key: cord-0749850-mgr75gt1
authors: Roth, Annika; Lütke, Steffen; Meinberger, Denise; Hermes, Gabriele; Sengle, Gerhard; Koch, Manuel; Streichert, Thomas; Klatt, Andreas R.
title: LL-37 fights SARS-CoV-2: The Vitamin D-Inducible Peptide LL-37 Inhibits Binding of SARS-CoV-2 Spike Protein to its Cellular Receptor Angiotensin Converting Enzyme 2 In Vitro
date: 2020-12-04
journal: bioRxiv
DOI: 10.1101/2020.12.02.408153
sha: af0876be7de96651a507d786311f86ff3ad1778a
doc_id: 749850
cord_uid: mgr75gt1

Objective Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is the pathogen accountable for the coronavirus disease 2019 (COVID-19) pandemic. Viral entry via binding of the receptor binding domain (RBD) located within the S1 subunit of the SARS-CoV-2 Spike (S) protein to its target receptor angiotensin converting enzyme (ACE) 2 is a key step in cell infection. The efficient transition of the virus is linked to a unique protein called open reading frame (ORF) 8. As SARS-CoV-2 infections can develop into life-threatening lower respiratory syndromes, effective therapy options are urgently needed. Several publications propose vitamin D treatment, although its mode of action against COVID-19 is not fully elucidated. It is speculated that vitamin D’s beneficial effects are mediated by up-regulating LL-37, a well-known antimicrobial peptide with antiviral effects. Methods Recombinantly expressed SARS-CoV-2 S protein, the extended S1 subunit (S1e), the S2 subunit (S2), the receptor binding domain (RBD), and ORF8 were used for surface plasmon resonance (SPR) studies to investigate LL-37’s ability to bind to SARS-CoV-2 proteins and to localize its binding site within the S protein. Binding competition studies were conducted to confirm an inhibitory action of LL-37 on the attachment of SARS-CoV-2 S protein to its entry receptor ACE2. Results We could show that LL-37 binds to SARS-CoV-2 S protein (LL-37/SStrep KD = 410 nM, LL-37/SHis KD = 410 nM) with the same affinity, as SARS-CoV-2 binds to hACE2 (hACE2/SStrep KD = 370 nM, hACE2/SHis KD = 370 nM). The binding is not restricted to the RBD of the S protein, but rather distributed along the entire length of the protein. Interaction between LL-37 and ORF8 was detected with a KD of 290 nM. Further, inhibition of the binding of SStrep (IC50 = 740 nM), S1e (IC50 = 170 nM), and RBD (IC50 = 130 nM) to hACE2 by LL-37 was demonstrated. Conclusions We have revealed a biochemical link between vitamin D, LL-37, and COVID-19 severity. SPR analysis demonstrated that LL-37 binds to SARS-CoV-2 S protein and inhibits binding to its receptor hACE2, and most likely viral entry into the cell. This study supports the prophylactic use of vitamin D to induce LL-37 that protects from SARS-CoV-2 infection, and the therapeutic administration of vitamin D for the treatment of COVID-19 patients. Further, our results provide evidence that the direct use of LL-37 by inhalation and systemic application may reduce the severity of COVID-19.

Severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) is one of seven known human coronavirus pathogens 1 . Most human coronavirus infections result in the common cold and account for up to 15% of such cases. In contrast, SARS-CoV, MERS-CoV, and SARS-CoV-2 infections can develop into life-threatening lower respiratory syndromes [2] [3] [4] . As of November 28, 2020, a total of 61,508,373 infected cases and more than 1,441,265 deaths (mortality ~2%) were reported (WorldOmeter https://www.worldometers.info/coronavirus/). Coronavirus disease 2019 has resulted in a global pandemic. An end of the pandemic will be reached if a majority of the population becomes immune after surviving the infection or through an effective vaccine. In addition to vaccine development, research focuses on therapy options. Attractive therapeutic approaches are the inhibition of SARS-CoV-2 fusion and entry, the disruption of virus replication, the suppression of excessive inflammatory response, and the treatment with convalescent plasma 5 .

