The present invention refers to a soluble receptor fragment (SRF) of the ACE-2 receptor, wherein the SRF comprises the peptidase domain (PD) of ACE-2 or a fragment and/or derivates thereof. Moreover, the present invention refers to SRF according to the present invention for use in a method for treatment of the human or animal body by surgery or therapy, as a vaccine or in diagnostic methods practiced on the human or animal body or with fluids or other material from the human or animal body. In addition, the present invention provides SRFs for use in a method of treating and/or preventing a virus infection, in particular a virus infection caused by Coronaviridae, more particularly caused by SARS coronavirus, SARS coronavirus-2, human coronavirus NL63 or SARS-CoV-2. Finally, the present invention relates to a method for capturing viral particles, the method comprises the steps of providing immobilized SRFs and contacting a liquid sample or fluid with the SRFs under conditions for allowing the SRFs to bind the viral particles.

In general, viruses infect a host cell via (1) attachment to a specific receptor on the target cell surface, (2) initiating viral uptake, (3) viral replication, and (4) the production of progeny virus for (5) onward release. The viral receptor on the target cell can be a protein or a carbohydrate or a lipid structure.

Thus, to combat a viral infection, these steps can be targeted by various strategies to prevent or treat a viral infection, whereas prevention also includes vaccination. Most of these strategies address steps of the viral infection cycle after entry into the human body. Examples for this are spike proteins or in general cell entry mechanisms as targets for treatment or vaccination strategies. Other treatment targets are e.g. essential viral enzymes necessary for proliferation e.g. proteases or replication enzymes. Vaccination strategies mostly involve viral surface structures, typically proteins on the viral surface e.g. spike proteins or capsid proteins that can be used to trigger an immune response.

However, drug or vaccine development can be time consuming and are often not successful. Thus, as many severe or even fatal problems occur after viruses have entered the human body, started replication and in human cells and thereafter spread through blood or lymphatic system, another alternative is to prevent the uptake of viruses into the body. To prevent the uptake of viruses, or at least to massively reduce the viral load entering the human body soluble fragments of host cell receptors can be applied e.g. in a washing solution according to the present invention for neutralising the viral receptors and allowing washing out of the neutralised viruses from the place of entry.

A dominant example for viruses having a big impact on human beings is Sars-CoV-2 causing COVID-19: SARS-CoV-2 or severe acute respiratory syndrome coronavirus 2 is the virus strain that causes a respiratory illness known as coronavirus disease 2019 (COVID-19). This virus belongs to the Coronaviridae, a family of lipid-enveloped positive-sensed RNA viruses. SARS- CoV-2 was previously referred to by its provisional name 2019 novel coronavirus (2019- nCoV). SARS-CoV-2 is contagious in humans, and the World Health Organization (WHO) has designated the ongoing pandemic of COVID-19 a Public Health Emergency of International Concern.

The main transmission route of Sars-CoV-2 in humans seems to be droplet infection. Theoretically, contact transfer, mainly in the surrounding of infected persons is also possible. The main transmission however, droplet infection, takes place via droplets with less than 5pm in diameter that arise when coughing and sneezing and are absorbed by the other person through the mucous membranes of the nose, mouth and possibly also the eye.

For Sars-CoV-2 the trimeric spike glycoprotein (S protein) on the virion surface is responsible for mediating receptor recognition and membrane fusion: During viral infection the S protein is cleaved into SI and S2 subunits. Subunit SI contains the receptor binding domain (RBD), which directly binds to the peptidase domain of angiotensin-converting enzyme 2 (ACE-2), whereas subunit S2 is responsible for membrane fusion. When SI binds to the host receptor ACE-2, another cleavage site on S2 is exposed and is cleaved by host proteases, a process that is critical for viral infection. The S protein of Sars-CoV-2 also exploits ACE2 for host infection. The structure of the S protein of Sars-CoV-2 was also solved and showed that the ectodomain of the Sars-CoV-2 S protein binds to the peptidase domain of ACE-2 with high affinity i.e. a dissociation constant (Kd) of~15nM (Wrapp et al. - 2020).

In fact, ACE2 serves as the entry point into cells for further coronaviruses including at least SARS coronavirus, SARS coronavirus-2 and human coronavirus NL63. Angiotensin-converting enzyme 2 (ACE-2) is an enzyme attached to the cell membranes of cells in the lungs, arteries, heart, kidney, and intestines and part of the renin- angiotensin system that maintains the homeostasis of blood pressure and the balance of fluids and salt in the body. ACE-2 lowers blood pressure by hydrolysing angiotensin II (a vasoconstrictor) into angiotensin (1-7) (a vasodilator). ACE-2 counters the activity of the related angiotensin-converting enzyme (ACE) by reducing the amount of angiotensin-II and increasing angiotensin (1-7) making it a promising drug target for treating cardiovascular diseases.

ACE-2 is a zinc containing metalloenzyme located on the surface of endothelial and other cells. ACE-2 protein consists of an N-terminal peptidase M2 (Carboxypeptidase) domain and a C- terminal collectrin renal amino acid transporter domain, which is unstructured. The C-terminal domain of ACE-2 is a membrane anchor, which leads to an exposure of the enzymatically active domain on the surface of cells in lungs and other tissues. The peptidase domain consists of two subdomains (I & II) which enclose the active site and are linked by an alpha helix (Towler et al. -2004). N-terminal subdomain I also contains a Zink ion. The extracellular domain of ACE- 2 is naturally cleaved from the transmembrane domain, and the resulting soluble protein is released into the blood stream and ultimately excreted into urine.

There are many strategies to combat Sars-CoV-2 at basically all stages of the viral life cycle. These strategies are summarised e.g. in the review by Zhang et al. (2020). These strategies summarise various treatment options as well as vaccination attempts. Most of the therapeutic strategies address viral life cycle steps after having entered a human cell. But some attempts also aim on preventing cell entry, either activating the immune system by vaccinating with inactivated viruses or parts of the virus or by blocking interaction of viral protein S with its cellular receptor ACE-2. In more details, said review summarizes hypothetical replication cycle of SARS-CoV-2 as follows: SARS-CoV-2 binds to the ACE2 receptor on the surface of cells using the Spike protein, which subsequently triggers endocytosis. On releasing the viral nucleocapsid to the cytoplasm, encapsidated positive-strand genomic RNA [(+)gRNA] serves as a template to translate polypeptide chains, which are cleaved to non- structural proteins including RNA-dependent RNA polymerase. The single negative strand RNA [(-)gRNA] synthesized from (+)gRNA template is employed to replicate more copies of viral RNAs. Subgenomic RNAs (sgRNAs) are synthesized by discontinuous transcription from the (+)gRNA template and then encode viral structural and accessary proteins, which are subsequently assembled with newly synthesized viral RNA to form new virions. The nascent virions are then transported in secretory vesicles to the plasma membrane and released by exocytosis. In addition, the review describes the following possible targets of anti-COVID-19 drugs: RhACE2, convalescent plasma and JAK inhibitor baricitinib could dampen the binding of the Spike protein on the surface of the SARS-CoV-2 to ACE2 expressed on the cell surface. Lopinavir/ritonavir and favipiravir inhibit the proteolysis of polypeptide chains. Remdesivir inhibits RNA-dependent RNA polymerase. EIDD-2801 could inhibit SARS-CoV-2 replication. iNO and Zinc might inhibit SARS-CoV-2 replication. Vitamin D might induce antimicrobial peptides to reduce SARSCoV-2 replication. Ivermectin could effectively block SARS-CoV-2 growth. Baricitinib could interrupt the passage of SARS-CoV-2 entering cells through inhibition of AAKl-mediated endocytosis. CQ and HCQ inhibit virus/cell fusion process. LHQW and IFNs could block the process of virus replication (RNAs transcription, protein translation, and post-translational modification). Abbreviations: AAK1, adaptor- associated kinase 1; CQ, chloroquine; ER, endoplasmic reticulum; HCQ, hydroxychloroquine sulfate; IFNs, interferons; iNO, inhaled nitric oxide; JAK, janus kinase; LHQW, Lianhua Qingwen; rhACE2, recombinant human angiotensin-converting enzyme 2; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2.

