可溶性ACE2受容体を投与することが有効かも知れない

カロリンスカ研究所のバネッサ・モンテイル氏らによってまとめられ、2020/4/2に生物学分野において権威ある学術雑誌「Cell」に掲載されました。

ウェーデンの研究者は、コロナ19が人間のACE2に結合する前に、偽のACE2（hrsACE2）をばらまく治療法を考え出しました。

その結果、偽ACE2を加えられたほうではコロナ19が騙され、感染能力が最大で5000分の1にまで低下していることがわかりました。

不思議なことに、マウスでは偽ACE2を使った治療に効果がなかったようです。

Inhibition of SARS-CoV-2 infections in engineered human tissues using clinical-grade soluble human ACE2

Vanessa Monteil1, Hyesoo Kwon2, Patricia Prado3, Astrid Hagelkrüys4, Reiner A. Wimmer4, Martin Stahl5, Alexandra Leopoldi4, Elena Garreta3, Carmen Hurtado del Pozo3, Felipe Prosper6, J.P. Romero6, Gerald Wirnsberger7, Haibo Zhang8, Arthur S. Slutsky8, Ryan Conder5, Nuria Montserrat3,9,10,*, Ali Mirazimi1, 2,*, Josef M. Penninger4,11,12*

https://www.cell.com/pb-assets/products/coronavirus/CELL_CELL-D-20-00739.pdf

Summary

We have previously provided the first genetic evidence that Angiotensin converting enzyme 2 (ACE2) is the critical receptor for SARS-CoV and that ACE2 protects the lung from injury, providing a molecular explanation for the severe lung failure and death due to SARS-CoV infections. ACE2 has now also been identified as a key receptor for SARS-CoV-2 infections and it has been proposed that inhibiting this interaction might be used in treating patients with COVID- 19. However, it is not known whether human recombinant soluble ACE2 (hrsACE2) blocks growth of SARS-CoV-2. Here we show that clinical grade hrsACE2 reduced SARS-CoV-2 recovery from Vero cells by a factor of 1,000-5,000. An equivalent mouse rsACE2 had no effect. We also show that SARS-CoV-2 can directly infect engineered human blood vessel organoids and human kidney organoids, which can be inhibited by hrsACE2. These data demonstrate that hrsACE2 can significantly block early stages of SARS-CoV-2 infections.

Results

Isolation of a SARS-CoV-2

To study potential therapeutic interventions for COVID-19, in early February 2020 we isolated the SARS-CoV-2 from a nasopharyngeal sample of a patient in Sweden with confirmed COVID-19. After successful culture on Vero E6 cells, the isolated virus was sequenced by Next-Generation Sequencing (Genbank accession number MT093571). Electron microscopy showed the prototypic coronal shape of viral particles of our SARS-CoV-2 isolate (Figure 1A). Phylogenetic analysis showed the virus belongs to the clad A3 (Figure 1B).

hrsACE-2 can inhibit SARS-CoV-2 infection in a dose dependent manner

hrsACE2 has already undergone clinical phase 1 and phase 2 testing (Khan et al., 2017) and is being considered for treatment of COVID-19 (Zhang et al., 2020b). Since ACE2 is the SARS- CoV-2 receptor, we wanted to provide direct evidence that clinical-grade hrsACE2 can indeed interfere with SARS-CoV-2 infections. To this end, we infected Vero-E6 cells (cells used for SARS-CoV-2 isolation) with different numbers of SARS-CoV-2: 103 plaque forming units (PFUs; MOI 0.02), 105 PFUs (MOI 2) and 106 PFUs (MOI 20). Viral RNA as a marker for replication was purified from cells and assayed by qRT-PCR (Figure 2A). Infection of cells in the presence of hrsACE2 during 1 hr, followed by washing and incubation without hrsACE2 significantly inhibited SARS-CoV-2 infections of Vero-E6 15 hours post infection (Figure 2A).

These data demonstrate that hrsACE2 inhibits the attachment of the virus to the cells. Importantly, as expected from a neutralizing agent, this inhibition was dependent on the initial quantity of the virus in the inoculum and the dose of hrsACE2 (Figure 2A), establishing dose-dependency. In contrast to hsrACE-2, the equivalent mouse recombinant soluble ACE2 (mrsACE2), produced in the same way as hrsACE2, did not inhibit the infection (Figure 2B). Finally, we performed experiments where cells were infected with SARS-CoV-2 in the presence of hrsACE2 or mrsACE2 for 15 hr, to capture any newly produced virus particles during the 15hr that could infect neighbouring cells. Again, we observed significantly reduced virus infections in the presence of hrsACE2 (Figure 2C), but not mrsACE2 (Figure 2D). Of note, addition of human or mouse rsACE2 was not toxic to the Vero-E6 cells, monitored for 15 hours (data not shown). These data show that hrsACE2 significantly reduces SARS-CoV-2 infections in vitro.

