key: cord-0946286-kb7eua2y
authors: Gottschalk, Gunnar; Keating, James F; Kesler, Kris; Knox, Konstance; Roy, Avik
title: Intranasal Administration of ACIS KEPTIDE™ Prevents SARS-CoV2-Induced Acute Toxicity in K18-hACE2 Humanized Mouse Model of COVID-19: A Mechanistic Insight for the Prophylactic Role of KEPTIDE™ in COVID-19
date: 2020-11-16
journal: bioRxiv
DOI: 10.1101/2020.11.13.378257
sha: 32777b0f10b95fec0ab0d4721c461ae3c445efe7
doc_id: 946286
cord_uid: kb7eua2y

Previously, we have demonstrated that ACIS KEPTIDE™, a chemically modified peptide, selectively binds to ACE-2 receptor and prevents the entry of SARS-CoV2 virions in vitro in primate kidney Cells. However, it is not known if ACIS KEPTIDE™ attenuates the entry of SARS-CoV2 virus in vivo in lung and kidney tissues, protects health, and prevent death once applied through intranasal route. In our current manuscript, we demonstrated that the intranasal administration of SARS-CoV2 (1*106) strongly induced the expression of ACE-2, promoted the entry of virions into the lung and kidney cells, caused acute histopathological toxicities, and mortality (28%). Interestingly, thirty-minutes of pre-treatment with 50 μg/Kg Body weight ACIS normalized the expression of ACE-2 via receptor internalization, strongly mitigated that viral entry, and prevented mortality suggesting its prospect as a prophylactic therapy in the treatment of COVID-19. On the contrary, the peptide backbone of ACIS was unable to normalize the expression of ACE-2, failed to improve the health vital signs and histopathological abnormalities. In summary, our results suggest that ACIS is a potential vaccine-alternative, prophylactic agent that prevents entry of SARS-CoV2 in vivo, significantly improves respiratory health and also dramatically prevents acute mortality in K18-hACE2 humanized mice. Highlights ACIS KEPTIDE stimulates the internalization of ACE-2 receptor (Fig. 2) and buffers the membrane localization of ACE-2 receptors (Fig. 2, 6 & 8). Intranasal inoculation of SARS-CoV2 upregulates the expression of ACE-2 in lung epithelium (Fig.6) and kidney tubular cells (Fig.8). ACIS KEPTIDE normalizes the expression of ACE-2 in the kidney tubular cells of virus-treated K18-hACE2mice (Fig. 8). ACIS KEPTIDE™ completely prevents the entry of SARS-CoV2 in Bronchiolar epithelium (Fig.6), alveolar parenchyma (Fig. 6), and kidney tubular cells (Fig.8). ACIS KEPTIDE™ improves the pulmonary (Fig. 5) and renal pathological changes (Fig. 7) caused by the SARS-CoV2 virus insult. Intranasal administration of 0.05% Beta-propiolactone (βPL)-inactivated SARS-CoV2 (1 *106) causes significant death (28%) in K18-hACE2 humanized mice after 24 hrs of intranasal inoculation (Supplemental videos) suggesting that SARS-CoV2 does not require its infective properties and genetic mechanism to be functional to cause mortality. The peptide backbone of ACIS KEPTIDE™ provides much less and insignificant protection in the prevention of pathological changes in Lungs (Fig.5 & 6) and Kidney (Fig.7 & 8). Peptide failed to normalize the upscaled expression of ACE-2 in kidney tubular cells (Fig.8) of SARS-CoV2-treated K18-hACE2 mice.

• ACIS KEPTIDE stimulates the internalization of ACE-2 receptor (Fig. 2) and buffers the membrane localization of ACE-2 receptors (Fig. 2, 6 

CoV2 upregulates the expression of ACE-2 in lung epithelium (Fig.6 ) and kidney tubular cells (Fig.8) . ACIS KEPTIDE normalizes the expression of ACE-2 in the kidney tubular cells of virus-treated K18-hACE2mice (Fig. 8 ).

