key: cord-0787779-x9lbmuk4
authors: Amruta, Narayanappa; Engler-Chiurazzi, Elizabeth B.; Murray-Brown, Isabel C.; Gressett, Timothy E.; Biose, Ifechukwude J.; Chastain, Wesley H.; Bix, Gregory
title: In-vivo Protection from SARS-CoV-2 infection by ATN-161 in k18-hACE2 transgenic mice
date: 2021-05-09
journal: bioRxiv
DOI: 10.1101/2021.05.08.443275
sha: 2df4efc47e36fb2ecd819d14974fcef9cfc5abd8
doc_id: 787779
cord_uid: x9lbmuk4

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an infectious disease that has spread worldwide. Current treatments are limited in both availability and efficacy, such that improving our understanding of the factors that facilitate infection is urgently needed to more effectively treat infected individuals and to curb the pandemic. We and others have previously demonstrated the significance of interactions between the SARS-CoV-2 spike protein, integrin α5β1, and human ACE2 to facilitate viral entry into host cells in vitro. We previously found that inhibition of integrin α5β1 by the clinically validated small peptide ATN-161 inhibits these spike protein interactions and cell infection in vitro. In continuation with our previous findings, here we have further evaluated the therapeutic potential of ATN-161 on SARS-CoV-2 infection in k18-hACE2 transgenic (SARS-CoV-2 susceptible) mice in vivo. We discovered that treatment with single- or repeated intravenous doses of ATN-161 (1 mg/kg) within 48 hours after intranasal inoculation with SARS-CoV-2 lead to a reduction of lung viral load, viral immunofluorescence and improved lung histology in a majority of mice 72 hours post-infection. Furthermore, ATN-161 reduced SARS-CoV-2-induced increased expression of lung integrin α5 and αv (an α5-related integrin that has also been implicated in SARS-CoV-2 interactions) as well as the C–X–C motif chemokine ligand 10 (Cxcl10), further supporting the potential involvement of these integrins, and the anti-inflammatory potential of ATN-161, respectively, in SARS-CoV-2 infection. To the best of our knowledge, this is the first study demonstrating the potential therapeutic efficacy of targeting integrin α5β1 in SARS-CoV-2 infection in vivo and supports the development of ATN-161 as a novel SARS-CoV-2 therapy.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the agent causative of coronavirus disease 2019 (COVID- 19) , is a highly transmissible respiratory pathogen in which an estimated 14% of all patients will develop serious conditions, with a subsequent mortality rate of 1.4 -3.4% [1] [2] [3] . Several non-pharmacological interventions have been implemented to slow down the spread of SARS-CoV-2 [4] [5] [6] [7] . However, to date there is no universally agreed direct therapy available to treat COVID-19. It is important to study effective therapeutic targets focusing on disrupting aspects of the viral replication process, including SARS-CoV-2 host cell entry [8] , which uses its spike proteins to bind to the angiotensin-converting enzyme 2 (hACE2) receptor on targeted cells to facilitate its entry and replication [9] . The virus infects the cells after the proteolytic cleavage of the spike protein by the transmembrane serine protease 2 (TMPRSS2) or Cathepsin B or L or FURIN is required for spike protein priming and virus infection [10] .

Integrins are family of cell adhesion receptors that may play an important role in SARS-CoV-2 host cell entry and infection due to the spike protein containing an integrin binding motif arginine-glycine-aspartate (RGD) sequence [11] [12] [13] [14] . Integrins are composed of non-covalently linked α and β subunits that recognize and bind to extracellular matrix (ECM) proteins and mediate cell survival, proliferation, differentiation, and migration [15] [16] [17] . Integrin dimers are expressed in most cells, including endothelial cells, and epithelial cells in the respiratory tract [18] , and are known to be involved in the infectious etiology of other viruses such as human cytomegalovirus [19] , Epstein-Barr virus [20] , rotavirus [21] , Kaposi's sarcoma-associated virus (HHV-8) [22] and Ebola [23] . Importantly, the β 1 family of integrins are closely associated with ACE2 [24] . A recent study reviewed novel mutation (K403R) in the spike protein that does not exist in other strains of the coronavirus, creating an RGD motif, which could be recognized by integrins [2] . Therefore, the new RGD motif in SARS-CoV-2 could increase the binding potency of ACE2-positive target cells in association with β 1 integrins as well as potentially facilitating infection of ACE2-negative cells [2] . This could help explain the faster and aggressive spread of virus as compared to SARS-CoV-1, which belongs to the same family of betacoronaviruses [25] .