hrsACE-2 inhibits SARS-CoV-2 infections of human capillary organoids

A primary site of SARS-CoV-2 infection appears to be the lung, which may be a source for viral spread to other tissues such as the kidney and intestine, where virus has been found (stool and urine) (Ling et al., 2020; Young et al., 2020). Moreover, viremia is established during the course of the disease, although viral RNA in blood is only infrequently observed (Peng et al., 2020; Wang et al., 2020). However, the virus has a size of 80-100nm indicating that viremic SARS-CoV-2 must first infect blood vessels prior to local tissue infections. To test this hypothesis, we established human capillary organoids from induced pluripotent stem cells (iPSCs) (Figure 3A) and infected them with our SARS-CoV-2 isolate. Of note, these organoids closely resemble human capillaries with a lumen, CD31+ endothelial lining, PDGFR+ pericyte coverage, as well as formation of a basal membrane (Wimmer et al., 2019). The capillary organoids were analysed by qRT-PCR for the presence of viral RNA at day 3 and 6 after primary SARS-CoV-2 exposure. Importantly, following infection, we could detect viral RNA in the blood vessel organoids with viral RNA increasing from day 3 to day 6 post infection (Figure 3B), indicating active replication of SARS- CoV-2.

Supernatant of infected organoids collected at day 6 post-infection could efficiently infect Vero E6 cells (Figure 3C), showing that the infected capillary organoids produced progeny virus. Importantly, addition of hrsACE2 markedly reduced SARS-CoV-2 infections of the engineered human blood vessels (Figure 3D). Of note, addition of human or mouse rsACE2 was not toxic to human blood-vessels, monitored for 3 days (data not shown). These data show that human capillary organoids can be infected with SARS-CoV-2 and this infection can be significantly inhibited by hrsACE2.

hrsACE-2 can inhibit SARS-CoV-2 infections of human kidney organoids

We and others have previously shown that ACE2 is strongly expressed in kidney tubules (Danilczyk and Penninger, 2006). Moreover, it has been reported that SARS-CoV-2 can be found in the urine (Young et al., 2020). To test whether SARS-CoV2 can directly infect human tubular kidney cells, we generated kidney organoids from human embryonic stem cells into 3D suspension culture, adapting our own protocol (Garreta et al., 2019). Importantly, kidney differentiation organoids demonstrated prominent tubular-like structures as detected by Lotus Tetraglobus Lectin (LTL) as a marker of proximal tubular epithelial cells (Figure 4A). Tubular-like cells also expressed the solute carrier SCL3A1 (Figure S1A) together with SCL27A2 and SCL5A12. Furthermore, LTL positive (LTL+) cell fractions from organoids expressed markers of proximal tubular identity (Figure S1B and S1C). Single cell profiling of kidney organoids showed the presence of cells expressing ACE2 in the proximal tubule and podocyte II cell clusters that express key marker genes of proximal tubular cells (SLC3A1, SLC27A2) and podocytes (PODXL, NPHS1, NPHS2), respectively (Figure S2). Thus, kidney organoids contain cell clusters that express ACE2 in a similar fashion to that observed in the native tissue (Lin et al. 2020).

Infections of kidney organoids were monitored 6 days after SARS-CoV-2 infection and assayed for the presence of viral RNA using q-RT-PCR. Progeny virus was determined as above using re- infections of Vero E6 cells. As expected from cells and tissues that express ACE2, SARS-CoV-2 replicated in kidney organoids (Figure 4B). Supernatant of infected kidney organoids collected at day 6 post-infection could efficiently infect Vero E6 cells (Figure 4C), showing that the engineered kidney organoids produced infectious progeny virus. Importantly, addition of hrsACE2 significantly reduced SARS-CoV-2 infections of the human kidney organoids in a dose dependent manner (Figure 4D). Of note, addition of human or mouse rsACE2 was not toxic to the kidney, monitored for 3 days (data not shown). These data indicate that besides blood vessels, engineered human kidney organoids can also be infected with SARS-CoV-2 and this infection can be inhibited by hrsACE2.

Discussion

ACE2 took centre stage in the COVID-19 outbreak as the key receptor for the spike glycoprotein of SARS-CoV-2, as demonstrated in multiple structural and biochemical interaction studies (Wrapp et al., 2020; Zhou et al., 2020b). Moreover, multiple drug development projects, including development of vaccines are focusing on the ACE2-SARS-CoV-2 Spike interactions. We initially identified mammalian ACE2 when we realized that flies carry two orthologues of ACE (Angiotensin-converting enzyme). Our first ace2 mutant mice then demonstrated that ACE2 is a

negative regulator of the renin-angiotensin system (RAS) and genetically controls cardiovascular function and damage of multiple organs such as the lung, liver, and kidney (Clarke and Turner, 2012; Crackower et al., 2002). ACE2 catalytically removes the last amino acid of angiotensin II, thereby counterbalancing ACE and Ang II actions and generating “beneficial” downstream peptides such as Ang1-7. ACE2 also catalytically acts on other peptides such as in the Apelin/APJ system (Clarke and Turner, 2012).