• ACIS KEPTIDE TM completely prevents the entry of SARS-CoV2 in Bronchiolar epithelium ( Fig.6 ), alveolar parenchyma (Fig. 6) , and kidney tubular cells (Fig.8 ).

• ACIS KEPTIDE TM improves the pulmonary ( • Intranasal administration of 0.05% Beta-propiolactone (βPL)-inactivated SARS-CoV2 (1 *10 6 ) causes significant death (28%) in K18-hACE2 humanized mice after 24 hrs of intranasal inoculation (Supplemental videos) suggesting that SARS-CoV2 does not require its infective properties and genetic mechanism to be functional to cause mortality.

• The peptide backbone of ACIS KEPTIDE TM provides much less and insignificant protection in the prevention of pathological changes in Lungs (Fig.5 & 6) and Kidney (Fig.7 & 8) . Peptide failed to normalize the upscaled expression of ACE-2 in kidney tubular cells (Fig.8 ) of SARS-CoV2-treated K18-hACE2 mice.

Abstract: Previously, we have demonstrated that ACIS KEPTIDE TM, a chemically modified peptide, selectively binds to ACE-2 receptor and prevents the entry of SARS-CoV2 virions in vitro in primate kidney Cells. However, it is not known if ACIS KEPTIDE TM attenuates the entry of SARS-CoV2 virus in vivo in lung and kidney tissues, protects health, and prevent death once applied through intranasal route. In our current manuscript, we demonstrated that the intranasal administration of SARS-CoV2 (1*10 6 ) strongly induced the expression of ACE-2, promoted the entry of virions into the lung and kidney cells, caused acute histopathological toxicities, and mortality (28%). Interestingly, thirty-minutes of pre-treatment with 50 μg/Kg Body weight ACIS normalized the expression of ACE-2 via receptor internalization, strongly mitigated that viral entry, and prevented mortality suggesting its prospect as a prophylactic therapy in the treatment of COVID-19. On the contrary, the peptide backbone of ACIS was unable to normalize the expression of ACE-2, failed to improve the health vital signs and histopathological abnormalities.

In summary, our results suggest that ACIS is a potential vaccine-alternative, prophylactic agent that prevents entry of SARS-CoV2 in vivo, significantly improves respiratory health and also dramatically prevents acute mortality in K18-hACE2 humanized mice.

Introduction: COVID-19, a severe acute respiratory disease [1, 2] , is primarily caused by a viral strain named as Coronavirus-2 (SARS-CoV2) [3] . As an entry mechanism of the virus into host cell, A recent study [4] has demonstrated that SARS-CoV2 employs its membrane-bound "Sglycoprotein" to attach with the host receptor ACE-2. Although, other mechanisms such as attachment of virus to the polysaccharide molecule heparan sulphate [5] in the cell membrane also facilitates the infectivity of the virus, until now, the interaction of SARS-CoV2 with ACE-2 protein has been considered as a primary mechanism of viral entry [6] [7] .

In our previous study [8] , we reported that ACIS KEPTIDE TM , a chemically modified peptide with a strong affinity towards ACE-2 receptor, efficiently nullified the interaction between ACE-2 and S-glycoprotein of SARS-CoV2. As a result, the entry of the SARS-CoV2 virions into host cells had been severely impaired. Based on the results derived from different experiments such as cytopathic effect assay, plaque assay and dual immunofluorescence analyses, we concluded that ACIS KEPTIDE TM indeed inhibited the attachment and entry of SARS-CoV2 virions in VEROE6 primate kidney cells. Moreover, our results also demonstrated that the chronic administration of KEPTIDE through intranasal pathway did not cause the loss of body weight, changes in vitals including body temperature, oxygen saturation, and heart rate nullifying any health-related toxicity due to KEPTIDE-treatment. Although that experiment was performed in aged BALB/C mice, understanding the toxicity of KEPTIDE is warranted in better animal model that express human ACE-2 receptor.

