (mainly liver) and neck (mainly salivary glands) regions. There was an elimination phase for the neck, thorax, and lower abdominal regions, whereas for the upper abdominal region there was accumulation of SP-NIRF after its distribution, instead of elimination. Peak levels were highest for the neck and upper abdominal regions, which may indicate greater uptake even though there was an elimination phase for the neck region but an accumulation phase for the upper abdominal region. SP-NIRF signals in the paw increased exponentially to a plateau and was almost constant thereafter. By extension, these data suggest that there will be differential multiorgan tropism of the virus in young mice, which could lead to organ dysfunction/failure in susceptible individuals, assuming SP mimics the viral distribution, as this is due mainly to SP-host cells interaction. SP uptake suppressed by anti-ACE2 and anti-SP antibodies. ACE2 receptor is widely distributed in the vasculature and in tissues, including the lungs, kidneys, heart, and possibly the cerebrovasculature [10] [11] [12] 14, 19, 32, 39, 40 . SP interaction with ACE2 receptor would indicate potential viral uptake regions and anti-ACE2 antibody will decrease viral-host cell interaction. Anti-SP antibody, as used in passive immunity, generated in active immunity, and produced by vaccines, will interact with the SP on SARS-CoV-2 to eliminate the virus and also reduce viral-host cell interaction. For some of the vaccines, the SP produced by the vaccine will be released from cells and distributed in the body before it can mount an immune response. Anti-SP antibody in the circulation can then interact with the SP on the virus. Thus, we tested the effect of anti-SP antibody and anti-ACE2 on the regional biodistribution, elimination kinetics, and organ uptake of an SP to establish whether there are differential effects. We confirm that the anti-ACE2 antibody interacts with mouse ACE2 (Supplementary Fig. 2a) . Also, we show that SP-555 interacts with mouse and human ACE2 at SP concentrations between 0.01 and 1.0 μg ml −1 (Supplementary Fig. 2b ). At lower SP-555 concentrations there were binding to human ACE2 (higher affinity), whereas there was little binding to mouse ACE2. In our IV studies, we administered 2.05 μg ml −1 of SP-555, and assuming that plasma volume is~1 ml, the plasma SP concentration should bẽ 2.0 μg ml −1 , at the peak, which may contribute to the results. In separate groups of mice, anti-ACE2 antibody (T1, 10 μg) or anti-SP antibody (T2, 10 μg) was injected 15 min prior to the SP-NRF injections. Although the general pattern of the ROIs intensity profile curves was similar, the values were lower than that for the young control mice (Fig. 2a, b, Supplementary Fig. 2b , c, and Fig. 1b) . The peak SP-NIRF signals were significantly higher in the control mice compared to the T1-treated mice, by 1.8-fold for the neck and thoracic regions but not significantly for the upper abdomen, lower abdomen, and the paw (Fig. 2c) . In contrast, for T2 treatment it was higher for the controls by twoto threefold for all ROIs (Fig. 2c) . The AUC was significantly greater in the control mice compared to the T1-or T2-treated mice by 1.8-to 2.5-fold in all the ROIs, except for T1 and the paw (Fig. 2d) . The intensities were transformed to the Ln scale and the elimination rate constants determined from the disappearance phase of the graph (Fig. 2e ). T1 and T2 increased the elimination rate constants for the neck by 2.0-to 2.5-fold but not for the thorax and upper lower abdomen compared to controls (Fig. 2f) . For the upper abdomen, the rate of SP-NIRF accumulation after its distribution was reduced by 5.5-fold with T2 but not by T1 compared to controls (Fig. 2g) . Also, T1 and T2 reduced SP-NIRF distribution (AUC) for the paw (Fig. 2h) . Collectively, these data show that both T1 and T2 reduced the biodistribution to the ROIs, with T2 (anti-SP antibody) more effective, in general. Interestingly, for the neck region (mainly the salivary glands, but also include the pharynx and larynx) T2 was effective in increasing the removal of SP-NIRF (higher elimination rate constant). Similarly, T2 was effective in reducing the accumulation phase of the upper abdomen (mainly the liver). For the paw T2 reduced SP-NIRF peak intensity and the AUC. In contrast, for the thorax and lower abdomen, there are many potential sources of the SP-NIRF signals, including lungs and heart for the thorax, and intestines and kidneys for the lower abdomen. Thus, by extension, therapies targeting ACE2 and the SP, and especially vaccines targeting SP are effective in suppressing the biodistribution of the virus to organs in this model. Ex vivo organ SP