Antibodies against SARS-CoV spike may offer some protection against SARS-CoV-2

The spike protein of SARS-CoV-2 is primed by TMPRSS2

The recent emergence of the novel, pathogenic SARS-coronavirus 2 (SARS-CoV-2) in China and its rapid national and international spread pose a global health emergency. Cell entry of coronaviruses depends on binding of the viral spike (S) proteins to cellular receptors and on S protein priming by host cell proteases. Unravelling which cellular factors are used by SARS-CoV-2 for entry might provide insights into viral transmission and reveal therapeutic targets. Here, we demonstrate that SARS-CoV-2 uses the SARS-CoV receptor ACE2 for entry and the serine protease TMPRSS2 for S protein priming. A TMPRSS2 inhibitor approved for clinical use blocked entry and might constitute a treatment option. Finally, we show that the sera from convalescent SARS patients cross-neutralized SARS-2-S-driven entry. Our results reveal important commonalities between SARS-CoV-2 and SARS-CoV infection and identify a potential target for antiviral intervention.

The spike (S) protein of coronaviruses facilitates viral entry into target cells. Entry depends on binding of the surface unit, S1, of the S protein to a cellular receptor, which facilitates viral attachment to the surface of target cells. In addition, entry requires S protein priming by cellular proteases, which entails S protein cleavage at the S1/S2 and the S2’ site and allows fusion of viral and cellular membranes, a process driven by the S2 subunit ( Figure 1 A). SARS-S engages angiotensin-converting enzyme 2 (ACE2) as the entry receptor () and employs the cellular serine protease TMPRSS2 for S protein priming (). The SARS-S/ACE2 interface has been elucidated at the atomic level, and the efficiency of ACE2 usage was found to be a key determinant of SARS-CoV transmissibility (). SARS-S und SARS-2-S share ∼76% amino acid identity. However, it is unknown whether SARS-2-S like SARS-S employs ACE2 and TMPRSS2 for host cell entry.

(C) Cell lines of human and animal origin were inoculated with pseudotyped VSV harboring VSV-G, SARS-S, or SARS-2-S. At 16 h postinoculation, pseudotype entry was analyzed by determining luciferase activity in cell lysates. Signals obtained for particles bearing no envelope protein were used for normalization. The average of three independent experiments is shown. Error bars indicate SEM. Unprocessed data from a single experiment are presented in Figure S1

(B) Analysis of SARS-2-S expression (upper panel) and pseudotype incorporation (lower panel) by western blot using an antibody directed against the C-terminal hemagglutinin (HA) tag added to the viral S proteins analyzed. Shown are representative blots from three experiments. β-Actin (cell lysates) and VSV-M (particles) served as loading controls (M, matrix protein). Black arrow heads indicate bands corresponding to uncleaved S proteins (S0) whereas gray arrow heads indicate bands corresponding to the S2 subunit.

(A) Schematic illustration of SARS-S including functional domains (RBD, receptor binding domain; RBM, receptor binding motif; TD, transmembrane domain) and proteolytic cleavage sites (S1/S2, S2′). Amino acid sequences around the two protease recognition sites (red) are indicated for SARS-S and SARS-2-S (asterisks indicate conserved residues). Arrow heads indicate the cleavage site.

A transmembrane serine protease is linked to the severe acute respiratory syndrome coronavirus receptor and activates virus entry.

Efficient activation of the severe acute respiratory syndrome coronavirus spike protein by the transmembrane protease TMPRSS2.

Evidence that TMPRSS2 activates the severe acute respiratory syndrome coronavirus spike protein for membrane fusion and reduces viral control by the humoral immune response.

In December 2019, a new infectious respiratory disease emerged in Wuhan, Hubei province, China (). An initial cluster of infections was linked to Huanan seafood market, potentially due to animal contact. Subsequently, human-to-human transmission occurred () and the disease, now termed coronavirus disease 19 (COVID-19) rapidly spread within China. A novel coronavirus, SARS-coronavirus 2 (SARS-CoV-2), which is closely related to SARS-CoV, was detected in patients and is believed to be the etiologic agent of the new lung disease (). On February 12, 2020, a total of 44,730 laboratory-confirmed infections were reported in China, including 8,204 severe cases and 1,114 deaths (). Infections were also detected in 24 countries outside China and were associated with international travel. At present, it is unknown whether the sequence similarities between SARS-CoV-2 and SARS-CoV translate into similar biological properties, including pandemic potential ().