Targeting therapies to disrupt the spike-binding event may also inhibit viral replication by halting host cell entry and offers a promising approach to treat COVID-19 [8] .

Our laboratory has previously shown that the SARS-CoV-2 spike protein is capable of binding to α 5β1 integrin in cell-free ELISA assays [26] , an observation which has since been repeated in epithelial cell-containing systems in vitro [27] . We further demonstrated that the small pentapeptide α 5β1 integrin inhibitor ATN-161 could inhibit this binding and also decrease SARS-CoV-2 infection and cytopathy in cultured vero-E6 cells [26] . ATN-161 has several properties that make it potentially attractive as a novel SARS-CoV-2 therapy; it is safe and welltolerated with no dose-limiting toxicity in phase I cancer clinical trials [28] , has demonstrated invivo efficacy in mice against a different beta coronavirus, porcine hemagglutinating encephalomyelitis virus (PHEV) [29] as well as other disease/injury models [30, 31] , and can also bind to and inhibit integrin α vβ3 which is similar to α 5β1 integrin that has also been implicated in SARS-CoV-2 infection [32, 33] . In the present study we examined the therapeutic potential of ATN-161 in a human ACE2 receptor-expressing K18 mouse model of SARS-CoV-2 infection.

Male heterozygous K18-hACE c57BL/6J mice (strain: 2B6.Cg-Tg(K18-ACE2)2Prlmn/J, 10weeks-old) were obtained from The Jackson Laboratory. Mice were housed in the animal facility at Tulane University School of Medicine. The Institutional Animal Care and Use Committee of Tulane University reviewed and approved all procedures for sample handling, inactivation, and removal from a BSL3 containment (permit number 3430 (#5)).

Mice were inoculated with either saline or SARS-CoV-2 via intranasal administration by the ABSL3-trained staff with a dose of 2x10 5 TCID 50 /mouse to induce viral infection in these animals [34, 35] . The infected mice were observed daily to record body weight and clinical signs of illness (e.g. fur ruffling, less activity). After 3 days post infection (dpi), the mice were euthanized by CO2 asphyxiation followed by cervical dislocation and lungs were harvested for histology, immunofluorescence and qRT-PCR analysis. [37, 34] .

The lung tissue was homogenized in Trizol Lysis Reagent (Invitrogen 

Fixed tissue samples were processed via indirect immunofluorescence assays (IFA) for the detection of the SARS-CoV-2 antigen. The slides were deparaffinized in xylenes and rehydrated through an ethanol series, followed by heat-induced antigen retrieval with high pH antigen Threshold and multiplex analyses were performed with HALO for quantitation.

Statistical tests were performed with GraphPad Prism, 8.4.3 version (GraphPad Software, San Diego, CA). Data are presented as mean ± SEM. Significant differences were designated using omnibus one-way ANOVA and, when significant, followed-up with two-group planned comparisons selected a priori to probe specific hypothesis-driven questions (saline vs SARS-CoV-2+vehicle; SARS-CoV-2+vehicle vs each of the various SARS-CoV-2+ATN-161 treated groups); the Holm-Sidak adjustment was applied to control for multiple comparisons. Nonparametric tests were employed in the event of violations of underlying ANOVA assumptions.

Statistical significance was taken at the p < 0.05 level.

Lung tissue expression of hACE2 and viral loads after 3dpi with SARS-CoV-2 + Saline or ATN-161 administered either once at 2 h or 48 h post-infection or daily, post-infection, were measured, analyzed and compared to Vehicle-treated/uninfected (no SARS-CoV-2 exposure) mice either given Saline or ATN-161 (daily) for 3dpi. (Fig 1A) . For hACE2, the omnibus oneway ANOVA was not significant ( Fig 1B) .