In addition to treatment or vaccination concepts as described above another strategy would be to reduce the viral load in mouth and nose by rinsing with suitable inactivation solution that inactivate and washout the virus. Animal experiments indicate that a short-term exposure to the virus is not enough for becoming sick. This is an interesting option, as there seem to be a certain viral load necessary for infection of a new host. Gargling or rinsing with an inactivation solution could be used to prevent infection in people having had short contact to infected patients, like medical doctors, nurses etc... Alternatively, an inactivation solution could be used to reduce the viral load in the mouth and nose of highly infectious patients to reduce the risk of virus transfer e.g. during medical procedures where a mask cannot be used.

Strategies to block viral entry focus either on antibodies or soluble recombinant human ACE-2 receptor. The authors of Monteil et al. (2020) expressed a large extracellular fragment of ACE- 2 (amino acids 1-740) purified this and could block viral entry into different cells. Others could show that a fusion protein consisting of these extracellular ACE-2 domains to the Fc-part of human IgGl is suited to neutralise the receptor-binding domains of Sars-CoV-2 spike protein in vitro (Lei et al. - 2020). However, use of soluble human recombinant ACE-2 protein in COVID-19 therapy is not without risk, as ACE-2 therapy is expected to result in angiotensin II depletion, therefore attention has to be paid to the impact on blood pressure and kidney function (Alhenc-Gelas et al - 2020).

Thus, there is a need for new and alternative concepts of inactivating viruses such as Sars-CoV-

2.

The problem to be solved by the present invention was thus to provide new means which can be used for inactivating and neutralising viruses such as Coronaviridae, in particular SARS coronavirus, SARS coronavirus-2, human coronavirus NL63 or SARS-CoV-2 including any mutations thereof such as variants from British lineage B.1.1.7, B.1.351 from South Africa, B.1.617 from India or variant B.1.1.28.1 from Brazil, possibly before the virus infects the subject’s cells. Moreover, the problem to be solved by the present invention was the provision of new means which are able to reduce the virus load without having significant side effects as e.g. an impact on blood pressure and kidney function. In addition, the problem to be solved by the present invention was the provision of new methods for detecting virus particles and for washing a liquid sample or fluid wherein the virus load in the liquid sample or fluid is reduced. Thus, the problem to be solved by the present invention is the provision of a preventive and/or therapeutic tool to stop the infection process with Coronaviridae such as SARS-CoV-2 by preventing the virus from entering a human cell. A further problem underlying the present invention is the provision of preventive and therapeutic means which are useful for any mutations of Coronaviridae, in particular SARS coronavirus, SARS coronavirus-2, human coronavirus NL63 or SARS-CoV-2 including variants from British lineage B.1.1.7, B.1.351 from South Africa, B.1.617 from India or variant B.1.1.28.1 from Brazil.

The problem underlying the present invention is solved by the subject matter defined in the claims.

The following figures serve to illustrate the invention.

Figure 1 shows binding of three SRF variants to the spike protein of Sars-CoV-2 immobilised on a biacore chip. Binding was observed for all three proteins tested, with variant 3_ACE2_19- 615_E375Q showing the highest binding response. Variant 4_ACE_2_19-103;301-365 is binding less efficiently and variant 9_ACE2_19-615 shows the lowest binding. Samples measured 1-3 were buffer; sample 4 is variant (3)_ACE2_19-615_E375Q; sample 5 is variant (4)_ ACE_2_19-103;301-365; sample 6 is variant (9)_ACE2_19-615.

Figure 2 shows neutralisation of Sars-Cov-2 virus particles by addition of SRF. Neutralisation was tested with 80 pfu/ml. Variant 9_ACE2_19-615 shows already at InM at minor reduction and a 50% reduction at 250nM. Application of variant 3_ACE2_19-615_E375Q in the neutralisation assay leads to a measurable reduction at all concentrations tested, with a reduction to about 40%.

Figure 3 shows neutralisation of Sars-Cov-2 virus particles by addition of SRF. Neutralisation was tested with 40 pfu/ml. Variant 9_ACE2_19-615, shows a 50% inhibition at 250nM and a full inhibition at 1.5 mM. Application of variant 3_ACE2_19-615_E375Q leads to a significant reduction in the concentration range of 250nM to ImM. At 1.5mM and 2mM an almost complete reduction is observed.

Figure 4 shows neutralisation of Sars-Cov-2 UK-variant (B.1.1.7)_virus particles by addition of SRFs. Neutralisation was tested with 80 pfu/ml. Both variants 9_ACE2_19-615 (A) and variant 3_ACE2_19-615_E375Q (B) show already at 250nM a massive reduction in pfu and a complete reduction at ImM.

The term „SARS-CoV-2“ as used herein refers to the severe acute respiratory syndrome coronavirus 2 which is the virus strain that causes a respiratory illness known as coronavirus disease 2019 (COVID-19). This virus belongs to the Corona viridae, a family of lipid-enveloped positive-sensed RNA viruses. SARS-CoV-2 was previously referred to by its provisional name 2019 novel coronavirus (2019-nCoV). As viruses show occurrence of mutations, the term “SARS-CoV-2” as used herein refers preferably to all variants of SARS-CoV-2 as described herein but also to all variants that show either single mutations, or multiple mutations or combinations of mutations, but are still named as SARS-CoV-2.

The terms “SARS-CoV-2 variant fromBritish lineage B.1.1.7”, “SARS-CoV-2 variant B.1.351 from South Africa” or “SARS-CoV-2 variant B.1.1.28.1 from Brazil” as used herein refer preferably to SARS-CoV-2 variants B.1.1.7, B.1.351 and B.1.1.28.1 as described e.g. in Singh et al. (2021).

The terms “SARS-CoV-2 B.1.617 from India” as used herein refers preferably to a variant having mutations Dll ID, G142D, L452R, E484Q, D614G and P681R, in the spike protein as described in Cherian et al. (2021).

The term “ACE-2” as used herein refers to Angiotensin-converting enzyme 2 (ACE-2). It is an enzyme attached to the cell membranes of cells e.g. in the lungs, arteries, heart, kidney, and intestines and part of the renin- angiotensin system that maintains the homeostasis of blood pressure and the balance of fluids and salt in the body. ACE-2 lowers blood pressure by hydrolysing angiotensin II (a vasoconstrictor) into angiotensin (1-7) (a vasodilator). ACE-2 counters the activity of the related angiotensin-converting enzyme (ACE) by reducing the amount of angiotensin-II and increasing angiotensin (1-7) making it a promising drug target for treating cardiovascular diseases. ACE-2 is a zinc containing metalloenzyme located on the surface of endothelial and other cells. ACE-2 protein consists of an N-terminal peptidase M2 (Carboxypeptidase) domain and a C-terminal collectrin renal amino acid transporter domain, which is unstructured. The C-terminal domain of ACE-2 is a membrane anchor, which leads to an exposure of the enzymatically active domain on the surface of cells in lungs and other tissues. The peptidase domain consists of two subdomains (I & II) which enclose the active site and are linked by an alpha helix (Towler et al. - 2004). The two subdomains are defined as follows (Towler et al. - 2004): The N terminus- and zinc-containing subdomain I, composed of residues 19-102, 290-397, and 417-430; and the C terminus-containing subdomain II, composed of residues 103-289, 398-416, and 431-615. N-terminal subdomain I also contains a Zink ion. The structure of ACE-2 and its peptidase and neck domain are preferably as described in Yan et al. (2020). Preferably ACE-2 comprises an amino acid sequence as shown in SEQ ID NO: 1. It comprises preferably a peptidase domain as shown in SEQ ID NO: 3 or 6 and a neck domain as shown in SEQ ID NO: 2.