The COVID-19 mortality rate is currently higher at northern latitudes, a possible role for vitamin D (1,25(OH) 2 D) in determining outcomes has been postulated 6 High-dose vitamin D supplementation should achieve and maintain optimal (range 40-60 ng/ml) serum 25(OH)D levels both for COVID-19 prevention and treatment 8 .

Vitamin D is not only known for its role in bone mineralization and calcium homeostasis, but is also involved in various pathophysiological mechanisms that occur during SARS-CoV-2 infection. It was suggested, that vitamin D increases the ratio of angiotensin converting enzyme (ACE)2 to ACE, thus increasing angiotensin II hydrolysis and reducing subsequent inflammatory cytokine response to pathogens and lung injury 6 . Also, many studies show that vitamin D has a decisive influence on the immune system and regulates antimicrobial innate immune responses [9] [10] [11] . The effects of vitamin D are mediated by its binding to the vitamin D receptor (VDR) and subsequent binding to vitamin D response elements (VREs) 9 . Such a consensus VRE is located in the promotor of the human cathelicidin antimicrobial peptide gene. Therefore, cathelicidin, the precursor of the well investigated human antimicrobial peptide LL-37, is strongly up-regulated by vitamin D 12, 13 .

LL-37 consists of 37 amino acids with an overall positive net charge (+6) and can thus eliminate microbes directly by electrostatic binding to negatively charged molecules on microbial membranes.

In addition, it has antiviral effects including inhibition of herpes simplex virus type one (HSV-1) replication, vaccinia virus replication, retroviral replication, and replication of some adenovirus serotypes 14 . The epidemiological evidence for a positive vitamin D-related immune effect includes many studies that feature enveloped viruses. This supports the notion that LL-37's antiviral effects may be partially mediated by envelope disruption 15 . SARS-CoV-2 is an enveloped single-stranded RNA virus harboring a genome of approximately 30 kb that encodes, among others, for the structural spike (S) glycoprotein that forms trimers at the viral surface. The S protein is important for viral attachment to the host cell via ACE2 and comprises two subunits, the S1 subunit containing the receptor binding domain (RBD) and the S2 subunit 16 . Furthermore, the genome codes for a total of six accessory proteins including open reading frame (ORF)8, which might function as a potential immune modulator to delay or attenuate the host immune response against the virus 17 . Interestingly, the S protein and the accessory protein ORF8 have a negative net charge of -8 and -4 (https://pepcalc.com/), respectively. Therefore, we speculated that LL-37 interacts with SARS-CoV-2 S protein and ORF8 and block viral entry.

In this study, we investigated the interaction between LL-37 and the SARS-CoV-2 S protein and ORF8 protein by surface plasmon resonance (SPR). Furthermore, we examined whether LL-37 inhibits the binding of the S protein to its cellular receptor hACE2.

The genes encoding ectodomain from the spike glycoprotein (MN908947; AA:1-1207; furin site mutated; K986P and V987P) and the S2 subunit (MN908947; AA:686-1207; K986P and V987P) including a C-terminal T4 foldon, the extended S1 subunit (MN908947; AA:14-722; furin site mutated) and the receptor binding domain (MN908947; AA:331-524) regions of the SARS-CoV-2 spike DNA as well as the ORF8 (MN908947; AA:16-121) and the human ACE2 (NM_001371415.1: AA:20-611) were amplified from synthetic gene plasmids using specific PCR-primers. After digestion with appropriate restriction enzymes the PCR products were cloned into modified sleeping beauty transposon expression vectors 18 that contain a double Strep II or a poly-histidine tag (Figure 1 A) and a thrombin cleavage site at the C-terminus. In the case of RBD, S1e, ORF8, and ACE2 the tag was added at the 5' end including a BM40 signal peptide sequence. The sleeping beauty transposon system was stably transfected into HEK293 

Synthetic LL-37 (Sequence: LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES) was purchased from Invivogen (USA). Lyophilized peptide was reconstituted with endotoxin-free water to reach a final concentration of 1 mg/ml, which was verified by BCA assay (Thermo Fischer Scientific, USA). Purity was ≥ 95%.