Therefore, in our current work, we included 6-8 weeks old K18-hACE2 mice. This strain is a humanized strain that express human ACE-2 receptor under the guidance of K18 promoter. K18-hACE2 mice display an acute lung and kidney injuries resulting significant mortality upon inoculation with SARS-CoV2 [9] [10] [11] . Therefore, to avoid significant mortality in these mice and for the safety of users, we inactivated virus with 0.05% beta-propiolactone (βPL). Although the treatment with 0.05% β-propiolactone has been reported to substantially reduce the infectivity of the virus, the ultrastructure of the external capsid remains unaltered and its antigenicity is also preserved [12, 13] that allows us to perform the entry experiment of SARS-CoV2 in lung tissue.

To explore the effect of ACIS on preventing the entry of virions, we performed an in vivo experiment with the intranasal treatment of ACIS for 30 minutes followed by the intranasal inoculation of SARS-CoV2 for another 24 hrs. Next, we performed an array of experiments in lungs and kidney of these humanized animals to confirm the role of ACIS KEPTIDE in the entry of virions through ACE-2 receptor, the regulation of ACE-2 receptor, amelioration of acute histopathological changes, and mortality.

Exploring toxicity in K18-hACE2 animals after chronic intranasal administration of ACIS KEPTIDE TM . Eight to 10 weeks old K18-hACE2 mice (n=8; 4 males and 4 females) were intranasally administered with 50 μg/Kg Bwt KEPTIDE every day for 10 days as described under method section. Average baseline body weights were 18.25±1.4 gms and 15.65± 2.3 gms for males and females, respectively. Each day animals were recorded for body weight (Fig. 1A) , body temperature ( Fig. 1B) , heart rate ( Fig. 1C) , and oxygen saturation (Fig. 1D ) as described previously [8] . As evident from figure 1A, both male and female mice maintained their body weights throughout the study. On average, there was a decrease in body weight (14.59 ± 2.32) recorded in female mice on day 9 that happened most likely due to the loss of water-weight. However, that drop was not statistically significant while comparing with the baseline value (p>0.05; =0.2015).

On contrary, male mice did not display any loss in body weights. Instead, we observed consistent increase in body weights from day 6. Again that increase was not statistically significant once compared with the baseline value (p>0.05; =0.0762). Daily administration of KEPTIDE did not alter body temperature (Fig. 1B) , heart rate ( Fig. 1C) , and oxygen saturation (Fig. 1D ). There was only partial and non-significant elevation of blood pressure on day 9 (baseline value of 448± 12.5 to 489± 10.7) in all females, otherwise all vital parameters were found to be stable. No mortality was observed across the study in any animal.

K18-hACE2 mice. Since ACIS did not display any mortality and health-related issue in K18-hACE2 mice, next we wanted to study if ACIS could also prevent the death and improve healthassociated toxicities in these mice upon exposure to SARS-CoV2 virions. Wuhan-standard SARS-CoV2 is highly infectious and therefore complete inactivation of virus is required in order to minimize the health risk associated with the administration procedure, perfusion, surgery, postsurgical processing of tissue, and histological analyses. Accordingly, the inactivation was performed with incubating viral stock with 0.05% βPL as described elsewhere [13] and also mentioned under method section. Based on our plaque assay ( Supplementary Fig. 1 ), we determined that 0.05% βPL indeed significantly inactivated the SARS-CoV2 virus.

In order to assess the protective role of ACIS KEPTIDE in the entry of SARS-CoV2 in lung Based on that result, we decided to pre-treat the mice (n=8 per group) with 50 μg/Kg Bwt KEPTIDE for 30 mins followed by the treatment with 1 *10 6 virus for 24 hrs. After another 24 hrs, mice were analyzed for the vital signs of health and mortality. After 24 hrs, we observed following.