For Genomic-N ( Fig 1C) , the omnibus one-way ANOVA was significant [F(5,20)=12.84, p<0.0001]. We followed up this significant analysis with two group planned comparisons using the Holm-Sidak correction. We found a significant increase in Genomic-N among SARS-CoV-2 + Saline mice compared to non-infected, Saline-treated mice [t(6)=5.98, p<0.0001]. Though we did not detect significant group differences between any other two-group comparison, visual inspection of the graph revealed that there was 1) heterogeneity among the ATN-161 treated groups, suggesting a dichotomy in this population with regards to response to the ATN-161 treatment with regard to the viral load and 2) a general trend towards reduced viral load among all ATN-161 treated groups, regardless of timepoint or number of injections. This indicated to us a potential for our analyses to be underpowered. Therefore, to increase power and reduce variability, we pooled all ATN-161 treated animals into a single group and re-analyzed the data by comparing lung viral load in this group to that of the SARS-CoV-2 + Saline-treated mice. We found a trend towards significance [t(18)=2.03, p<0.06] such that ATN-161-treated mice had lower Genomic N viral load in lungs than SARS-CoV-2-infected mice. Then we analyzed the data using the Mann-Whitney U nonparametric test to account for a significant (p<0.01) violation of the homogeneity of variance assumption (though ANOVA is robust to this type of violation), the effect remained marginal (p=0.08). We also dichotomized ATN-161-treated animals into 'responder' and 'non-responder' groups such that mice were considered nonresponders if they displayed Genomic N values > 2x10 9 (one power of 10 lower than the lowest value observed in the SARS-CoV-2 group), Sgm-N values > 1x10 5 (one power of 10 lower than the lowest value observed in the SARS-CoV-2 group), and viral immunohistology staining counts >0.7 (one power of 10 lower than the lowest value observed in the SARS-CoV-2 group).

Ns of responders for each ATN-161 treated group were 3, 3, and 2 for ATN-161-2hr, ATN-161daily, and ATN-161-48hr groups respectively. When we re-analyzed the data by comparing lung viral load in these groups to that of the SARS-CoV-2 + Saline-treated mice, we found a 

Given the observations in Figure 

Representative images of hematoxylin and eosin staining of lung sections from saline/ATN-161 (Fig. 3Ai-Aii) ; SARS-CoV-2+vehicle (Fig. 3Bi-ii) ; Responders (Fig. 3Ci-ii) and

non-responders (Fig. 3Di-ii) with SARS-CoV-2+ATN-161 (either 2h, daily, or 48h) administration post-SARS-CoV-2 intranasal inoculation are shown in Figure 3 .

Histopathological observations indicated that multifocal lesion, moderate interstitial pneumonia ( Fig. 3Bii , Dii, black frames), infiltration of lymphocytes (Fig. 3Bii , Dii, green arrow), fibrin exudation (Fig. 3Bii, receptor. Interestingly, an RDG integrin-binding motif is a novel feature of SARS-CoV-2 spike protein, which is not seen in other coronaviruses [41, 11] . While this feature may have enhanced viral infectivity of SARS-CoV-2, it remains unknown how variant strains of SARS-CoV-2 may affect binding to integrins, with the resultant feature of affecting viral entry and propagation.

However, it is worth noting that to the best of our knowledge, none of the currently characterized SARS-CoV-2 variants have directly mutated the RGD motif; This suggests the possibility that the RGD motif is of evolutionary advantage to the virus by supporting its ability to infect hosts.

Integrins are cell surface receptors that may bind to the SARS-CoV-2 spike protein interaction [42, 43, 11, 33] . In our previous study, we demonstrated by ELISA that the SARS-CoV-2 spike protein and ACE2 could bind to α 5β1, and that ATN-161 could disrupt both of these interactions [26] . We further demonstrated that ATN-161 could significantly reduce SARS- [26] . In this study, we extend the understanding of the role of integrins and explored the therapeutic role of ATN-161 against SARS-CoV-2 infection in vivo in the k18-hACE2 mice model.

K18-hACE2 mice provide a platform for the rigorous screening of candidate drugs before their evaluation in other animal models [44] . This mouse model is widely used to evaluate the pathogenicity of viruses such as SARS-CoV-2 that require or prefer the human form of ACE2

(versus mouse ACE2) to readily infect mice and can be used to study potential therapies [45, 35] .

Previous studies provided the evidence that SARS-CoV-2 infection could cause typical interstitial pneumonia and develop respiratory disease in hACE2-expressing mice resembling what is commonly seen in COVID-19 patients [46, 45, 47, 48] .

We measured genomic-N (Nucleocapsid (N) protein) and Sgm-N in lungs to assess SARS-CoV-2viral infectivity and replication. Our results showed that SARS-CoV-2-infected hACE2 mice had significantly higher SARS-CoV-2 genomic-N. Similarly, viral sgmRNA copies were detected predominantly in the lung as compared to uninfected saline treated mice.