distributions. To better understand which organ was associated with the SP-NIRF, at the end of the experiment, these (brain, lungs, liver, kidneys, spleen, intestine, and spine) were removed, washed in cold phosphate-buffered saline (PBS), and imaged using the same parameters as in the in vivo imaging. There were no significant differences in the intensities between the dorsal and ventral surfaces of the brain even though there are more visible surface blood vessels on the ventral surface ( Supplementary Fig. 2d ). As these organs vary in weight, signal intensities were standardized per unit wet weight. SP-NIRF uptake into the lungs, kidney, and heart was 8.0-to 12-fold greater than that of brain ( Supplementary Fig. 2e) . The brain had the lowest SP-NIRF uptake, whereas the lungs had the highest. SP-NIRF signals for the spleen was low, but could contributed to the upper abdominal ROI ( Supplementary Fig. 2f, g) . Likewise, the intestine contributed to the SP-NIRF signal for the lower abdomen. Interestingly, SP-NIRF signal for the small intestine was mainly detected in the duodenum ( Supplementary Fig. 2h ). After 60 min, there was very low SP-NIRF levels for the salivary glands ( Supplementary Fig. 2i ). Other tissues may contribute to signal in the neck region, such as the trachea, tongue, and larynx and pharynx. T1 and T2 reduced the SP-NIRF uptake by the liver, lungs, and kidney by four-to sevenfold, but not for the heart and brain compared to control young mice (Fig. 2i ). T1 and T2 had no significant effect on the SP-NIRF uptake by the brain. These data show that anti-SP antibodies can effectively reduce SP multiorgan biodistribution and, thus, by extension should reduce the virus effects on these regions, in this model. Ex vivo analysis of tissue sections. To establish whether the SP was taken up within the organs we analyzed SP-555 uptake and compared it to a reference protein of similar molecular weight (ovalbumin, OA-488). Sagittal sections of the head show that most of the SP-555 and OA-488 were present in peripheral tissues, with limited entry, if any, into the brain parenchyma ( Fig. 3a ) compared to that of brain sections from non-injected mice ( Supplementary Fig. 3a, b) . However, there were SP and OA signals in the region associated with pituitary gland, which lacks a blood-brain barrier (BBB), and the CP, but not across the cribriform plate (Fig. 3a-c) . Coronal brain sections confirmed the absence of detectible SP-555 signal in brain parenchyma, although there were signals for both SP-555 and OA-488 associated with the CP compared to samples from non-injected mice ( Fig. 3d and Supplementary Figs. 3b and 4a ). There was no significant association of SP with the olfactory bulb, blood vessels, or neurons ( Supplementary Fig. 4b-d) . In contrast, the spinal cord had detectible signals for both SP-555 and OV-488, compared to samples from non-injected mice ( Fig. 3f and Supplementary Figs. 3c and 4e, f), possibly due to a greater permeability of the spinal cord capillaries than that of brain 41 . Thus, the low brain SP-NIRF levels could be due mainly to the CP and possibly areas lacking BBB, such as the pituitary gland. Thus, the central nervous system (CNS) vascular barriers seem to be effective in limiting SP entry into brain parenchyma, in young healthy brains. For the peripheral tissues, there were significant distribution of SP-555 and OA-488 in whole-section analysis for the lungs, liver, kidney, and heart ( However, the level of auto fluorescence was high in these tissues, which range from almost all of the signal (as seen in the heart) to about 40% for 555 nm signals. Thus, in whole section, the analysis of fluorescence level at these wavelengths (555 and 488 nm) may not be as relevant in these studies. However, intensity plots show that there were higher levels locally, such as that associated with the airways (bronchioles), liver lobule, kidney tubules, and heart tissues ( Fig. 4a-d) . The intensity plots of the primary bronchus showed similar distribution as the smaller bronchiole (Fig. 4f) . Collectively, these data indicate that the CNS vascular barriers restrict the entry of SF-555 and OA-488 but not for peripheral tissues (such as lungs, liver, kidneys, and heart). The biodistribution and tissue uptake may be due to ACE2 receptors in these tissues, and to other facilitators, such nonspecific entry mechanisms. Liver maybe involved in the degradation/elimination of proteins, such as SP. Both of these molecules will be filtered and excreted by the kidneys. SP may be reabsorbed by the kidney tubules, including the proximal tubules 24, 25 . SP-555 uptake by the isolated CP. To confirm the in vivo data on SP uptake by the CP, the isolated