A Novel Coronavirus from Patients with Pneumonia in China, 2019.

A familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: a study of a family cluster.

A Novel Coronavirus from Patients with Pneumonia in China, 2019.

Clinical features of patients infected with 2019 novel coronavirus in Wuhan.

Several members of the family Coronaviridae constantly circulate in the human population and usually cause mild respiratory disease (). In contrast, the severe acute respiratory syndrome coronavirus (SARS-CoV) and the Middle East respiratory syndrome coronavirus (MERS-CoV) are transmitted from animals to humans and cause severe respiratory diseases in afflicted individuals, SARS and MERS, respectively (). SARS emerged in 2002 in Guangdong province, China, and its subsequent global spread was associated with 8,096 cases and 774 deaths (). Chinese horseshoe bats serve as natural reservoir hosts for SARS-CoV (). Human transmission was facilitated by intermediate hosts like civet cats and raccoon dogs, which are frequently sold as food sources in Chinese wet markets (). At present, no specific antivirals or approved vaccines are available to combat SARS, and the SARS pandemic in 2002 and 2003 was finally stopped by conventional control measures, including travel restrictions and patient isolation.

Isolation and characterization of viruses related to the SARS coronavirus from animals in southern China.

WHO Summary of probable SARS cases with onset of illness from 1 November 2002 to 31 July 2003.

[Coronaviruses as the cause of respiratory infections].

Convalescent SARS patients exhibit a neutralizing antibody response directed against the viral S protein (). We investigated whether such antibodies block SARS-2-S-driven entry. Four sera obtained from three convalescent SARS patients inhibited SARS-S- but not VSV-G-driven entry in a concentration-dependent manner ( Figure 5 ). In addition, these sera also reduced SARS-2-S-driven entry, although with lower efficiency compared to SARS-S ( Figure 5 ). Similarly, rabbit sera raised against the S1 subunit of SARS-S reduced both SARS-S- and SARS-2-S-driven entry with high efficiency, and again inhibition of SARS-S-driven entry was more efficient. Thus, antibody responses raised against SARS-S during infection or vaccination might offer some level of protection against SARS-CoV-2 infection.

Pseudotypes harboring the indicated viral surface proteins were incubated with different dilutions of sera from three convalescent SARS patients or sera from rabbits immunized with the S1 subunit of SARS-S and subsequently inoculated onto Vero cells in order to evaluate cross-neutralization potential. The average of three independent experiments performed with triplicate samples is shown. Error bars indicate SEM. Statistical significance was tested by two-way ANOVA with Dunnett posttest.

Two-year prospective study of the humoral immune response of patients with severe acute respiratory syndrome.

We next analyzed whether TMPRSS2 usage is required for SARS-CoV-2 infection of lung cells. Indeed, camostat mesylate significantly reduced MERS-S-, SARS-S-, and SARS-2-S- but not VSV-G-driven entry into the lung cell line Calu-3 ( Figure 4 C) and exerted no unwanted cytotoxic effects ( Figure S3 C). Similarly, camostat mesylate treatment significantly reduced Calu-3 infection with authentic SARS-CoV-2 ( Figure 4 D). Finally, camostat mesylate treatment inhibited SARS-S- and SARS-2-S- but not VSV-G-driven entry into primary human lung cells ( Figure 4 E). Collectively, SARS-CoV-2 can use TMPRSS2 for S protein priming and camostat mesylate, an inhibitor of TMPRSS2, blocks SARS-CoV-2 infection of lung cells.