The terms “peptidase domain of ACE-2”, or “PD of ACE-2” as used herein refers to the peptidase domain of ACE-2. The PD of ACE-2 lacks the neck domain of ACE-2. Preferably, the PD of ACE-2 has an amino acid sequence as shown in SEQ ID NO: 3 or 6. The PD of ACE- 2 may comprise alpha helices, beta-sheets and/or beta-turns. The PD of ACE-2 has an active site. The amino acid residues of PD of ACE-2 as listed in the following table are involved in the activity:

Residues involved in binding (with reference the PD sequence as shown in SEQ ID NO:3):

The term „soluble“ as used herein in connection with PD or SRF means preferably that the protein is in solution at a concentration of about 1 pg/ml to 1000 mg/mL in a physiological buffer e.g. PBS at a temperature of about 20°C to about 40°C. More preferably, it is in solution at a concentration of at least about 2 pg/mL, 5 pg/mL, 10 pg/ml, 20 pg/ml, 30 pg/ml, 40 pg/ml, 50pg/ml, 60 pg/ml, 70 pg/ml, 80 pg/ml, 90 pg/ml, 100pg/ml, 110pg/ml, 120pg/ml, 150pg/ml or 200pg/ml.

The term “polypeptide” or “protein” as used herein refers in particular to a polymer of amino acids linked by peptide bonds in a specific sequence. The amino acid residues of a polypeptide may be modified by e.g. covalent attachments of various groups such as carbohydrates and phosphate. Other substances may be more loosely associated with the polypeptide, such as heme or lipid, giving rise to conjugated polypeptides which are also comprised by the term “polypeptide” or “protein” as used herein. The term "polypeptide" or “protein” does encompass embodiments of polypeptides or proteins which exhibit optionally modifications typically used in the art, e.g. biotinylation, acetylation, pegylation, chemical changes of the amino-, SH- or carboxyl-groups (e.g. protecting groups) etc. The term "polypeptide" or “protein”, as used herein, is not limited to a specific length of the amino acid polymer chain, but typically the polypeptide or protein will exhibit a length of more than about 50 amino acids, more than about 100 amino acids or even more than about 150 amino acids. Usually, but not necessarily, a typical polypeptide of the present invention will not exceed about 850 amino acids in length. The term “polypeptide” or “protein” as used herein may also comprises dimer fusions of polypeptides or proteins. In case of dimer fusions, a typical polypeptide of the present invention will usually not exceed about 1600 or 1700 amino acids in length.

The term “derivative”, as used herein, refers to an amino acid sequence which exhibits, in comparison to the respective reference sequence, one or more additions, deletions, insertions, and/or substitutions and/or combinations thereof. This includes for example combinations of deletions/insertions, insertions/deletions, deletions/additions, additions/deletions, insertion/ additions, additions/insertions etc. A person skilled in the art will however understand that the presence of an amino acid residue at a certain position of the derivative sequence which is different from the one that is present at the respective same position in the reference sequence is not a combination of, for example, a deletion and a subsequent insertion at the same position but is a substitution as defined herein. Rather, if reference is made herein to combinations of one or more of additions, deletions, insertions, and substitutions, then combination of changes at distinct positions in the sequence are intended, e.g. an addition at the N-terminus and an intrasequential deletion. Such derived sequence will exhibit a certain level of sequence identity with the respective reference sequence, for example a given SEQ ID NO, which is preferably at least 60%, such as at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%. Preferred derivatives are fragments of the parent molecule, for example a given SEQ ID NO, retaining the activity of the parent molecule, i.e. exhibiting on a general level same activity as the respective parent molecule if not otherwise stated. However, said activity can be the same, higher or lower as the respective parent molecule. Also preferred derivatives are those resulting from conservative amino acid substitutions within the parent sequence, for example a given SEQ ID NO, again retaining the activity of the parent molecule on a general level.

As used herein, the term "% sequence identity", has to be understood as follows: Two sequences to be compared are aligned to give a maximum correlation between the sequences. This may include inserting "gaps" in either one or both sequences, to enhance the degree of alignment. A % identity may then be determined over the whole length of each of the sequences being compared (so-called global alignment), that is particularly suitable for sequences of the same or similar length, or over shorter, defined lengths (so-called local alignment), that is more suitable for sequences of unequal length. In the above context, an amino acid sequence having a "sequence identity" of at least, for example, 95% to a query amino acid sequence, is intended to mean that the sequence of the subject amino acid sequence is identical to the query sequence except that the subject amino acid sequence may include up to five amino acid alterations per each 100 amino acids of the query amino acid sequence. In other words, to obtain an amino acid sequence having a sequence of at least 95% identity to a query amino acid sequence, up to 5% (5 of 100) of the amino acid residues in the subject sequence may be inserted or substituted with another amino acid or deleted. Methods for comparing the identity and homology of two or more sequences are well known in the art. The percentage to which two sequences are identical can for example be determined by using a mathematical algorithm. A preferred, but not limiting, example of a mathematical algorithm which can be used is the algorithm of Karlin et al. (1993), PNAS USA, 90:5873-5877. Such an algorithm is integrated in the BLAST family of programs, e.g. BLAST or NBLAST program (see also Altschul et al, 1990, J. Mol. Biol. 215, 403-410 or Altschul et al. (1997), Nucleic Acids Res, 25:3389-3402), accessible through the home page of the NCBI at world wide web site ncbi.nlm.nih.gov) and FASTA (Pearson (1990), Methods Enzymol. 83, 63-98; Pearson and Lipman (1988), Proc. Natl. Acad. Sci. U. S. A 85, 2444-2448.). Sequences which are identical to other sequences to a certain extent can be identified by these programmes. Furthermore, programs available in the Wisconsin Sequence Analysis Package, version 9.1 (Devereux et al, 1984, Nucleic Acids Res., 387-395), for example the programs BESTFIT and GAP, may be used to determine the % identity between two polypeptide sequences. BESTFIT uses the "local homology" algorithm of (Smith and Waterman (1981), J. Mol. Biol. 147, 195-197.) and finds the best single region of similarity between two sequences. If herein reference is made to an amino acid sequence sharing a particular extent of sequence identity to a reference sequence, then said difference in sequence is preferably due to conservative amino acid substitutions. Preferably, such sequence retains the activity of the reference sequence, e.g. albeit maybe at a slower rate. In addition, if reference is made herein to a sequence sharing "at least" at certain percentage of sequence identity, then 100% sequence identity are preferably not encompassed.

"Conservative amino acid substitutions", as used herein, may occur within a group of amino acids which have sufficiently similar physicochemical properties, so that a substitution between members of the group will preserve the biological activity of the molecule (see e.g. Grantham, R. (1974), Science 185, 862-864). Particularly, conservative amino acid substitutions are preferably substitutions in which the amino acids originate from the same class of amino acids (e.g. basic amino acids, acidic amino acids, polar amino acids, amino acids with aliphatic side chains, amino acids with positively or negatively charged side chains, amino acids with aromatic groups in the side chains, amino acids the side chains of which can enter into hydrogen bridges, e.g. side chains which have a hydroxyl function, etc.)· Conservative substitutions are in the present case for example substituting a basic amino acid residue (Lys, Arg, His) for another basic amino acid residue (Lys, Arg, His), substituting an aliphatic amino acid residue (Gly, Ala, Val, Leu, lie) for another aliphatic amino acid residue, substituting an aromatic amino acid residue (Phe, Tyr, Trp) for another aromatic amino acid residue, substituting threonine by serine or leucine by isoleucine. Further conservative amino acid exchanges will be known to the person skilled in the art.