SgmRNA levels of the virus is an adequate surrogate assay for detection of replicating virus (replicating virus separated from the total genome, and form dimers as the virus is replicating its machinery which can continue to produce protein) [34] . Thus, we are confident that our mice were successfully infected with SARS-CoV-2 [35] .

We found that ATN-161-treated mice had lower Genomic-N viral load in lungs than This suggests a lack of statistical power due to low Ns among individual ATN-treated groups;

follow-up studies with additional subjects will be conducted to replicate these findings. infected hACE2 mice [35, 49] . Furthermore, we observed that viral immunohistology staining counts were negative in responders for each ATN-161 treated group with n's of 3, 3, and 2 for ATN-161-2 h, ATN-161-daily, and ATN-161-48 h groups, respectively. The infected K18-hACE2 mice did not lose body weight after only 3dpi as expected, but we observed high levels of viral copies and infectious virus in the lungs given as demonstrated by other studies in K18-hACE2 mice [35, 49, 50] .

In regards to an inflammatory response to SARS-CoV-2 infection, we observed that

Cxcl10 mRNA expression was significantly upregulated in the lungs of SARS-CoV-2 infected mice as has previously been reported [50] . In patients having rapid early viral replication, this is followed by inflammatory responses that contribute to pathology [51] . Post-mortem analysis of human COVID-19 patients showed immune cell accumulation in the lungs [52] . In our study, Our results suggest a critical role of integrins as an additional receptor to SARS-CoV-2 spike protein cell entry [59, 60] . We observed induced expression of integrin α 5 and integrin α v in the lungs of SARS-CoV-2 infected K18-hACE2 mice whereas lung expression of hACE2 levels did not vary in SARS-CoV-2+Saline or ATN-161 treated mice, suggesting that SARS-CoV-2 infection and / or pathogenesis involves these, and perhaps other integrins, that activate downstream signaling to induce lung pathology [33, 61, 62] . Indeed, a recent study showed that increased integrin α 5β1 and α vβ3 levels in cardiac myocytes, obtained from heart failure patients, correlates with ACE2 expression [63] . This suggests that the concomitant elevation of these integrins and the upregulation of ACE2 in an organ may render it more susceptible to SARS-CoV-2 infection. Hence, as observed in the present study, decreasing the expressions of and angiogenesis [30] . ATN-161 also binds to integrin α vβ3 [30] , an additional integrin that has been implicated in SARS-CoV-2 pathogenesis [33, 32] , and is safe, well-tolerated in human clinical trials (cancer) with no dose limiting toxicity [28, 66] , and can be administered i.v., i.p., and intranasally [31, 67] ; The latter may support a more readily accessible means of COVID-19 treatment as well as afford a prophylactic approach which is currently under investigation in our laboratory.

To the best of our knowledge, this study is the first to demonstrate that integrin blockade Our results further support the hypothesis that integrins play an important role in SARS-CoV-2 infection as well as support the further investigation of ATN-161, and potentially other antiintegrin agents, as novel therapies for COVID-19.

Although several studies have predicted the potential for SARS-CoV-2 to bind integrins and thereby infect host cells with or without associated ACE2 interaction, for the first time we present evidence here that inhibition of integrin α 5β1 (and Masson's Trichrome-stained sections (Ai-Dii) of lung sections from Saline/ATN-161 (Ai-Aii); SARS-CoV-2+vehicle (Bi-ii); Responders (Ci-ii) and Non-responders (Di-ii) with SARS-CoV-2+ATN-161(either 2h, daily, or 48h) administration post SARS-CoV-2 intranasal inoculation with tissue analysis at 3 dpi using the protocol described in Figure 1 . Histopathological observations indicated that showing multiple intra-arteriolar microthrombi (black arrows), intraalveolar microthrombi (green arrows), large interstitial hemorrhagic area (yellow arrow). Each image is representative of a group of Control n=3; ATN-161 n=3; SARS-CoV-2 (5 mice for vehicle, 5 mice for each 3 ATN-161 groups). Scale bar: 500 μ m (Ai, Bi, Ci, and Di) and 200 μ m (Aii, Bii, Cii, and Dii). 

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The authors have declared that no conflict of interest exists.