In order to determine whether SARS-CoV-2 can use CatB/L for cell entry, we initially employed ammonium chloride, which elevates endosomal pH and thereby blocks CatB/L activity. 293T cells (TMPRSS2, transfected to express ACE2 for robust S protein-driven entry) and Caco-2 cells (TMPRSS2) were used as targets. Ammonium chloride blocked VSV-G-dependent entry into both cell lines whereas entry driven by Nipah virus F and G proteins was not affected ( Figure S3 A; data not shown), consistent with Nipah virus but not VSV being able to fuse directly with the plasma membrane (). Ammonium chloride treatment strongly inhibited SARS-2-S- and SARS-S-driven entry into TMPRSS2293T cells ( Figure S3 A), suggesting CatB/L dependence. Inhibition of entry into TMPRSS2Caco-2 cells was less efficient compared to 293T cells ( Figure S3 A), which would be compatible with SARS-2-S priming by TMPRSS2 in Caco-2 cells. Indeed, the clinically proven serine protease inhibitor camostat mesylate, which is active against TMPRSS2 (), partially blocked SARS-2-S-driven entry into Caco-2 ( Figure S3 B) and Vero-TMPRSS2 cells ( Figure 4 A). Full inhibition was attained when camostat mesylate and E-64d, an inhibitor of CatB/L, were added ( Figure 4 A; Figure S3 B), indicating that SARS-2-S can use both CatB/L as well as TMPRSS2 for priming in these cell lines. In contrast, camostat mesylate did not interfere with SARS-2-S-driven entry into the TMPRSS2cell lines 293T ( Figure S3 B) and Vero ( Figure 4 A), which was efficiently blocked by E-64d and therefore is CatB/L dependent. Moreover, directed expression of TMPRSS2 rescued SARS-2-S-driven entry from inhibition by E-64d ( Figure 4 B), confirming that SARS-2-S can employ TMPRSS2 for S protein priming.

The average of three independent experiments conducted with triplicate or quadruplicate samples is shown in (A–E). Error bars indicate SEM. Statistical significance was tested by two-way ANOVA with Dunnett posttest. Cells that did not receive inhibitor served as reference in (A), (C), (D), and (E) whereas cells transfected with empty vector and not treated with inhibitor served as reference in (B).

(E) In order to investigate whether serine protease activity is required for SARS-2-S-driven entry into human lung cells, primary human airway epithelial cells were incubated with camostat mesylate prior to transduction.

(D) Calu-3 cells were pre-incubated with camostat mesylate and infected with SARS-CoV-2. Subsequently, the cells were washed and genome equivalents in culture supernatants were determined by quantitative RT-PCR.

(C) Calu-3 cells were pre-incubated with the indicated concentrations of camostat mesylate and subsequently inoculated with pseudoparticles harboring the indicated viral glycoproteins.

(B) To analyze whether TMPRSS2 can rescue SARS-2-S-driven entry into cells that have low CatB/L activity, 293T cells transiently expressing ACE2 alone or in combination with TMPRSS2 were incubated with CatB/L inhibitor E-64d or DMSO as control and inoculated with pseudotypes bearing the indicated viral surface proteins.

(A) Importance of activity of CatB/L or TMPRSS2 for host cell entry of SARS-CoV-2 was evaluated by adding inhibitors to target cells prior to transduction. E-64d and camostat mesylate block the activity of CatB/L and TMPRSS2, respectively (additional data for 293T cells transiently expressing ACE2 and Caco-2 cells are shown in Figure S3 ).

(C) Absence of cytotoxic effects of camostat mesylate. Calu-3 cells were treated with camostat mesylate identically as for infection experiments and cell viability was measured using a commercially available assay (CellTiter-Glo, Promega).

(A and B) Importance of endosomal low pH (A) and activity of CatB/L or TMPRSS2 (B) for host cell entry of SARS-CoV-2 was evaluated by adding inhibitors to target cells prior to transduction. Ammonium chloride (A) blocks endosomal acidification while E-64d and camostat mesylate (B) block the activity of CatB/L and TMPRSS2, respectively. Entry into cells not treated with inhibitor was set as 100%.

Protease Requirement for SARS-2-S-Driven Entry and Absence of Unwanted Cytotoxicity of Camostat Mesylate, Related to Figure 4

Membrane fusion tropism and heterotypic functional activities of the Nipah virus and Hendra virus envelope glycoproteins.