The term “deletion” as used herein refers preferably to the absence of 1, 2, 3, 4, 5 (or even more than 5) continuous amino acid residues in the derivative sequence in comparison to the respective reference sequence, either intrasequentially or at the N- or C-terminus. A derivative of the present invention may exhibit one, two or more of such deletions.

The term “insertion” as used herein refers preferably to the additional intrasequential presence of 1, 2, 3, 4, 5 (or even more than 5) continuous amino acid residues in the derivative sequence in comparison to the respective reference sequence. A derivative of the present invention may exhibit one, two or more of such insertions.

The term “addition” as used herein refers preferably to the additional presence of 1, 2, 3, 4, 5 (or even more than 5) continuous amino acid residues at the N- and/or C-terminus of the derivative sequence in comparison to the respective reference sequence.

The term “substitution” as used herein refers to the presence of an amino acid residue at a certain position of the derivative sequence which is different from the amino acid residue which is present or absent at the corresponding position in the reference sequence. A derivative of the present invention may exhibit 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more of such substitutions. As mentioned above, preferably such substitutions are conservative substitutions.

The term “mutation” in respect of Coronaviridae, SARS coronavirus, SARS coronavirus-2, human coronavirus NL63 or SARS-CoV-2 refers to any mutations of any of said viruses including variants from British lineage B.1.1.7, variant B.1.351 from South Africa, variant B.1.617 from India or variant B.1.1.28.1 from Brazil. In particular, the term “mutation” of Coronaviridae, SARS coronavirus, SARS coronavirus-2, human coronavirus NL63 or SARS- CoV-2 refers to any mutations of the respective viruses. Said mutations may comprise both single mutations or multiple mutations. Said mutations may occur in the spike protein and/or another viral protein and/or other viral proteins. The mutation or mutations may be in any part of the virus and may be silent mutations having no effect or non- silent mutations having an effect on viral properties like host cell binding, replication, viral stability etc.

The term “infection” as used herein preferably means that the virus has entered a host cell, in particular a mammalian or a human host cell, and is ready to replicate.

The term “host cell” as used herein refers preferably to any eukaryotic, animal, mammalian or human cell. It further refers preferably to any eukaryotic, animal, mammalian or human cell having an ACE-2 receptor. In particular, the term „host cell“ as used herein refers to human, cats, tiger, poultry and mouse cells having an ACE-2 receptor and all host cells which are able to be infected by SARS-CoV-2/COVID-19 or any of the mutations or variants thereof as described herein as e.g. described in Stout et al. (2020).

As used herein, the term “tag” refers to an amino acid sequence, which is typically in the art fused to or included in another amino acid sequence for a) improving expression of the overall amino acid sequence or polypeptide, b) facilitating purification of the overall amino acid sequence or polypeptide, c) facilitating immobilisation of the overall amino acid sequence or polypeptide, and/or d) facilitating detection of the overall amino acid sequence or polypeptide. Examples for tags are His tags, such as His5-tags, His6-tags, His7-tags, His8-tags, His9-tags, HislO-tags, Hisl 1-tags, Hisl2-tags, Hisl6-tags and His20-tags, Strep-tags, Avi-tags, Myc-tags, GST-tags, JS-tags, cystein-tags, FLAG-tags, HA-tags, thioredoxin or maltose binding proteins (MBP), CAT, GFP, YFP, etc. The person skilled in the art will know a vast number of tags suitable for different technical applications. The tag may for example make such tagged polypeptide suitable for e.g. antibody binding in different ELISA assay formats or other technical applications.

The term "comprising" as used herein shall not be construed as being limited to the meaning "consisting of" (i.e. excluding the presence of additional other matter). Rather, "comprising" implies that optionally additional matter may be present. The term "comprising" encompasses as particularly envisioned embodiments falling within its scope "consisting of" (i.e. excluding the presence of additional other matter) and "comprising but not consisting of' (i.e. requiring the presence of additional other matter), with the former being more preferred.

The abbreviation “aa” as used herein refers preferably to the term “amino acids” and is in particular used in respect of the position of certain amino acid in a given amino acid sequence. For example, “aa 19-103 of SEQ ID NO: 3” refers to the amino acid sequence starting with the 19 ^th and ending with the 103th amino acid residue of the amino acid sequence according to SEQ ID NO: 3.

In a first object of the present invention it is envisaged to provide a soluble receptor fragment (SRF) of the ACE-2 receptor, wherein the SRF comprises the peptidase domain (PD) of ACE- 2. Alternatively, the SRF according to the present invention comprises a fragment and/or derivative of the PD of ACE-2, in particular one, two or more fragment(s) of the PD of ACE- 2. If the SRF according to the present invention comprises two or more fragments, the terms “fragment combination of two or more fragments” or simply “fragment combination” may be used in the present disclosure to describe this embodiment. In a further preferred embodiment of the present invention, the SRF comprises a derivative of the PD of ACE-2 or a derivative of the fragment or fragment combination of the PD of ACE-2. In a preferred embodiment of the present invention the SRF consists of the PD of ACE-2 or one, two or more fragment(s) thereof or a derivative of the PD of ACE-2 or a fragment thereof. In a further preferred embodiment, the SRF comprises or consists of the PD of ACE-2 or one, two or more fragment(s) thereof or a derivative of the PD of ACE-2 or a fragment thereof wherein the active site is inactivated by one or more mutation.

The fragment, the fragment combination and/or derivate of the PD of ACE-2 of the SRF according to the present invention has basically the same or a higher binding affinity or property of binding to the receptor-binding cleft of a virus spike protein, in particular a virus spike protein S, as the wild-type full length PD of ACE-2. Basically, the same means that the binding affinity or property of binding to the receptor-binding cleft of the same virus spike protein is in the range of about 70% to about 150%, more preferably of about 80% to about 130%, or of about 90% to about 120% of the wild-type full length PD of ACE-2 as e.g. shown in SEQ ID NO: 3. In a preferred embodiment of the present invention any fragment, fragment combination and/or derivative of the PD of ACE-2 which effectively binds to the receptor-binding cleft of a virus spike protein S of a SARS coronavirus, a SARS coronavirus-2, a human coronavirus NL63 or a SARS-CoV-2 or any mutation thereof is a fragment, fragment combination or derivative of an SRF according to the present invention. Binding may be considered to be effective if the binding affinity of the tested derivative, fragment or fragment combination is as least 50%, 60%, 70%, 80%, 90%, 100%, 110% or 120% of the binding affinity of (9)_ACE2_19-615 according to SEQ ID NO: 12 as tested in the Examples herein.

The inventors of the present invention have surprisingly found that SRFs according to the present invention bind to the receptor-binding cleft of a virus spike protein, in particular to the receptor-binding cleft of a virus spike protein S of Coronaviridae. In a preferred embodiment, the SRF binds to the receptor-binding cleft of a virus spike protein S of a SARS coronavirus, a SARS corona virus-2, a human coronavirus NL63 or a SARS-CoV-2. In an especially preferred embodiment the SRF binds to the receptor binding cleft of a virus spike protein S of SARS- CoV-2. By said binding the virus is prevented from entering a host cell, preferably a human cell. The consequence is that virus replication cannot start and thus, no extended numbers of viral particles can be produced by the host cells as e.g. human cells.

Preferably, the PD of ACE-2 of the SRF according to the present invention is a soluble expressed isolated PD of ACE-2 or a soluble expressed isolated fragment, fragment combination and/or derivates thereof. Using a fragment or fragment combination of the full- length PD of ACE-2 may result in increasing the solubility of the SRF. Said fragment or fragment combination is preferably designed by deleting one, two three, four, five or more structural elements of the PD of ACE-2 which are not essential for the binding activity of the PD to the receptor-binding cleft of the virus spike protein, in particular of the virus spike protein S. Structural elements of the PD of ACE-2 are for example those which are required forming the alpha helices, beta-sheets and beta-turns. Alternatively or in addition, the solubility of the PD of ACE-2 or a derivative or fragment thereof may be increased by 1, 2, 3, 4, 5 or more mutations such as insertions, substitutions, deletions or additions. Alternatively or in addition, the solubility of the PD of ACE-2 or a derivative or fragment thereof may be increased by removing glycosylation sites.