Importantly, we reported that ACE2 protects from lung injury, based on its catalytic domain, and that ACE2 is the critical in vivo SARS-CoV spike glycoprotein receptor (Imai et al., 2005; Kuba et al., 2005). Initially two receptors had been identified for SARS-CoV in cell lines, namely ACE2 (Li et al., 2003) and the lectin L-SIGN (Jeffers et al., 2004). The severity of SARS could be partially explained by SARS-CoV Spike protein binding to ACE2 at a molecular interaction site that does not interfere with its catalytic activity (Li et al., 2005), which then leads to endocytosis of the virus and loss of ACE2 (Kuba et al. 2005), establishing a vicious circle of viral infection and local loss of lung injury protection. This led to the initiation of a drug development program – the development of soluble recombinant human ACE2, a drug that has undergone phase 1 testing in healthy volunteers and phase 2 testing in some patients with acute respiratory distress syndrome (ARDS) (Haschke et al., 2013; Khan et al., 2017; Treml et al., 2010). Our data now show that this clinical-grade human ACE2 molecule - but not mouse soluble ACE2 - can significantly inhibit SARS-CoV-2 infections and reduce viral load by a factor of 1,000-5,000. However, as observed in antibody neutralizing experiments of many viruses, the inhibition is not complete, though clearly dose-dependent. This may be due to the fact that there might be other co-receptors/auxiliary proteins or even other mechanisms by which viruses can enter cells, as had been initially proposed for SARS (Jeffers et al., 2004; Qi et al., 2020). Such a second receptor has been also suggested based on clinical data: SARS transmissibility was very low possibly due to the low level expression of ACE2 in the upper respiratory tract (Bertram et al., 2012; Hamming et al., 2004). Transmissibility of SARS-CoV-2 is much greater than that of SARS-CoV, suggesting that SARS- CoV-2 might use a co-receptor and/or other factors which allow infection of ACE2 expressing cells in the upper respiratory tract (Lukassen et al., 2020). Most importantly, our results demonstrate that hrsACE2 significantly blocks SARS-CoV-2 infections, providing a rationale that soluble ACE2 might not only protect from lung injury but also block the SARS-CoV-2 from entering target cells.

Pathology due to SARS, MERS, and now COVID-19 is not limited to the lung; damage can occur in multiple organs (Gu et al., 2005; Wu and McGoogan, 2020; Yeung et al., 2016). ACE2 is expressed in various tissues including the heart, kidney tubules, the luminal surface of the small

intestine, and blood vessels (Crackower et al., 2002; Danilczyk and Penninger, 2006; Ding et al., 2004; Gu et al., 2005; Hamming et al., 2004; Zhang et al., 2020b), suggesting that SARS-CoV-2 could also infect these tissues. We now show that blood vessels as well as kidney organoids can be readily be infected by SARS-CoV-2. SARS-CoV-2 must enter the blood stream to infect other tissues. However, the size of the infectious viral particles is about 80-100nm (Wrapp et al., 2020). Thus, unless there is already tissue damage, the virus must enter vascular endothelial cells to migrate into the organs. Our data in engineered human capillary organoids now suggest that SARS- CoV-2 could directly infect blood vessel cells. Infected blood vessel organoids also shed progeny viruses. Importantly, hrsACE2 markedly inhibited SARS-CoV-2 infections of the vascular organoids.

ACE2 is strongly expressed in kidney tubules, controlling a local RAS circuit (Clarke and Turner, 2012; Hashimoto et al., 2012). As an infection model, we therefore engineered human kidneys organoids from stem cells differentiated to contain tubular networks (Garreta et al., 2019). We now show that SARS-CoV-2 can infect such human kidney organoids, resulting in infectious viral progeny, inhibited by hrsACE2. Clinically, SARS-CoV-2 has been found in the urine (Peng et al., 2020) and many patients with COVID-19 present with cardiovascular and renal dysfunctions (Huang et al., 2020; Yang et al., 2020; Zhang et al., 2020a; Zhou et al., 2020a). Whether direct viral infection of the vasculature and kidneys directly contribute to the observed multi-organ damage in COVID-19 patients needs to be established. Given the fact that cardiac cells express high levels of ACE2, and heart alterations were the first phenotype observed in our ace2 mutant mice (Crackower et al. 2002), it will be important to expand on our studies to heart and in particular lung organoids to better understand the multi-organ dysfunction in patients with COVID-19.

Our study has limitations. The design of our studies focused on the early stages of infection, demonstrating that hrsACE2 can block early entry of SARS-CoV-2 infections in host cells. As such, we cannot make any predictions with respect to the effect of hrsACE2 in later stages of the disease process. Secondly, we did not study lung organoids, and the lung is the major target organ for COVID-19. Finally, the RAS system represents a complex network of pathways which are influenced by external processes which are not simulated in our model systems. To address these issues, further studies are needed to illuminate the effect of hrsACE2 at later stages of infection in vitro and in vivo.