First, there was significant mortality (28%; 2 out of 7 animals) in virus-treated animals. Two females (66%; 2 out of 3 females) died (Supplementary video 1). Second, virus-treatment caused significant loss of body temperature (Fig. 3A) . Third, another striking feature is abrupt loss of body weight (Fig.3D) . One male showed 8 gm loss of body weight (26 gm day 0 to 18 gm day post inoculation; tail mark1 male). Otherwise, across the virus-treatment group there was average 3.04 ± 2.54 gms of body weight loss (Fig. 3D) . However, there was no statistical significance (p>0.05) while measuring significance of mean between groups with paired t test as because the sample size changed due to mortality. All virus-treated mice are moribund, with low body temperature ( results suggest that even after significant attenuation of infectivity, βPL-treated SARS-CoV2 virus is still able to cause acute health issues and mortality.

and kidneys of K18-hACE2 mice. Since intranasal inoculation of βPL-inactivated virus caused significant health toxicity and pre-treatment of ACIS KEPTIDE, but not peptide, dramatically prevented the health issues, next we wanted to analyze the histopathological abnormalities in lungs and kidneys in virus-treated animals. We were also interested to analyze if ACIS KEPTIDE prevented these abnormalities. Therefore, we first performed hematoxylin & eosin (H&E) staining in paraffin-embedded lungs and kidney sections of vehicle-, virus-, virus +KEPTIDE, and virus+peptide-treated groups. A detailed analysis in H&E-stained slides demonstrated that intranasal inoculation of βPL-treated virus, but not βPL-treated uninfected VERO sup (vehicle; & Hiv) and pulmonary hemorrhagia.

Is this KEPTIDE-mediated protection of acute toxicity due to the prevention of viral entry in lung epithelium? To address this concern, we performed a dual immunohistochemical analyses of ACE2 and Spike glycoprotein (Fig. 6) . Surprisingly, twenty-four hour treatment with SARS-CoV2 significantly stimulated the expression of ACE-2 in the outer (Fig. 6 Bc) and inner layers (Fig.6 Bd) of bronchiolar epithelium compared to vehicle treatment (Fig, 6Aa & 6 Ab).This upregulation of ACE-2 expression by SARS-CoV2 was unexpected and might facilitate the entry of virions through lung epithelium [14] . Thirty-minutes pretreatment with ACIS KEPTIDE (Fig. 6Ce &  6Cf ), but not peptide ( Fig. 6Dg & 6Dh) , strongly downregulated the expression of ACE-2 and normalized its expression to the basal level. Accordingly, we observed the infiltration of SARS-CoV2 in the bronchiolar parenchyma (brown arrow) in virus-and virus + peptide-treated groups, whereas KETIDE treatment significantly protected the entry of virions (indicated with brown arrow). The result was corroborated with the quantification of numbers of ACE-2-immunoreactive (ir) cells in bronchiolar epithelium in all four groups (Supplementary Fig. 2A) . To further evaluate if these brown signals were not artifacts, we confirmed the size of these particles in reference to eosin-stained nuclei. However, these viral particles were inactivated and therefore lost the proliferative properties. As a result, the load of virions was not overwhelming in lung parenchyma.

According to a recent report [15] , tubular cells of Kidney strongly express ACE-2 receptors. Next, we explored pre-treatment of ACIS was able to nullify the entry of SARS-CoV2 virions in renal tubular cells. We observed that SARS-CoV2 ACIS (Fig. 7A ), but not vehicle ( (Fig. 8C ), but not peptide (Fig. 8D) , normalized the expression of ACE-2 ( Supplementary Fig. 2B ). In addition to that, we also observed that pre-treatment with KEPTIDE significantly inhibited the entry of SARS-CoV2-promoted entry of virions in kidney tubular cells and also through Bowman's capsule of glomerulus.