We next investigated protease dependence of SARS-CoV-2 entry. SARS-CoV can use the endosomal cysteine proteases cathepsin B and L (CatB/L) () and the serine protease TMPRSS2 () for S protein priming in cell lines, and inhibition of both proteases is required for robust blockade of viral entry (). However, only TMPRSS2 activity is essential for viral spread and pathogenesis in the infected host whereas CatB/L activity is dispensable ().

TMPRSS2 Contributes to Virus Spread and Immunopathology in the Airways of Murine Models after Coronavirus Infection.

A transmembrane serine protease is linked to the severe acute respiratory syndrome coronavirus receptor and activates virus entry.

Efficient activation of the severe acute respiratory syndrome coronavirus spike protein by the transmembrane protease TMPRSS2.

Evidence that TMPRSS2 activates the severe acute respiratory syndrome coronavirus spike protein for membrane fusion and reduces viral control by the humoral immune response.

The Cellular Serine Protease TMPRSS2 Primes SARS-2-S for Entry, and a Serine Protease Inhibitor Blocks SARS-CoV-2 Infection of Lung Cells

In order to elucidate why SARS-S and SARS-2-S mediated entry into the same cell lines, we next determined whether SARS-2-S harbors amino acid residues required for interaction with the SARS-S entry receptor ACE2. Sequence analysis revealed that SARS-CoV-2 clusters with SARS-CoV-related viruses from bats (SARSr-CoV), of which some but not all can use ACE2 for host cell entry ( Figure 2 A; Figure S2 ). Analysis of the receptor binding motif (RBM), a portion of the receptor binding domain (RBD) that makes contact with ACE2 (), revealed that most amino acid residues essential for ACE2 binding by SARS-S were conserved in SARS-2-S ( Figure 2 B). In contrast, most of these residues were absent from S proteins of SARSr-CoV previously found not to use ACE2 for entry ( Figure 2 B) (). In agreement with these findings, directed expression of human and bat (Rhinolophus alcyone) ACE2 but not human DPP4, the entry receptor used by MERS-CoV (), or human APN, the entry receptor used by HCoV-229E (), allowed SARS-2-S- and SARS-S-driven entry into otherwise non-susceptible BHK-21 cells ( Figure 3 A). Moreover, antiserum raised against human ACE2 blocked SARS-S- and SARS-2-S- but not VSV-G- or MERS-S-driven entry ( Figure 3 B). Finally, authentic SARS-CoV-2 infected BHK-21 cells transfected to express ACE2 cells but not parental BHK-21 cells with high efficiency ( Figure 3 C), indicating that SARS-2-S, like SARS-S, uses ACE2 for cellular entry.

The average of three independent experiments conducted with triplicate samples is shown in (A–C). Error bars indicate SEM. Statistical significance was tested by two-way ANOVA with Dunnett posttest. Cells transfected with empty vector served as reference in (A) whereas cells that were not treated with antibody served as reference in (B).

(C) BHK-21 cells transfected with ACE2-encoding plasmid or control transfected with DsRed-encoding plasmid were infected with SARS-CoV-2 and washed, and genome equivalents in culture supernatants were determined by quantitative RT-PCR.

(B) Untreated Vero cells as well as Vero cells pre-incubated with 2 or 20 μg/mL of anti-ACE2 antibody or unrelated control antibody (anti-DC-SIGN, 20 μg/mL) were inoculated with pseudotyped VSV harboring VSV-G, SARS-S, SARS-2-S, or MERS-S. At 16 h postinoculation, pseudotype entry was analyzed (normalization against untreated cells).

(A) BHK-21 cells transiently expressing ACE2 of human or bat origin, human APN, or human DPP4 were inoculated with pseudotyped VSV harboring VSV-G, SARS-S, SARS-2-S, MERS-S, or 229E-S. At 16 h postinoculation, pseudotype entry was analyzed (normalization against particles without viral envelope protein).

(B) Alignment of the receptor binding motif of SARS-S with corresponding sequences of bat-associated betacoronavirus S proteins, which are able or unable to use ACE2 as cellular receptor, reveals that SARS-CoV-2 possesses crucial amino acid residues for ACE2 binding.