Protein samples were reduced with dithiothreitol, mixed with LDS Sample Buffer (Thermo Fischer Scientific, USA), and heated to 90°C for 10 min. SDS-PAGE was performed in a 4-12% Bis-Tris polyacrylamide gel (Thermo Fischer Scientific, USA). Proteins were visualized using Coomassie Brilliant Blue R 250 (Merck, Germany) or using the SilverQuest silver staining kit (Invitrogen Thermo Fisher Scientific, USA). For immunoblotting, the proteins were transferred to a polyvinylidene fluoride (0.45 µm) membrane (Thermo Fischer Scientific, USA). After blocking the membrane for 1h in PBS containing 0.05% Tween and 3% BSA the membrane was incubated with Strep-Tactin®-HRP conjugate (IBA Lifescience, Germany) diluted 1:100,000 in blocking solution. The membrane was then treated with Amersham TM ECL TM Prime Western Blotting Detection Reagent (Thermo Fisher Scientific, USA) and signals were visualized using the ChemiDoc XRS+ System (BioRad, Germany).

Interaction studies using surface plasmon resonance (SPR) SPR experiments were performed as described previously 19,20 using a BIAcore 2000 system (BIAcore AB, Uppsala, Sweden). SARS-CoV-2 proteins were coupled to CM5 sensor chips using the Experiments were performed in quadruplicate.

Binding competition studies using surface plasmon resonance

For competition experiments SARS-CoV-2 S Strep was immobilized at 9000 RUs and 0-2560 nM LL-37 was injected followed by a subsequent 10 nM injection of hACE2 ("coinject" mode). Competition experiments with SARS-CoV-2 RBD, SARS-CoV-2 S1e, and SARS-CoV-2 S2 were performed by injecting 0-2560 nM LL-37 followed by an immediate injection of 640 nM hACE2 ("coinject" mode). Competition effects were detected as the percentage loss of signal compared to prior buffer injection.

To study the interaction of LL-37 with SARS-CoV-2 we used recombinantly expressed SARS-CoV-2 S Strep (139 kDa), S His (136 kDa), S1e (83 kDa), RBD (26 kDa), S2 (65 kDa), and the accessory protein ORF8 (16 kDa) (Figure 1 A) . Recombinant hACE2 was used as a positive control. Integrity and purity of the proteins was checked by SDS-PAGE following Coomassie staining (Figure 1 B) Interaction studies using surface plasmon resonance (SPR)