In a particularly preferred embodiment of the present invention the SFR may comprise any fragment of the PD of ACE-2 which binds to the receptor-binding cleft of the virus spike protein, in particular of the virus spike protein S and inhibits the binding of the spike protein S to a host cell. Preferably, the SFR comprises a fragment or a first fragment of the PD of ACE-2 having a length of at least 80, 81, 82, 83, 84, 85, 110, 111, 112, 113, 114, 580, 581, 582, 583, 584, 593, 594, 595, 596 or 597 amino acid residues. Preferably the SRF comprises a fragment of the PD of ACE-2 having a length of at most 614, 615, 616, 617, 618, 619, 620, 582, 583, 584, 585, 586, 587, 595, 596, 597, 598, 599 or 600 amino acid residues. In a preferred embodiment, the SFR comprises a fragment or a first fragment of the PD of ACE-2 having a length of about 80 to about 600 amino acids, more preferably of about 84 to about 597 amino acids.

If the SFR according to the present invention comprises two or more fragments of the PD of ACE-2, the first fragment has preferably a length of about 80 to 120 amino acid residues, or of about 84 to about 114 amino acid residues. Particularly preferred fragments have a length of 80, 81, 82, 83, 84, 85, 86, 87, 110, 111, 112 113, 114, 115 or 116 amino acid residues.

If the SFR according to the present invention comprises a second fragment of the PD of ACE- 2, said second fragment has preferably a length of about 50 to about 150, about 60 to about 130 amino acid, or about 64 to about 125 amino acid residues. Particularly preferred fragments have a length of 60, 61, 62, 63, 64, 65, 66, 67, 82, 83, 84, 85, 86, 87, 120, 121, 122, 123, 124, 125, 126, 127 or 128 amino acid residues.

In a preferred embodiment of the present invention, the SRF comprises at least 5, 6, 7, 8, 9, 10, 11, 12, 13 or all 14 of the Sars-CoV-2 contacting residues Q24, T27, F28, D30, K31, H34, E35, E37, D38, Y41, Q42, L79, M82, Y83 of SEQ ID NO: 3. Preferably, the SRF comprises at least 60%, 70%, 80% or 90% of the above listed residues.

In a further preferred embodiment of the present invention, the SRF comprises at least 5, 6, 7, 8, 9, 10, 11, 12 or all 13 the Sars-CoV contacting residues Q24, T27, F28, K31, H34, E37, D38, Y41, Q42, F45, F79, M82, Y83 of SEQ ID NO: 3.

In a further preferred embodiment of the present invention, the SRF comprises 3, 4, 5, 6, 7, 8, 9 or all 10 of the alpha-helical structures of the PD of ACE-2 as shown in SEQ ID NO: 34 - 43 or a derivative thereof. A derivative of any of the amino acid sequences according to SEQ ID NO: 34-43 comprises preferably 1, 2, 3, 4 or 5 deletions or mutations under the provision that it is still able to form an alpha-helical structure. The deletions may be within the given amino acid sequences and/or at one or both ends. The derivative of SEQ ID NO: 34, comprises preferably at least 5, 6, 7, 8, 9, 10 or all 11 of the Sars-CoV-2 contacting residues Q24, T27, F28, D30, K31, H34, E35, E37, D38, Y41 and Q42 of SEQ ID NO:3. The derivative of SEQ ID NO: 40 comprises preferably the Sars-CoV-2 contacting residue N330 of SEQ ID NO:3 and/or 1, 2 or 3 of the Sars-CoV contacting residues Q325, E329 and N330 of SEQ ID NO:3.

In a preferred embodiment of the present invention, the SRF comprises at least the alpha-helical structures of aa 21-53 of SEQ ID NO: 3, as shown in SEQ ID NO: 34 or a derivative thereof as defined above, aa 56-80 of SEQ ID NO: 3, as shown in SEQ ID NO: 35 or a derivative thereof as defined above, and aa 91-100 of SEQ ID NO: 3, as shown in SEQ ID NO: 36 or a derivative thereof as defined above.

Examples of such SFRs are SFRs comprising an amino acid sequence as shown in SEQ ID NO: 8 or 13 but also as shown in SEQ ID NO: 4-14, 17-22, 25-27 and 29-31. In a further preferred embodiment of the present invention, the SRF comprises additionally a further alpha-helical structure, namely aa 110-129 of SEQ ID NO: 3, as shown in SEQ ID NO: 37 or a derivative thereof as defined above. Examples of such SFRs are SFRs comprising an amino acid sequence as shown in SEQ ID NO: 9 or 14 but also shown in SEQ ID NO: 4-6, 10, 12 and 17-19.

If the SRF of the present invention comprises a second fragment of the PD of ACE-2, said second fragment comprises preferably an alpha-helical structure of aa 304-318 of SEQ ID NO: 3, as shown in SEQ ID NO: 39 or a derivative thereof as defined above and/or aa 325-330 of SEQ ID NO: 3, as shown in SEQ ID NO: 40 or a derivative thereof as defined above.

An example of such a second fragment is shown in SEQ ID NO: 15. Examples of SRFs comprising such a second fragment are shown in SEQ ID NO: 4-7, 10-12, 17-22 and 25-27. In a further preferred embodiment of the present invention, the second fragment of the SRF comprises an alpha-helical structure namely aa 366-385 of SEQ ID NO: 3, as shown in SEQ ID NO: 41 or a derivative thereof as defined above. An example of such a second fragment is shown in SEQ ID NO: 23. Examples of SRFs comprising such a second fragment are shown SEQ ID NO: 4-6, 10-12, 17-22, 25-27 and 29-31. In a further preferred embodiment of the present invention, the second fragment of the SRF comprises an alpha-helical structure of aa 400-412 of SEQ ID NO: 3, as shown in SEQ ID NO: 42 or a derivative thereof as defined above and/or aa 415-421 of SEQ ID NO: 3, as shown in SEQ ID NO: 43 or a derivative thereof. An example of such a second fragment is an amino acid sequence as shown in SEQ ID NO: 28. Examples of SRFs comprising such a second fragment are shown in SEQ ID NO: 4-6, 10, 12, 17-19, 25-27 and 29-31.

In a preferred embodiment of the present invention, the SRF further comprises 1, 2, 3, 4, 5 or 6 of the Sars-CoV-2 contacting residues N330, K353, G354, D355, R357, R393 of SEQ ID: 3.

In a further preferred embodiment of the present invention, the SRF comprises 1, 2, 3, 4, 5, 6 or 7 of the Sars-CoV contacting residues Q325, E329, N330, K353, G354, D355, R357 of SEQ ID NO: 3.

In a particularly preferred embodiment, the SFR according to the present invention comprise at least aa 19-103 of the PD of ACE-2 (with respect to the amino acid sequence as shown in SEQ ID NO: 3). Accordingly, the SFR according to the present invention comprises at least an amino acid sequence as shown in SEQ ID NO: 8 or a derivative thereof. Said derivative comprises preferably at least 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 of residues Q6, T9, F10, D12, K13, H16, E17, E19, D20, Y23, Q24, L61, M64, Y65 of SEQ ID NO: 8 and preferably at least the alpha- helical structures of

SEQ ID NO: 34 or a derivative thereof,

SEQ ID NO: 35 or a derivative thereof, and SEQ ID NO: 36 or a derivative thereof.