Kidney epithelial cells. Bronchiolar epithelium is composed of tightly attached epithelial cells that are known to express wide-range of pattern recognition receptors dedicated to innate immune response for providing defense against invading microbes [16] . Apart from immune protection against microbes, these epithelial cells also strongly express ACE-2 [17] , which is known for its vasodilative action. Recent studies indicate that ACE-2 is a selective receptor for the entry of SARS-CoV2 virions in lung epithelial cells. Upon binding to ACE-2, SARS-CoV2 virus gets internalized to the cytosol of the epithelial cell to initiate the early pathogenic events [17, 18] .

ACIS KEPTIDE was designed to selectively inhibit the binding of S-glycoprotein of SARS-CoV2 with ACE2 and subsequently block the entry of SARS-CoV2 through ACE-2 receptor [8] .

However, the effect of ACIS on the regulation of ACE-2 receptor is not known. In our present manuscript, we explored the effect of ACIS on the expression and turn-over of ACE-2 receptors in lung and kidney epithelial cells. Combing all results, we confirmed that ACIS is a selective ACE-2 inhibitor that binds, internalizes, and balances the expression of ACE-2 preventing the entry of SAR-CoV2 in the host cell. Surprisingly, KEPTIDE-treatment prevented the degeneration of these epithelial cells in virusinfected K18-hACE2 mice. We also observed increased vacuolization in kidney cortex with glomerular swelling after twenty-four hours of virus treatment, which is frequently observed in degenerative kidney [19, 20] . ACIS KEPTIDE significantly protected the kidney parenchyma with decreased vacuolization and also protected glomerular swelling.

One most important highlight of this paper is to identify a new mechanism of SARS-CoV2mediated death, which is entirely independent of its infective property. In our current project, we adopted βPL-mediated attenuation of infectivity in virus. This process strongly minimizes the risk of infection in users without compromising the purpose of our experiment, which is to measure the interaction and entry of the virus through ACE-2 receptor. These processes require the preservation of the 3D-structure of surface S-glycoprotein, that was not altered by βPL-treatment.

SARS-CoV2 virions were thoroughly inactivated with 0.05% βPL-treatment. for 18 hrs in 4°C.

After 18 hrs, the residual activity of βPL was neutralized with additional incubation at 37°C for 2 hrs. That process completely attenuated the infective property of virus. In accordance with the previous finding [13] , our CPE experiment demonstrated that after βPL-treatment, virus completely lost its infective property. Interestingly, intranasal inoculation of that inactivated virus (1 *10 6 ) significantly caused mortality in 28% of treated animals (2 out of 7) just after 24 hrs. The death was observed only in female mice. In addition to that, we observed reduced body weight, lowered body temperature, dropped oxygen saturation, and decreased heart rate in surviving animals. This dramatic and unexpected toxic effect after 24 hrs of inoculation with inactivated virus offered a paradigm shift in the molecular action of virus. Bpl-treated Inactivated virus does not have abilities to infect, replicate and spread, even though our results have demonstrated that it can cause substantial toxicity. Hence, our study identifies that the binding and modulation of ACE-2 receptor with spike protein seem to be the most critical step of SARS-CoV2-mediated death and toxicity. Upon binding, SARS-CoV2 might alter the physiological activity of ACE-2 receptor that potentially cause cytotoxicity and death. Our current report suggests that SARS-CoV2 does not require its traditional infective property to cause death.

infected K18-hACE2 mice. Several findings in this paper demonstrated that ACIS KEPTIDE TM displayed much stronger effect than its peptide backbone in terms of amelioration of lung and kidney pathologies, improving vital parameters of health, and preventing acute death response. In our previous report [8] , we justified the significance of biochemical modification in the core peptide backbone of ACIS. Our cell-free assays demonstrated that these modifications are critical in order to enhance its binding affinity to ACE-2, physiological longevity, and robust absorption through intranasal path. Combining our previous findings with current results, we conclude that biochemical modifications of ACIS peptide backbone has significantly enhanced its protective effect in preventing SARS-CoV2-mediated toxicities.