To investigate the binding kinetics and interaction affinity of LL-37 with SARS-CoV-2 S protein by SPR, S Strep and S His were immobilized on a CM5 sensor chip (Figure 2 ). LL-37 showed consistent and specific binding to both spike ectodomain preparations S Strep (K D = 410 nM) and S His (K D = 420 nM) which excluded tag-specific effects. Binding of S proteins to hACE2As was measured as a positive control (Figure 2 B, D) .. The binding affinities of S Strep to hACE2 (K D = 370 nM) and S His to hACE2 (K D = 370 nM) were comparable to the binding affinities seen for LL-37. To define the binding site of LL-37 within the S protein the recombinant proteins RBD, S1e, and S2, representing different subregions of the S protein (Figure 3) , were immobilized on a CM5 sensor chip and binding of LL-37 was analyzed. LL-37 bound to RBD (K D = 220 nM), S1e (K D = 200 nM), and S2 (K D = 230 nM) with the same binding affinities. As a control, we analyzed the binding of hACE2 to RBD (K D = 940 nM), S1e (K D = 610 nM) and S2. As expected, only S2, which possesses no RBD, did not show an interaction with hACE2, demonstrating the specificity of the binding of RBD and S1e to hACE2, and of RBD, S1e, and S2 to LL-37. The accessory protein ORF8 is regarded as a virulence factor. Therefore, we also analyzed the interaction of ORF8 and LL-37 ( Figure 4) . Binding between LL-37 and ORF8 was detected with a K D of 290 nM. A Strep-Tactin-HRP conjugate was used to verify the functionality of the ORF8 coupled sensor chip, as ORF8 carries a double Strep II tag. Binding competition studies using surface plasmon resonance Competition studies were performed to investigate the bindingof S protein, S1e, and RBD to hACE2 by LL-37. Therefore, sensor chips were immobilized with S Strep , RBD, and S1e, respectively, and hACE2 was injected with a constant concentration directly after various concentrations of LL-37 ( Figure 5 ). Calculated half maximal inhibitory concentrations (IC 50 ) for the binding of S protein, S1e, and RBD to hACE2 by LL-37 were determined. LL-37 was able to reduce the binding of S Strep (IC 50 = 740 nM), S1e (IC 50 = 170 nM), and RBD (IC 50 = 130 nM) to hACE2. In contrast to RBD and S1e, the full-length S protein possesses additional binding sites for LL-37, which are not involved in hACE2 binding. Therefore, higher concentrations of LL-37 are needed to block the binding site of hACE2. to the N-terminal helix of hACE2 26 . Surprisingly, LL-37 did not only bind to RBD and proteins containing the RBD, but also to S2, lacking the RBD. Therefore, we hypothesize that binding of LL-37 to the S protein does not occur through to interactions with a special protein domain, but are rather due to electrostatic or hydrophobic interactions or its amphipathicity. This assumption is further supported by the fact that the antiviral effectivity of LL-37 against influenza infection in vitro or in vivo was not altered by replacing L-amino acids with D-amino acids, indicating that its action is not dependent on specific receptor interactions 27 . Interestingly, peptide binding to non-RBD regions can also reduce SARS-CoV-2 S protein attachment to hACE2 28 . Therefore, the here demonstrated binding of LL-37 to S1e and S2 may also hinder viral binding to its host receptor. In summary, we could show that LL-37 reduces attachment of the S protein to hACE2. The calculated IC 50 values for the LL-37 inhibition of the binding of S protein and its subdomains to the hACE2 ranged from 130 nM (IC 50 RBD) to 740 nM (IC 50 S Strep ). In contrast, the minimal inhibitory concentration (MIC) of LL-37 against different gram-positive and gram-negative bacteria is reported to range from 3.6 µM to 6.9 µM 29 .

In vitro experiments predicted cytotoxicity of LL-37. However, in plasma up to 90% of LL-37 is bound to apolipoprotein A-I, which attenuates its cytotoxic effects in vivo 30 31, 32 . A study of infants hospitalized due to bronchiolitis associated low LL-37 levels with increased severity of disease 33 . Furthermore, after cyclic stretch, typically caused by mechanical ventilation, down-regulation of the cathelicidin gene was detected in human bronchial epithelial cell lines.

Treatment with vitamin D counteracted this effect on the mRNA level as well as on the protein level and protected against ventilator-induced injury 34 . In human bronchial epithelial cells, a 10-fold induction of cathelicidin mRNA levels was achieved by vitamin D stimulation 11 . Concerning a direct therapeutic application of antimicrobial peptides like LL-37, it is noteworthy that their pharmaceutical use has received approval for clinical trials 25, 35 . This could help to accelerate the use of LL-37 for the treatment of COVID-19.