The PD of ACE-2 or a fragment, fragment combination and/or derivative thereof of the SRF according to the present invention may be expressed in any suitable expression systems such as in Baculovirus cells, HEK293 cells, CHO or other eukaryotic cells. Alternatively, the SRF proteins may be expressed in E.coli. For purifying the expressed SRF proteins affinity tags may be used such as His-Tag or Strep-Tag or other standard purification techniques. Accordingly, in a preferred embodiment of the present invention the SRF according to the present invention may comprise a tag, preferably an affinity tag such as a His-Tag, Strep-Tag, MBP-tag or any other tag used for standard purification techniques.

In a further preferred embodiment of the present invention, the SRF may additionally comprise a methionine as translation start signal.

The PD of ACE-2 has preferably an amino acid sequence according to SEQ ID NO: 3 or 6. In a preferred embodiment of the present invention the SRF consists of or comprises an amino acid sequence according to SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6 or a fragment, fragment combination or derivative of the PD of ACE-2 or a fragment thereof. A SRF consisting of or comprising an amino acid sequence according to SEQ ID NO: 4 is a preferred example of a SRF consisting of or comprising a fragment of the amino acid sequence according to SEQ ID NO: 3 as it lacks amino acids 1-18 of SEQ ID NO: 3. An example for a SRF comprising a sequence as shown in SEQ ID NO: 4 is a SRF consisting of or comprising a sequence as shown in SEQ ID NO: 12. The SRF having a sequence as shown in SEQ ID NO: 12 comprises in addition to a sequence as shown in SEQ ID NO:4 a methionine as translation start signal and a tag, namely a His-tag.

An SRF consisting of or comprising an amino acid sequence according to SEQ ID NO: 6 is a further preferred example of an SRF consisting of or comprising a fragment of the amino acid sequence according to SEQ ID NO: 3 as it consists of amino acids 2-615 of SEQ ID NO: 3 and therefore lacks amino acids 1 of SEQ ID NO: 3, i.e. it lacks methionine as translation start signal.

A SRF consisting of or comprising an amino acid sequence according to SEQ ID NO: 8 is a further preferred example of a SRF consisting of or comprising a fragment of the amino acid sequence according to SEQ ID NO: 3 as SEQ ID NO: 8 consists of amino acids 19-103 of SEQ ID NO: 3 and therefore lacks amino acids 1-18 and 104-615 of SEQ ID NO: 3. An example for a SRF comprising a sequence as shown in SEQ ID NO: 8 is a SRF consisting of or comprising a sequence as shown in SEQ ID NO: 13. The SRF having a sequence as shown in SEQ ID NO: 13 comprises in addition to a sequence as shown in SEQ ID NO: 8 a methionine as translation start signal and a tag, namely a His-tag. A SRF consisting of or comprising an amino acid sequence according to SEQ ID NO: 9 is a further preferred example of a SRF consisting of or comprising a fragment of the amino acid sequence according to SEQ ID NO: 3 as SEQ ID NO: 9 consists of amino acids 19-132 of SEQ ID NO: 3 and therefore lacks amino acids 1-18 and 133-615 of SEQ ID NO: 3. An example for a SRF comprising a sequence as shown in SEQ ID NO: 9 is a SRF consisting of or comprising a sequence as shown in SEQ ID NO: 14. The SRF having a sequence as shown in SEQ ID NO: 14 comprises in addition to a sequence as shown in SEQ ID NO:9 a methionine as translation start signal and a tag, namely a His-tag.

A SRF consisting of or comprising an amino acid sequence according to SEQ ID NO: 17 is a further preferred example of a SRF consisting of or comprising a fragment of the amino acid sequence according to SEQ ID NO: 3 as SEQ ID NO: 17 consists of amino acids 19-602 of SEQ ID NO: 3 and therefore lacks amino acids 1-18 and 603-615 of SEQ ID NO: 3. Examples for a SRF comprising a derivative of the fragment as shown in SEQ ID NO: 17 are SRFs consisting of or comprising a sequence as shown in SEQ ID NO: 18 or 19. The SRF having a sequence as shown in SEQ ID NO: 18 has a substitution Glu375Gln, i.e. a substitution of glutamic acid to glutamine at position 375 in respect of the sequence as shown in SEQ ID NO: 3 i.e. at position 357 of SEQ ID NO: 18. The SRF having a sequence as shown in SEQ ID NO: 19 comprises in addition to a sequence as shown in SEQ ID NO: 18 a methionine as translation start signal and a tag, namely a His-tag.

In a further preferred embodiment of the present invention, the SRF comprises more than one fragment of the amino acid sequence according to SEQ ID NO: 3, i.e. a fragment combination of two, three or more fragments of the amino acid sequence according to SEQ ID NO: 3. In a particularly preferred embodiment the SRF according to the present invention comprises two fragments of the amino acid sequence according to SEQ ID NO: 3. The fragments of such a fragment combination can be directly bound to each other or they may be linked together by a linker. Said linker should be flexible enough to allow binding to subunits of a trimeric spike or to subunits of different spike trimers. The linker may be a linker as it is already included in the ACE-2 receptor linking structural modules like alpha-helices in the full-length protein. Alternatively, it may be a linker designed to be stable, flexible enough for allowing the variant to fold into a native-like structure and resistant against protease cleavage to increase half-life of the variant. Such a linker has preferably a length of about 5 to about 50 amino acid residues, about 10 to about 30 amino acid residues or about 15 to 25 amino acid residues. In a preferred embodiment, the SRF according to the present invention comprises a fragment combination of a first and a second fragment of SEQ ID NO: 3, wherein the first fragment is selected from the amino acid sequences as shown in SEQ ID NO: 8 or a derivative thereof and the second fragment is selected from the amino acid sequences as shown in SEQ ID NO: 15, 23, 28 and 32 or each a derivative thereof. In a further preferred embodiment, the SRF according to the present invention comprises a fragment combination of a first and a second fragment of SEQ ID NO: 3, wherein the first fragment is selected from the amino acid sequences as shown in SEQ ID NO: 9 or a derivative thereof and the second fragment is selected from the amino acid sequences as shown in SEQ ID NO: 15, 23, 28 and 32 or each a derivative thereof. In a further preferred embodiment, the second fragment according to SEQ ID NO: 23, 28 and 32 comprises the substitution Glu375Gln, i.e. a substitution of glutamic acid to glutamine at position 375 in respect of the sequence as shown in SEQ ID NO: 3.

Preferably, the first fragment and the second fragment of the fragment combination are linked by a linker selected from the amino acid sequences as shown in SEQ ID NO: 16, 24 and 33.

In a preferred embodiment, the SRF according to the present invention comprises a fragment combination of a first and a second fragment of SEQ ID NO: 3, wherein the first fragment consists of an amino acid sequence according to SEQ ID NO: 8 or a derivative thereof and the second fragment consists of an amino acid sequence according to SEQ ID NO: 15 or a derivative thereof. In a further preferred embodiment, said first and second fragment are linked by linker having an amino acid sequence according to SEQ ID NO: 16. In a further preferred embodiment the SRF comprises or consists of an amino acid sequence according to SEQ ID NO: 7 or a derivative thereof. An example for a SRF comprising a sequence as shown in SEQ ID NO: 7 is a SRF consisting of or comprising a sequence as shown in SEQ ID NO: 11. The SRF having a sequence as shown in SEQ ID NO: 11 comprises in addition to a sequence as shown in SEQ ID NO: 7 a methionine as translation start signal and a tag, namely a His-tag.

In a further preferred embodiment, the SRF according to the present invention comprises a fragment combination as shown in SEQ ID NO: 20, 21, 22, 25, 26, 27, 29, 30, 31 or each a derivative thereof. A derivative in terms of SEQ ID NO: 3-14, 17-22, 25-27, 29-31 refers preferably to a derivative having at least 60, 70, 80, 90, 95 or 98% sequence identity with the given sequence, wherein said derivative comprises preferably at least 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 of Q24, T27, F28, D30, K31, H34, E35, E37, D38, Y41, Q42, L79, M82, Y83 of SEQ ID NO: 3 and at least the alpha-helical structures of

SEQ ID NO: 34 or a derivative thereof,

SEQ ID NO: 35 or a derivative thereof, and SEQ ID NO: 36 or a derivative thereof.

