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bioRxiv · 10.1101/2020.11.13.378257

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

Gottschalk, G.·Keating, J. F.·Kesler, K.·Knox, K.·Roy, A.
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Abstract

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 g/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. HighlightsO_LIACIS 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). C_LIO_LIACIS KEPTIDE completely prevents the entry of SARS-CoV2 in Bronchiolar epithelium (Fig.6), alveolar parenchyma (Fig. 6), and kidney tubular cells (Fig.8). C_LIO_LIACIS KEPTIDE improves the pulmonary (Fig. 5) and renal pathological changes (Fig. 7) caused by the SARS-CoV2 virus insult. C_LIO_LIIntranasal administration of 0.05% Beta-propiolactone ({beta}PL)-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. C_LIO_LIThe 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. C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=122 SRC="FIGDIR/small/378257v1_fig2.gif" ALT="Figure 2"> View larger version (51K): org.highwire.dtl.DTLVardef@15c9911org.highwire.dtl.DTLVardef@453819org.highwire.dtl.DTLVardef@65f8a0org.highwire.dtl.DTLVardef@a602d8_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFig. 2.C_FLOATNO Effect of KEPTIDE on the Expression of ACE-2 Receptor on the Membrane of CALU-3 Human Lung Cells. Human lung epithelial cells. CALU-3 cells were grown in complete DMEM cells for 2 days until it reached 70% confluency followed by starving with serum for 2 hrs. After that, 25 M of ACIS KEPTIDE were treated for 30 mins, 1 hr, 2 hrs and 6 hrs. After each time point cells were fixed and stained for ACE-2 (Green; Rabbit anti-ACE-2 antibody; Abcam; 1:250 dilution) and KEPTIDE (blue). Thirty minutes of KEPTIDE treatment significantly stimulated the internalization of ACE-2 along with KEPTIDE. Subsequent incubation periods displayed significant down-regulation of ACE-2 receptors. Experiments were confirmed after three different experiments. C_FIG O_FIG O_LINKSMALLFIG WIDTH=154 HEIGHT=200 SRC="FIGDIR/small/378257v1_fig6.gif" ALT="Figure 6"> View larger version (98K): org.highwire.dtl.DTLVardef@104bd7forg.highwire.dtl.DTLVardef@34ecc1org.highwire.dtl.DTLVardef@a3ac74org.highwire.dtl.DTLVardef@18f0570_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFig. 6.C_FLOATNO Protective effect of ACIS KEPTIDE on the expression of ACE-2 and the entry of SARS-CoV2 virions in lung of SARS-CoV2-insulted K18-hACE2 mice. (A-D) Dual IHC staining of ACE-2 (red) and SARS-CoV2 (brown) in bronchiolar epithelium in (A) vehicle-treated (0.05% PL-inactivated VEROE6 sup, (B)virus, (C) virus + KEPTIDE, and (D) virus + peptide-treated K18-hACE2 mice (n= 7-8). (a-h) Magnified views of outer layers and inner layers of bronchiolar epithelia of respective images enclosed in a dotted squares. Arrows were justified in the bottom of each image. Results are confirmed after three independent experiments. C_FIG O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/378257v1_fig8.gif" ALT="Figure 8"> View larger version (87K): org.highwire.dtl.DTLVardef@ae105aorg.highwire.dtl.DTLVardef@1b39472org.highwire.dtl.DTLVardef@d6e3e9org.highwire.dtl.DTLVardef@ce6d6_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFig. 8.C_FLOATNO Protective effect of ACIS KEPTIDE on the expression of ACE-2 and prevention of SARS-CoV2 entry in 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 g/kg Bwt KEPTIDE or 50 g/kg Bwt peptide for 30 mins followed by inoculation with PL-inactivated 1*106 virus. In group1 mice (n=8) were inoculated with vehicle only (0.05% PL-treated VEROE6 sup); in group 2, mice (n=7) were treated with virus only (0.05% PL-inactivated; in group 