We also tested LL-37s' ability to bind to the negatively charged, secreted accessory protein ORF8, which is unique to SARS-CoV-2 and thus linked to efficient pathogen transmission 36, 37 . Multiple functions of ORF8 have been proposed including disruption of antigen presentation via down-regulation of MHC I expression and inhibition of interferon I signaling 38,39 . Furthermore, a 382-nucleotide deletion in the ORF8 region of the genome led to lower concentrations of proinflammatory cytokines, chemokines, and growth factors that are strongly associated with severe COVID-19 2,40 . Therefore, binding of LL-37 to ORF8 may mitigate these effects leading to a less serious course of the disease.

LL-37 has not only antimicrobial and antiviral activities but also immunomodulatory properties. It helps to maintain homeostasis of the immune system by regulating chemokine, cytokine production.

Immune responses are promoted through an enhanced antigen presentation by supporting the maturation of monocytes to dendritic cells, in this manner bridging innate and adaptive immunity 41,42 . The peptide is also able to augment the release of pro-inflammatory cytokines by immunocompetent cells and human bronchial epithelial cells 43, 44 . On the other hand, it can diminish the secretion of proinflammatory factors induced by bacterial lipopolysaccharides 45 Meanwhile, vaccines against SARS-CoV-2 have been developed. With extensive vaccination the pandemic will end, but the threat of COVID-19 remains. It is for several reasons challenging to reach herd immunity. The vaccine will not be available to everyone, some individuals have contraindications or are not willing to be vaccinated and others will not develop effective immunity after vaccination. For those patients, prophylactic and therapeutic agents directed against COVID-19 are urgently needed. The combined antiviral, antimicrobial, and immunomodulatory properties are advantages of LL-37 compared to antibodies from reconvalescent donors and should be considered as COVID-19 patients are prone to develop bacterial superinfections. Furthermore, the number of plasma donors limits the quantity of reconvalescent plasma, and the transfer of human material carries the risk of infection. In contrast, LL-37 can be produced synthetically with high purity and in unlimited quantities. Also, the use of antibodies from reconvalescent donors did not result in clinical benefits or in reduction in all-cause mortality 47, 48 . Therefore, the use of vitamin D as an infection prophylaxis and the therapeutic administration of vitamin D for the treatment of COVID-19 patients, and the direct use of LL-37 by inhalation or systemic application could be good alternatives.

In conclusion, we have revealed a biochemical link between vitamin D, LL-37, and COVID-19 severity. SPR analysis demonstrated that LL-37 binds to SARS-CoV-2 S protein and inhibits binding to its receptor hACE2, and most likely viral entry into the cell. This study supports the prophylactic use of vitamin D to induce LL-37 to protect from SARS-CoV-2 infection, and the therapeutic administration of vitamin D for the treatment of COVID-19 patients. Further, our results provide evidence that the direct use of LL-37 by inhalation and systemic application might reduce the severity of COVID-19.

There are no competing financial and/or non-financial interests in relation to the work to report. The authors declare no competing interests.

Coronaviruses post-SARS: update on replication and pathogenesis

Clinical features of patients infected with 2019 novel coronavirus in Wuhan

A Novel Coronavirus Associated with Severe Acute Respiratory Syndrome

Isolation of a Novel Coronavirus from a Man with Pneumonia in Saudi Arabia

Updated Approaches against SARS-CoV-2

Perspective: Vitamin D deficiency and COVID-19 severity -plausibly linked by latitude, ethnicity, impacts on cytokines, ACE2 and thrombosis

Effect of calcifediol treatment and best available therapy versus best available therapy on intensive care unit admission and mortality among patients hospitalized for COVID-19: A pilot randomized clinical study

Vitamin D and COVID-19: It is time to act

Vitamin D and innate immunity

Cutting edge: 1,25-dihydroxyvitamin D3 is a direct inducer of antimicrobial peptide gene expression

Induction of cathelicidin in normal and CF bronchial epithelial cells by 1,25-dihydroxyvitamin D(3)

Human cathelicidin antimicrobial peptide (CAMP) gene is a direct target of the vitamin D receptor and is strongly up-regulated in myeloid cells by 1,25-dihydroxyvitamin D3