Several lines of our paper demonstrated that ACIS KEPTIDE could be a vaccine alternative. First, the pre-treatment with 50 μg/Kg Bwt of ACIS KEPTIDE for thirty minutes significantly protected the acute toxicity by SARS-CoV2. Surprisingly, KEPTIDE treatment not only protected the loss of body weight, temperature, heart rat, and oxygen saturation; but also prevented mortality.

Second, the pre-treatment with the peptide backbone of ACIS KEPTIDE was found to worsen the vital signs of health in SARS-CoV2 infected animals. Although, no mortality was observed in peptide-treated group, 50% mice were moribund with severe motor impairment. Third, our immunohistochemical analyses of lung tissue revealed that the pre-treatment of ACIS KEPTIDE significantly prevented virus-driven histopathological alterations such as swelling of alveolar walls, integrity of bronchiolar epithelial membrane and hemorrhagic response in pulmonary vessels. Fourth, pre-treatment of ACIS KEPTIDE was also observed to protect glomerular integrity with decreased vacuolization in kidney cortices in SARS-CoV2-treated K18-hACE2 mice.

In summary, our current manuscript highlights the preventive role of ACIS KEPTIDE in the protection of acute toxicity and death caused by SARS-CoV2 virus and also demonstrates a novel mode of toxicity of SARS-CoV2, which does not require its genetic mechanism to be functional. hrs, the virus-containing media was harvested, aliquoted, and kept at -80°C. Virus stock was titrated with a routine plaque assay on Vero E6 cells as described previously [8, 21] .

severe respiratory stress and pneumonia if not inactivated. Our aim is to explore if KEPTIDE blocks the entry of virus in lung cells. To perform this entry assay, the virus need not be in its infective state. Additionally, the inactivation also minimizes the health risk of users. Therefore, before virus administration, we performed the inactivation assay. Briefly, viral cells will be treated with 0.05% βPL for 18 hrs in 4°C. After 18 hrs, the cells will be kept at 37°C for 2 hrs for complete hydrolysis of βPL. That process will completely attenuate the infective property of virus and also completely nullifies the risk of handling SARS-CoV2 virus. This protocol has been optimized and reproduced by different scientific groups including KK in her CDC-approved CLEA-certified laboratory. The entire procedure of virus inactivation was performed in BSL-3 laboratory at Coppe laboratories. Coppe Lab has a dedicated Biosafety level 3 laboratory suite with one dedicated hoods, centrifuge, cell culture microscope, hot-water bath, two incubators, and a sink built in a secured containment room. Passage to that lab has entry through two sets of doors from access corridors. A clothing change room (i.e. anteroom) was located in the passageway between the two self-closing doors. Once inactivated, these virus particles (1*10 6 ; stock conc = 2.5*10 7 particles/ mL) were stored at 4°C overnight. On the day of experiment, mice were anesthetized with ketamine (50 mg/kg) and xylazine (5-7 mg/kg) mixture followed by the treatment with 50 μg/Kg Bwt KEPTIDE or peptide through intranasal route. After 30 minutes, we performed inoculation of 1*10 6 virus in the nasal cavity. The virus stock was loaded in a micropipette and then gently disposed in both the nostrils by micropipette. After 24 hrs, animals were recorded for their vital health signs and then perfused with PBS followed by 4% PFA.