CP was incubated with SP-555 (50 nM) and the reference molecule (OA-488), in the presence and absence of T1 or T2 for 60 min. Images confirmed that SP-555 was associated with the CP (Fig. 5a-c) . SP-555 location was not always colocalized with OA-488, perhaps due to SP association with epithelial cell and resident leukocytes. Both molecules were present along vessels (Fig. 5a) . In general, a similar pattern was seen for the in vivo studied, even though the CP was too folded to analyze accurately (Fig. 3e) . Intensity plot across the SP-555 (red) spots revealed high intensity signal (Fig. 5d ). T1 reduced the association of SP-555 with the CP, perhaps due to blocking ACE2 receptor-binding sites (Fig. 5e, f) . However, T2 was less effective in reducing SP-555 association with the CP (Fig. 5g-j) . This may be due to IgG/SP-555 binding to Fc receptors on leukocytes of the CP. Collectively, these data show that SP binds to the CP and indicated that T1 altered the SP-555 distribution in the CP (Fig. 5i, j) . Our data show that SP-NIRF, administered intravenously into young adult mice (equivalent to young mature humans), was differentially distributed to multiple organs and eliminated slowly from these regions, except the upper abdominal region (mainly liver), which showed a slow accumulation after its distribution. For the paw, SP-NIRF was shown to exponentially increased to a plateau. At 60 min post injection, regional levels were higher than that of plasma, which was undetected. Brain showed the lowest levels, whereas the lung had the highest levels followed by the kidney, heart, and liver. Anti-SP antibody was more effective in reducing SP-NIRF multiorgan tropism than anti-ACE2, possibly due to its higher affinity for SP. These antibodies had no effects on brain SP-NIRF levels. In contrast, SP-NIRF uptake was significantly reduced with both antibodies for the lung, liver, and kidney. Although there was no significant presence of SP-555 within the brain parenchyma, blood vessels, or neuron, the CP was labeled with the injected SP-555, which may contribute to the signal detected in the isolated whole brain, but there was no detectible signal in CSF. Interestingly, both SP-555 and OA-488 was associated with the spinal cord. Thus, the brain vascular barriers (BBB and CP) restrict the entry of this viral SP into brain parenchyma, at least within 60 min. We used the receptor-binding domain (RBD) of the SP as a surrogate of the virus (SARS-CoV-2), as RBD plays a major role in viral entry into host cells. Also, there was no significant differences in the interaction and effects of different molecular sizes of SP once they contain the RBD 32 . In addition, the transfer constant for two types of SP1 influx into brain was similar 33, 34 . The RBD may have a higher affinity for ACE2 than SP of higher molecular weight 42 . We injected 57 pmol of SP, which was about 34 × 10 12 monomeric particles using Avogadro's number. As there are about 24 SP trimers (72 monomeric forms) on a SARS-CoV-2 virus 43 , an estimated 5 × 10 11 viruses were injected into blood as SP. Intravenous clearance of viruses was studied at (1.6 × 10 9 to 1.6 × 10 11 ) 44 . Thus, we have used an optimum condition to study SP biodistribution, organ uptake, and elimination. SARS-CoV-2. SARS-CoV-2 consists of a viral lipid envelop, which encloses the nucleocapsid bound RNA 45 . The lipid membrane has structural proteins, including the SP, which forms a heterodimer (S1-S2) that is assembled as a trimer protruding from the viral surface that gives the crown-like appearance 10, 45 . The S1 unit contains a RBD, which promotes attachment to the extracellular peptidase domain on ACE2 receptors on the host cell plasma membranes 10, 42, 43 . TMPRESS 2 (transmembrane protease, serine 2) on the host cells cleaves the SP to facilitate viral entry to cells 14, 46 . There are other receptors/facilitators on the cell surface that mediate the entry of SARS-CoV-2, including sialic acid 47 and CD147 48 . SARS-CoV-2 enters the body mainly during inhalation. In severe COVID-19 cases, the infection is manifested as cardiopulmonary symptoms 1, 4, 6, 49, 50 . However, clinical manifestations of COVID-19 have revealed multiple organs are affected 51 , include the heart 21-23 , kidneys 24, 25 , liver [26] [27] [28] [29] , intestine 27,28,52 , and brain 3, 30, 31, 34, 35, 37, [53] [54] [55] [56] . The distribution of SARS-CoV-2 RNA in autopsy tissue samples from severely infected COVID-19 patients shows evidence of multiorgan tropism 51, 56 . ACE2 is widely expressed on the cell membranes, including the gastrointestinal tract, kidneys, CP, heart, lungs, oral mucosa, and bladder 10, 11, [13] [14] [15] 39, 57 . In the brain, ACE2 