Convergence of IL-1beta and VDR activation pathways in human TLR2/1-induced antimicrobial responses

Immunomodulatory Role of the Antimicrobial LL-37 Peptide in Autoimmune Diseases and Viral Infections

Vitamin D and the anti-viral state

Novel Immunoglobulin Domain Proteins Provide Insights into Evolution and Pathogenesis of SARS-CoV-2-Related Viruses

Optimized Sleeping Beauty transposons rapidly generate stable transgenic cell lines

Targeting of bone morphogenetic protein growth factor complexes to fibrillin

A new model for growth factor activation: type II receptors compete with the prodomain for BMP-7

The structure of the antimicrobial human cathelicidin LL-37 shows oligomerization and channel formation in the presence of membrane mimics

Identification of a New Region of SARS-CoV S Protein Critical for Viral Entry

Bromelain Inhibits SARS-CoV-2 Infection in VeroE6 Cells. bioRxiv : the preprint server for biology

Investigation of ACE2 N-terminal fragments binding to SARS-CoV-2 Spike RBD. bioRxiv

Preliminary evaluation of the safety and efficacy of oral human antimicrobial peptide LL-37 in the treatment of patients of COVID-19, a small-scale, single-arm, exploratory safety study. medRxiv

An in Silico Scientific Basis for LL-37 as a Therapeutic and Vitamin D as Preventive for Covid-19. chemRxiv

Antiviral activity and increased host defense against influenza infection elicited by the human cathelicidin LL-37

Enhanced Binding of SARS-CoV-2 Spike Protein to Receptor by Distal Polybasic Cleavage Sites

The peptide antibiotic LL-37/hCAP-18 is expressed in epithelia of the human lung where it has broad antimicrobial activity at the airway surface

Apolipoprotein A-I attenuates LL-37-induced endothelial cell cytotoxicity

Antimicrobial peptide LL-37 is upregulated in chronic nasal inflammatory disease

Increased levels of antimicrobial peptides in tracheal aspirates of newborn infants during infection

Clinical infectious diseases : an official publication of the Infectious Diseases Society of America

Cyclic mechanical stretch down-regulates cathelicidin antimicrobial peptide expression and activates a pro-inflammatory response in human bronchial epithelial cells

Antimicrobial peptides under clinical investigation

Accurate Diagnosis of COVID-19 by a Novel Immunogenic Secreted SARS-CoV-2 orf8 Protein

A unique view of SARS-CoV-2 through the lens of ORF8 protein. bioRxiv

The ORF8 Protein of SARS-CoV-2 Mediates Immune Evasion through Potently Downregulating MHC-I. bioRxiv

The ORF6, ORF8 and nucleocapsid proteins of SARS-CoV-2 inhibit type I interferon signaling pathway

Effects of a major deletion in the SARS-CoV-2 genome on the severity of infection and the inflammatory response: an observational cohort study

Antimicrobial Peptide LL-37 Internalized by Immature Human Dendritic Cells Alters their Phenotype

The cationic antimicrobial peptide LL-37 modulates dendritic cell differentiation and dendritic cell-induced T cell polarization

Host defence peptide LL-37 induces IL-6 expression in human bronchial epithelial cells by activation of the NF-kappaB signaling pathway

Significance of LL-37 on Immunomodulation and Disease Outcome

Modulation of the TLR-Mediated Inflammatory Response by the Endogenous Human Host Defense Peptide LL-37

Cytokine Storm in COVID-19: The Current Evidence and Treatment Strategies

Convalescent plasma in the management of moderate covid-19 in adults in India: open label phase II multicentre randomised controlled trial (PLACID Trial)

Convalescent plasma is ineffective for covid-19

Funding for this study was provided by the Deutsche Forschungsgemeinschaft (DFG) via FOR2722/ C2 to G.S. and FOR2722/ B2 to M.K..

Pending

There are no competing financial and/or non-financial interests concerning to the work to report. The authors declare no competing interests.