A derivative in terms of SEQ ID NO: 34-43 refers preferably to a derivative comprising 1, 2, 3, 4 or 5 deletions or mutations as e.g. substitutions, in particular conservative amino acid substitutions, under the provision that the derivative it is still able to form an alpha-helical structure.

Preferably, the SRF according to the present invention comprises in addition to any of the fragments or derivatives of the PD of ACE-2 as described herein no further domains or sequences of ACE2, or parts of at least 10, at least 15, at least 20, at least 30, at least 40 at least 50, at least 60 or at least 70 consecutive amino acids thereof. In particular, the SFR according to the present invention does preferably not comprise further domains or sequences of aa 620- 805 of ACE2 as shown in SEQ ID NO: 1 or parts of at least 10, at least 15, at least 20, at least 30, at least 40 at least 50, at least 60 or at least 70 consecutive amino acids thereof.

Therefore, the term “comprising” in connection with the definition of the SFR according to the present invention means that the SFR may comprise in addition to any of the fragments or derivatives of the PD of ACE-2 as described herein in particular one or more of the following components:

- methionine as translation start signal

- one or more tags

- one or more linkers

- proteins, antibodies or fragments thereof, other compounds or molecules as further defined below but no further domains or sequences of ACE2 as defined above. In a preferred embodiment of the present invention, the SFR according to the present invention does not comprise in addition to any of the fragments or derivatives of the PD of ACE-2 as described herein more than 14, 18, more preferably more than 31, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550 or 600 amino acids. Preferably none of these additional amino acids comprise domains or sequences of ACE2 as defined above.

The inventors of the present invention have surprisingly found out that Coronaviridae such as the Sars-CoV-2 virus can be inactivated or neutralised with soluble receptor fragments (SRF) of the ACE-2 receptor. These SRF consist of or comprises the soluble expressed isolated peptidase domain (PD) of ACE-2 or a fragment, fragment combination or derivative thereof only, were the neck domain described in the structure by Yan et al. (2020) was cleaved off. Accordingly, the SFRs according to the present invention do not comprise an amino acid sequence as shown in SEQ ID NO: 2. To avoid impact on blood pressure and kidney function the active site of the PD is preferably rendered inactive, as the goal according to the present invention is to block the receptor-binding cleft of Coronaviridae such as the Sars-CoV-2 spike protein S without a significant impact on blood pressure or kidney function. Moreover, to prevent the uptake of viruses, or at least to massively reduce the viral load entering the human body, the inventors have found that soluble fragments of host cell receptors, in particular the SRF according to the present invention, can be applied in a washing solution according to the present invention for neutralising the viral receptors and preferably allowing washing out of the neutralised viruses from the place of entry.

Thus, in a highly preferred embodiment of the present invention the derivative, fragment and/or fragment combination of the PD is a PD, fragment or fragment combination of the PD where the active site of the PD is inactivated preferably by 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more mutations such as insertions, additions, deletions or substitutions. In particular, PD’s activity of hydrolysing angiotensin II (a vasoconstrictor) into angiotensin (1-7) (a vasodilator) is inactivated by said mutation/s. An inactivated PD, inactivated fragment or inactivated fragment combination of the PD has preferably an activity of hydrolysing angiotensin II into angiotensin which is in comparison to the active PD, active fragment or active fragment combination of the PD reduced by about 60% to about 100%, more preferably by about 70% to about 100%, by about 80% to about 100% or by about 90% to about 100%. In a preferred embodiment the PD of the SRF according to the present invention is inactivated by 1, 2, 3, 4, 5 or more suitable mutations such as insertions, additions, deletions or substitutions of the amino acid as listed in the following table. Suitable mutations are those which are suitable to inactivate the PD. Suitable mutations can be readily determined by those skilled in the art e.g. by routine cloning techniques.

In an especially preferred embodiment of the present invention the SRF according to the present invention comprises a PD of ACE-2 or a fragment, fragment combination or derivatives thereof having the substitution at position 375 as shown in SEQ ID NO: 3. In particular the substitution Glu375Gln is preferred. Said substitution results preferably in the inactivation of the active site of the PD. In a more preferred embodiment the SRF comprises a fragment of the inactivated PD as e.g. shown in SEQ ID NO: 5 or a fragment, fragment combination and/or derivative thereof. An example for an SRF comprising a sequence as shown in SEQ ID NO: 5 is an SRF consisting of or comprising a sequence as shown in SEQ ID NO: 10. The SRF having a sequence as shown in SEQ ID NO: 10 comprises in addition to a sequence as shown in SEQ ID NO:5 a methionine as translation start signal and a tag, namely a His-tag. Further examples of SRFs according to the present invention comprising an inactivated PD of ACE-2, an inactivated fragment thereof or an inactivated fragment combination thereof are shown SEQ ID NO: 18, 19, 21, 22, 26, 27, 30 and 31.

The SRFs having a sequence as shown in SEQ ID NO: 21, 26 and 30 have each a substitution Glu375Gln, i.e. a substitution of glutamic acid to glutamine at position 375 in respect of the sequence as shown in SEQ ID NO: 3 i.e. at position 175 of SEQ ID NO: 21 and 26 and at position 144 of SEQ ID NO: 30.

In a further preferred embodiment, the PD of the SRF according to the present invention comprises in addition to a mutation at position 375, in particular the substitution Glu375Gln, one, two, three, four or five further mutations such as insertions, additions, deletions or substitutions of the amino acid as listed in the table shown above.

Depending on the application of the SRF according to the present invention, the SRF and the SRF’s PD, fragment, fragment combination or derivative thereof, respectively, is preferably able to dimerize. Dimerization may increase stability of the SRF and/or may increase the binding avidity for protein S. Alternatively, the SRF’s PD, fragment, fragment combination or derivative thereof comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more mutations such as insertions, additions, deletions or substitutions to monomerize the PD. Monomerization may increase the distribution velocity in solution.

Appling a solution containing SRF according to the present invention as e.g. a gargling solution for the mouth or rinsing solution for the nose the binding pockets of the spike proteins (e.g. protein S) of free Coronaviridae such as Sars-CoV-2 present in these parts of the human body will be bound by SRF and no longer capable of infecting cells of the subject. These solutions can be also applied prophylactically to prevent infection in advance. The SRFs according to the present invention can be also applied therapeutically to inactivate the viral particles at the place of infection in the human body, - without significant impact of the SRF on blood pressure and kidney function.

Alternatively, the SRFs can be immobilised on beads or other surfaces to capture viral particles e.g. to improve washing effects by generating avidity effects and thereby facilitating washing out of viral particles from infected areas.

Alternatively, the SRFs can be immobilised on beads or other surfaces to capture viral particles e.g. to improve diagnostic quality, or washing effects. This includes also the use of dialysis application with SRFs coupled to the dialysis membrane or beads to filter out viral particles. Alternatively, the SRFs can be immobilised or fused to proteins e.g. the Fc part of IgGl for application inside the human body.

Alternatively, the SRFs can be immobilised or fused to proteins for application inside the human body allowing for dimerization of the PDs and thus increasing avidity.

Thus, in a preferred embodiment of the present invention the SRF is immobilised, bound, coupled or linked on beads, membranes such as dialysis membranes or other surfaces. In a further preferred embodiment, the SRF is immobilised, bound, coupled, linked or fused to proteins, antibodies, antibody fragments as e.g. the Fc part of IgGl or other compounds or molecules.