mRNA is present in the cortex, striatum, hippocampus, and brain stem [16] [17] [18] . It is mainly expressed in brain regions associated with regulation of the cardiovascular system, blood pressure, and the autonomic nervous system 19 . Brain endothelium expresses ACE2 as the protein 32 and as the RNA-sequencing 11, 58 , and the SARS-CoV-2 protease cathepsin B 40 . ACE2 is expressed on pericytes 12 . The wide expression of ACE2 receptor suggests that many organs will be affected by SARS-CoV-2. SP biodistribution from the blood to brain. The mammalian CNS is unique in that it is enclosed within its vascular barriers and has CSF continuously circulating around it [59] [60] [61] [62] . The vascular barriers at the blood-brain interface (the BBB) and blood-CSF interface (blood-CSF barrier; the CPs) restrict the diffusion of polar molecules and large molecules into and out of the brain [59] [60] [61] [62] . The physical sites of the BBB and blood-CSF barrier are the tight junctions between endothelial cells and epithelial cells, respectively 63,64 , which restrict paracellular diffusion. The endothelium of the cerebrovasculature is at the interface between the blood and brain 59, 60 , and SP needs to interact with the luminal surface of the endothelium to enter brain from blood. CSF is mainly produced within the cerebral ventricles by the CPs and circulate from the lateral ventricles to the subarachnoid space, around the brain, spinal cord, and within the spinal canal, before ultimately draining at multiple outflow sites into blood 61, 62, [65] [66] [67] [68] . A major CSF drainage pathway is via the olfactory bulb, across the cribriform plate and towards the cervical lymphatic nodes, and the spinal cord 38, 61, 62 . The endothelium of the CP is fenestrated, and allows entry of proteins into the stroma, which is restricted from entering CSF due to the tight junctions between the epithelial cells at the apical surfaces 59, 61, 62 . Following intravenous SP-NIRF injection, it will be distributed to all organs via blood, and uptake determined by the degree of restriction offered by the endothelium of the vasculature 69, 70 . Continuous endothelium of the CNS cerebrovasculature offers the greatest restriction to protein flux, except at the CP 59-62 . Thus, both SP-555 and OA-488 will cross the fenestrated endothelium of the CP, but not the epithelium layer, which has tight junctions between these cells at the apical side 61 , as seen in our data. Although the CP was labeled with SP-555, there was no detection of SP-555 in CSF, as the epithelium, likely, restricts its entry. However, there was SP distribution to the spinal cord, which may reflect the greater permeability of the blood-spinal barrier to large (albumin) and small (inulin) molecules compared to brain 41 . Thus, the brain vascular barriers at the BBB and CP seem to restrict the entry of SP into brain and CSF from blood. There is a report that SP interacts with the brain endothelial cells using in vitro models of the BBB 32 . In addition, there is a report that SP enters the murine brain from blood 33, 34 . Similar to this report, we found that the SP-NIRF brain levels were not dependent on ACE2. The time point for these reports and our study was similar. In our study, the low levels of SP-NIRF in brain is likely associated with the CP. Thus, our data support the finding that CP is a potential target of SARS-COV-2. SARS-CoV-2 was shown to be associated with the CP, which can damage the epithelium 71 , and SARS-CoV-2 transcripts were associated with the CP 54 . We found that SP-555 also binds to the apical surface of the CP using the isolated CP from mice, which may also limit SARS-CoV-2 in CSF. The highly convoluted apical surface of the CP and its folding make it difficult to analyze tracer distribution. However, there are spots of high intensity SP-555 not associated with OA-488. In addition, there are reports both for the presence and absence of SARS-CoV-2 in the CSF 5, 53, 72, 73 . Further work is needed. The exact mechanism of SARS-CoV-2 neuroinvasion is unclear. Viruses could enter the brain by retrograde transport via sensory nerve endings within the nasal and buccal cavities [74] [75] [76] [77] , and possibility via the gastrointestinal tract 53, 78 . It is possible that SARS-CoV-2 enters the brain as a consequence of pneumonia-induced hypoxia. However, there are reports of encephalitis, which was not due to COVID-19-induced hypoxia 3,79,80 . The virus was detected in frontal lobe brain tissue, which may suggest access via the nasal route 81 . There are also reports of the presence of SARS-CoV-2 in autopsy brain samples and especially in the olfactory mucosal region of severely infected patients [75] [76] [77] 82 . However, this is not always the case, as SARS-CoV-2 RNA was not detected in the nasopharyngeal swab of a patient but was detected in CSF 5 . In a COVID-19 patient with meningitis the virus RNA was detected in the CSF 3 . Viral particles are associated with the cerebrovasculature and the endothelium of other organs [83] [84] [85] [86] [87] . However, it is unclear whether these clinical neurological presentations seen in severe COVID-19 patients are due to the virus entering brain or as a consequence of cardio-respiratory, multiorgan failure, and systemic inflammation. It is possible that the viral invasion of the lungs and the subsequent inflammation will dominate and drive the outcomes of the infection. Thus, further studies are needed to support significant neuroinvasion as the explanation for the neurological symptoms associated with COVID-19. SP biodistribution from the blood to peripheral organs. We performed in vivo dynamic imaging in regions (ROIs) that were known to be affected by COVID-19. Although these ROIs are representing multiple organs/tissues, in some of them signals were associated with a predominant organ. These included the neck region where the SP-NIRF signal was predominantly from the salivary gland and the upper abdomen where it was from the liver. For the thoracic ROI, the NIRF signal was most likely from the lungs and heart, whereas the signals from the lower abdomen are from mainly the intestine and kidneys. Signals from the paw are due to the hairless skin. There is a continuous endothelium with tight junction between endothelial cells for the lung which should limit protein flux 88-90 . However, unlike the brain, the lungs had the highest SP-NIRF uptake from blood. This may be due to the heterogeneity of the endothelial cells and SP-NIRF binding to the glycocalyx and possibly ACE2 receptor 70, [88] [89] [90] . For the liver, the sinusoidal endothelium and Kupffer cells clear viruses and proteins 44, [91] [92] [93] [94] . ACE2 is expressed on many parts of the nephron, including the luminal brush border of the proximal tubule [95] [96] [97] [98] . Thus, the kidney likely takes up a significant level of SP. Also, ACE2 is expressed in the heart [21] [22] [23] 97, 99 . Our findings that SP is distributed to the salivary glands may support suggestions that these glands are a potential reservoir of SAR-CoV-2, which may lead to parotitis-like syndrome 100, 101 . Interestingly, within the gastrointestinal tract, blood-borne SP-NIRF was mostly located at the duodenum 27, 28, 52, 102, 103 . The intestine expresses ACE2 and this has been suggested to contribute to COVID-19-related gastrointestinal tract effects 27, 28, 52, 102 . Our data confirm multiorgan tropism seen in clinical manifestations of COVID-19, with the exception of brain in young healthy mice. Role of anti-ACE2 and anti-SP antibodies in SP biodistribution. Although the antibodies were injected 15 min prior to the tracer injection, they will remain in the circulation for a longer time as the half-life is very long, about 18-20 hrs 104 . In mice, many organs are involved in IgG clearance, including the liver, kidney, muscle, skin, and spleen 104, 105 . The rationale for the current study is that for the anti-ACE2 antibody, it will likely interact with ACE2 receptor and reduce SP binding to the host cells 106 . In contrast, anti-SP antibody will interact with the SP and reduce the free SP levels, which in turn will reduce SP binding to tissues. Our data show that the anti-SP antibody was effective in reducing SP biodistribution and uptake into peripheral organs 107 . In contrast, there was no effect of these antibodies on brain SP-NIRF uptake. In addition, although mouse ACE2 may not bind SP as efficiently as humanized ACE2 [108] [109] [110] , at the higher SP concentrations it seems to interact with mouse ACE2. This is a likely explanation for anti-ACE2 antibody less effectiveness in mice. It is possible that IgG-SP could bind to Fc receptors on cells, especially leukocytes. The CP has resident immune cells, including macrophages and CD4 cells 111, 112 , which could have a greater effect on SP-NIRF uptake by the isolated CP, which may not be seen in vivo due to clearance by the circulation and immune cells. Limitations of the present study. We used the RBD of the SP as a surrogate of the virus (SARS-CoV-2) to study its biodistribution and elimination, as RBD plays a major role in the viral entry into host cells. However, the distribution pattern might be different for the actual virus and the molecular weight of SP would influence filtration at the kidneys and convective flow into peripheral organs, such as muscles. Thus, a lower molecular weight RBD (