3, mice (n=8) were treated with virus +KEPTIDE; and, in group 4 mice (n=8) were treated with virus + PEPTIDE. (Results are confirmed after three independent experiments. (A-D) Dual IHC of ACE-2 (red) and SARS-CoV2 (brown) in tubular epithelium in (A) vehicle, (B) Virus, (C) Virus + KEPTIDE-, and (D) Virus + peptide-treated groups. (a) showed magnified view of kidney cortex vehicle-treated mouse with basal expression of ACE-2 (red), (b) magnified view of kidney of virus-tread animal. Upscaled expression of ACE-2(red) with degenerated Bowmans capsule and invasion of SARS CoV2 (Brown arrow). (c) Magnified view of Glomerulus of Virus + KEPTIDE-treated group. Significantly less ACE-2 expression (red arrow) and no virus-infiltration were noted. (d) Elevated expression of ACE-2 in virus +Peptide-treated group. Results were confirmed after three different experiments in 7-8 animals. C_FIG O_FIG O_LINKSMALLFIG WIDTH=190 HEIGHT=200 SRC="FIGDIR/small/378257v1_fig5.gif" ALT="Figure 5"> View larger version (123K): org.highwire.dtl.DTLVardef@1072b3aorg.highwire.dtl.DTLVardef@1a70702org.highwire.dtl.DTLVardef@bd227corg.highwire.dtl.DTLVardef@12335bd_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFig. 5.C_FLOATNO ACIS KEPTIDE protects acute histopathological changes in Lungs 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 g/kg Bwt KEPTIDE or 50 g /kg Bwt peptide for 30 mins followed by inoculation with PL-inactivated 1*106 virus. In group1 mice (n=8) were inoculated with vehicle only (0.05% PL-treated media); in group 2, mice (n=7 were treated with virus only (0.05% PL-inactivated; in group 3, mice (n=8) were treated with virus +KEPTIDE; and, in group 4 mice (n=8) were treated with virus + PEPTIDE. (A-D) Hematoxylin Background staining of small airway alveolar parenchyma. (E-H) H & E staining of Bronchiolar epithelium, surrounding cartilaginous and alveolar parenchyma. Magnified views of bronchiolar epithelia of (Ei) Control (orange arrow indicates intact epithelial lining: orange star demonstrates preserved connective tissue), (Fii) Virus only (thin red arrow indicates the degenerated bronchiolar epithelium), (Giii) Virus + KEPTIDE (thin blue arrow indicates protected epithelium), and (Hiv) Virus + Peptide-treated (thin green arrow indicates degenerated epithelium). Results are confirmed after three different experiments in 7-8 animals. C_FIG O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=179 SRC="FIGDIR/small/378257v1_fig7.gif" ALT="Figure 7"> View larger version (150K): org.highwire.dtl.DTLVardef@9d3c17org.highwire.dtl.DTLVardef@d40a51org.highwire.dtl.DTLVardef@f17652org.highwire.dtl.DTLVardef@8f3230_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFig. 7.C_FLOATNO Effect of ACIS KEPTIDE on the protection of acute histopathological changes in 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 g/kg Bwt KEPTIDE or 50 g/kg Bwt peptide for 30 mins followed by inoculation with PL-inactivated 1*106 virus. In group1 mice (n=8) were inoculated with vehicle only (0.05% PL-treated VEROE6 sup); in group 2, mice (n=7) were treated with virus only (0.05% PL-inactivated; in group 3, mice (n=8) were treated with virus +KEPTIDE; and, in group 4 mice (n=8) were treated with virus + PEPTIDE. (A-D) H & E staining of kidney cortex with detailed structures of glomeruli, tubular epithelium, and Bowmans capsule in all 4 groups. Results are confirmed after three independent experiments. C_FIG

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Gottschalk, G., Keating, J. F., Kesler, K., Knox, K., Roy, A.. 2020-11-16. 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. https://doi.org/10.1101/2020.11.13.378257

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