(A) The S protein of SARS-CoV-2 clusters phylogenetically with S proteins of known bat-associated betacoronaviruses (see Figure S2 for more details).

Differential sensitivity of bat cells to infection by enveloped RNA viruses: coronaviruses, paramyxoviruses, filoviruses, and influenza viruses.

Isolation and characterization of a bat SARS-like coronavirus that uses the ACE2 receptor.

Replication-defective VSV particles bearing coronavirus S proteins faithfully reflect key aspects of coronavirus host cell entry (). We employed VSV pseudotypes bearing SARS-2-S to study cell entry of SARS-CoV-2. Both SARS-2-S and SARS-S were robustly incorporated into VSV particles ( Figure 1 B), allowing a meaningful side-by-side comparison; although, formally, comparable particle incorporation of the S1 subunit remains to be demonstrated. We first asked which cell lines were susceptible to SARS-2-S-driven entry, using a panel of well-characterized cell lines of human and animal origin, respectively. All cell lines were readily susceptible to entry driven by the glycoprotein of the pantropic VSV (VSV-G) ( Figure 1 C; Figure S1 ), as expected. Most human cell lines and the animal cell lines Vero and MDCKII were also susceptible to entry driven by SARS-S ( Figure 1 C). Moreover, SARS-2-S facilitated entry into an identical spectrum of cell lines as SARS-S ( Figure 1 C), suggesting similarities in choice of entry receptors.

The indicated cells lines were inoculated with pseudoparticles harboring the indicated viral glycoprotein or harboring no glycoprotein (no protein) and luciferase activities in cell lysates were determined at 16 h posttransduction. The experiment was performed with quadruplicate samples, the average ± SD is shown.

Mutations in the Spike Protein of Middle East Respiratory Syndrome Coronavirus Transmitted in Korea Increase Resistance to Antibody-Mediated Neutralization.

The goal of our study was to obtain insights into how SARS-2-S facilitates viral entry into target cells and how this process can be blocked. For this, we first asked whether SARS-2-S is robustly expressed in a human cell line, 293T, commonly used for experimentation because of its high transfectability. Moreover, we analyzed whether there is evidence for proteolytic processing of the S protein because certain coronavirus S proteins are cleaved by host cell proteases at the S1/S2 cleavage site in infected cells ( Figure 1 A). Immunoblot analysis of 293T cells expressing SARS-2-S protein with a C-terminal antigenic tag revealed a band with a molecular weight expected for unprocessed S protein (S0) ( Figure 1 B). A band with a size expected for the S2 subunit of the S protein was also observed in cells and, more prominently, in vesicular stomatitis virus (VSV) particles bearing SARS-2-S ( Figure 1 B). In contrast, an S2 signal was largely absent in cells and particles expressing SARS-S ( Figure 1 B), as previously documented (). These results suggest efficient proteolytic processing of SARS-2-S in human cells, in keeping with the presence of several arginine residues at the S1/S2 cleavage site of SARS-2-S but not SARS-S ( Figure 1 A). In contrast, the S2′ cleavage site of SARS-2-S was similar to that of SARS-S.

S protein of severe acute respiratory syndrome-associated coronavirus mediates entry into hepatoma cell lines and is targeted by neutralizing antibodies in infected patients.

Evidence that TMPRSS2 activates the severe acute respiratory syndrome coronavirus spike protein for membrane fusion and reduces viral control by the humoral immune response.

Discussion

The present study provides evidence that host cell entry of SARS-CoV-2 depends on the SARS-CoV receptor ACE2 and can be blocked by a clinically proven inhibitor of the cellular serine protease TMPRSS2, which is employed by SARS-CoV-2 for S protein priming. Moreover, it suggests that antibody responses raised against SARS-CoV could at least partially protect against SARS-CoV-2 infection. These results have important implications for our understanding of SARS-CoV-2 transmissibility and pathogenesis and reveal a target for therapeutic intervention.

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In sum, this study provided key insights into the first step of SARS-CoV-2 infection, viral entry into cells, and defined potential targets for antiviral intervention.