The removal of biohazard waste product was performed using proper institutional safety procedures. The perfusion waste was collected in a container that was thoroughly soaked with bleach and half-filled with concentrated bleach solution. For disposal of animal carcass, BSL-3 containment is strictly adhered to with proper bleach sanitization, locked in bleach-soaked biohazard bag, sealed with sealing threads, followed by placing into a potentially infectious material red waste container in the dedicated morgue. Data collection, audiovisual recordings, and statistical analysis. Six to eight weeks old K18-hACE2 mice (n=8; 4 males and 4 females) were included to explore the toxic effect of KEPTIDE TM treatment. Mice were administered intranasally with 50 μg/Kg bwt KEPTIDE every day for 10 days and health vitals were measured as described previously [8] . Animals were tail-marked with marker pen before the treatment. Data of body weight, body temperature, heart rate, and O2 saturation were recorded in the notebook with date. Data were plotted in GraphPad Prism 8 software as XY scatter plots with X axis of "days" and Y axis of health variables. Eight to ten weeks old K18-hACE2 mice were intranasally administered with 50 μg/Kg Bwt KEPTIDE or 50 μg/Kg Bwt peptide for 30 mins followed by inoculation with 1*10 6 inactivated virus. Virus was inactivated with 0.05% β-propiolactone as mentioned under method section. In group1 mice (n=7) were inoculated with virus only; in group 2, mice (n=8) were treated with virus +KEPTIDE; and, in group 3, mice (n=8) were treated with virus +peptide. After 24 hrs, significant mortality ( 2 females of 7 mice) was observed in group 1, but not other groups. (A-C) Body temperature was monitored.

Both virus only-and virus+peptide-treated animals have significantly low body temperature once compared with the baseline body temperature recorded one day before the procedure. (D-F) Body weight was monitored in three groups one day before and after of procedure. No significant change in body weight observed in group1 and 2, however peptide treatment could not protect the loss of body weight due to virus inoculation. Mortality is the possible confounder in group1 while measuring body weight. Results are mean ± SEM of 7-8 animals. Related p value with descriptive t statistics were mentioned below each histogram.

2 Humanized Mice Infected with SARS-CoV2 Virus for 24 hrs. Eight to ten weeks old K18-hACE2 mice were intranasally administered with 50 μg/Kg Bwt KEPTIDE or 50 μg/Kg Bwt peptide for 30 mins followed by inoculation with βPL-inactivated 1*10 6 virus. In group1 mice (n=7) were inoculated with virus only; in group 2, mice (n=8) were treated with virus + KEPTIDE; and, in group 3, mice (n=8) were treated with virus + peptide. (A-C) Body temperature was monitored after 24 hrs. Both virus only-and virus+peptide-treated animals have significantly low heart rate once compared with the baseline heart rate recorded one day before the procedure. (D-F) Oxygen (O2) saturation was monitored in three groups one day before and after of procedure.

Significant change in O2 saturation observed in group1 and 3. Interestingly, KEPTIDE treatment protected the respiratory health even after virus inoculation. Results are mean ± SEM of 7-8 animals. Related p value with descriptive t statistics were mentioned below each histogram. Kidney of K18-hACE2 mice twenty-four hours post SARS-CoV2 inoculation. Eight to ten weeks old K18-hACE2 mice (n=7-8 per group) were intranasally administered with 50 μg/kg Bwt KEPTIDE or 50 μg/kg Bwt peptide for 30 mins followed by inoculation with βPL-inactivated 1*10 6 virus. In group1 mice (n=8) were inoculated with vehicle only (0.05% βPL-treated VEROE6 sup); in group 2, mice (n=7) were treated with virus only (0.05% βPL-inactivated; in group 3, mice inoculation. Eight to ten weeks old K18-hACE2 mice (n=7-8 per group) were intranasally administered with 50 μg/kg Bwt KEPTIDE or 50 μg/kg Bwt peptide for 30 mins followed by inoculation with βPL-inactivated 1*106 virus. In group1 mice (n=8) were inoculated with vehicle only (0.05% βPL-treated VEROE6 sup); in group 2, mice (n=7) were treated with virus only (0.05% βPL-inactivated; in group 3, mice (n=8) were treated with virus +KEPTIDE; and, in group 

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