For example, the SRF according to the present invention may additionally comprise a tag, linker, protein, compound or molecule which may facilitate the stability, the solubility, the distribution and the half-life in the human body or other features of the SRF needed to address for its respective application. In general, the SRF may be modified to address the challenges of the application addressed, like this is known for therapeutic proteins (for example summarised in the review by Dellas et al. (2021). For example, the SRF may additionally comprise a maltose binding protein (MBP) for increasing the stability and or solubility of the SRF or a Fc-fusion for dimerization.

The present invention, therefore, also provides SRF according to all embodiments of the present invention as outlined above for use in a method for treatment of the human or animal body by surgery or therapy, as a vaccine or in diagnostic methods practiced on the human or animal body or with fluids or other material from the human or animal body.

More particularly, the present invention provides SRF according to all embodiments of the present invention as outlined above for use in a method of treating and/or preventing a virus infection in a subject, in particular a virus infection caused by Coronaviridae, more particularly caused by SARS corona virus, SARS corona virus-2, human coronavirus NL63 or SARS-CoV- 2 including any mutation thereof such as variants from British lineage B.1.1.7, B.1.351 from South Africa, B.1.617 from India or variant B.1.1.28.1 from Brazil. In particular, SRFs according to the present invention are particularly effective for treating and/or preventing a virus infection in a subject if said virus mutations or variants still allow host cell receptor binding, in particular wherein the mutated virus or virus variant still binds the ACE-2 receptor of the potential host cell. To test whether any potential mutation or variant allows host cell receptor binding, in particular ACE-2 receptor binding, affinity measurements on a Biacore chip or by MicroScale Thermophoresis as described in the Examples below can be used. If such a test results in a stronger binding than the standard deviation of a negative control, the tested virus may be considered as a mutation or variant of Coronaviridae, SARS coronavirus, SARS coronavirus-2, human coronavirus NL63 or SARS-CoV-2 which can be effectively treated or viral infection can be prevented by the SRFs according to the present invention.

Preventing a virus infection with SRFs according to the present invention may comprise inactivating or neutralizing the virus, in particular by blocking the binding pockets of the spike proteins of the virus, more particularly by binding the SRFs to the binding proteins of the virus. Preferably, preventing a virus infection with SRFs according to the present invention may comprise inactivating or neutralizing the virus, in particular by blocking the binding pockets of the spike proteins (protein S) of the virus, more particularly by binding the SRFs to the binding proteins S of the virus.

Replication of viruses is known to also produce variations or mutations. These mutations can be with or without effect on the viral proteins. Mutations in the viral receptor protein can also occur, but only mutations that still allow host cell receptor binding will replicate. This means that the binding pocket of the viral receptor protein is relatively stable against mutations that impact binding to the host cell, otherwise the binding to the host cell might get lost and viruses bearing those mutations will not be replicated. Human beings consist of 100 trillion cells. Naturally, those cells do not mutate a receptor that has a biological function in order to avoid a viral infection. Thus, the docking receptor of the host cells, in particular of human cells, is highly conserved. As a consequence, there is high pressure on the coronaviruses to not mutate their host cell receptor or spike protein away from recognizing the host cell docking receptor with significant affinity. Variants from British lineage B.1.1.7 (spike mutation N501Y (that has a higher affinity to ACE-2), plus other mutations in the spike ) or B.1.351 from South Africa (multiple mutations in the spike protein, including K417N, E484K, N501Y) although bearing mutations in the spike protein show binding of the spike protein to the host cell docking receptor ACE-2 so the SRF according to the present invention are effective against said variants as well.

The SRFs according to the present invention provides a blocking tool which blocks the viral spike protein disabling the virus infectious ability. The SRFs pretend to be the human cell lock, the viral docking key is looking for. Once the virus docked in, it cannot infect the human cells and can therefore not replicate. Inactivated virus, i.e. virus docked to SRFs according to the present invention, will be destroyed by the immune system. The SRFs, especially SRFs with no enzymatic activity that do not impact the blood pressure system according to the present invention, do not interact with living cells and therefore protect the human natural microbiome.

The subject to be treated is preferably a human or an animal, in particular a mammal, most preferably a human.

In a preferred embodiment the SRFs for use according to the present invention are administered in an amount sufficient to reduce the virus load of viruses which are capable of infecting cells of the subject and/or to inactivate the viral particles at the place of infection in the subject’s body. The effective amount of the compound to be administered can be readily determined by those skilled in the art during pre-clinical trials and clinical trials by methods familiar to physicians and clinicians.

In a further preferred embodiment, the SRF for use according to the present invention are fused to proteins, wherein the fused SRFs are administered in an amount sufficient to increase avidity by allowing for dimerization of the PDs.

In accordance with all embodiments of the present invention, an effective amount of SRFs for use in a method of treating and/or preventing a virus infection may be administered to the subject in need thereof by any of a number of well-known methods for administering pharmaceutical compounds. The compound may be administered locally or systemically, wherein local administration is preferred. The route of administration may be nasal, oral, intraocular, topical, systemic, intravenous, by inhalation, by injection or locally or any other suitable route of administration. Thus, the SRF for use according to the present invention are preferably formulated for nasal, oral, intraocular, topical, systemic, intravenous or wound flushing administration. A further aspect of the present invention refers to a pharmaceutical composition or medical product comprising SRF according to all embodiments of the present invention together with a pharmaceutical or physiological acceptable excipient and/or carrier.

The pharmaceutical composition according to the present invention may additionally contain one or more conventional additive(s). Some examples of such additives include a solubilizer such as, for example, glycerol; an antioxidant such as for example, bcnzalkonium chloride, benzyl alcohol, chloretone or chlorobutanol; and/or an isotonic agent.

The pharmaceutical composition or medical product may be formulated as a tablet, a lozenge, a bonbon, a drop, a chewing gum, a lollipop, a spray, in particular a nasal, oral, mouth, throat or wound spray, a rinsing solution, in particular a nasal, oral, wound or eye rinsing solution, an injection solution, a balm, an ointment, as eyedrops, or as mouth or throat washes.

The administration of the SRFs according to the present invention and/or the pharmaceutical composition according to the present invention as outlined above may in particular be formulated or designed to prevent the uptake of viruses, or at least to reduce the viral load entering the human body. Thus, the administration is preferably performed for allowing a washing effect or as a washing solution for neutralising the viral receptors and preferably allowing washing out of the neutralised viruses from the place of entry.

In addition, the present invention also provides a pharmaceutical pack comprising one or more compartments, wherein at least one compartment comprises SRFs according to the present invention or the pharmaceutical composition according to the present invention.

In another specific embodiment of the present invention the SRFs according to the present invention and/or the pharmaceutical composition of the present invention is used in the manufacture of a medicament for the treatment or prevention of a virus infection, in particular a virus infection caused by Coronaviridae, more particularly caused by a SARS coronavirus, a SARS coronavirus-2, a human coronavirus NL63 or SARS-CoV-2, most preferably caused by SARS-CoV-2 including any mutation thereof such as variants from British lineage B.1.1.7, B.1.351 from South Africa, B.1.617 from India or variant B.1.1.28.1 from Brazil. In a specific embodiment of the present invention SRFs according to the present invention and/or the pharmaceutical composition of the present invention is used as a medicament for the treatment or prevention of a virus infection, in particular a virus infection caused by Coronaviridae, more particularly caused by a SARS coronavirus, a SARS coronavirus-2, a human coronavirus NL63 or SARS-CoV-2, most preferably caused by SARS-CoV-2 including any mutation thereof such as variants from British lineage B.1.1.7, B.1.351 from South Africa, B.1.617 from India or variant B.1.1.28.1 from Brazil.

A further aspect of the present invention is a method of treating or preventing a virus infection, in particular a virus infection caused by Coronaviridae, more particularly caused by a SARS coronavirus, a SARS coronavirus-2, a human coronavirus NL63 or SARS-CoV-2, most preferably caused by SARS-CoV-2 including any mutation thereof such as variants from British lineage B.1.1.7, B.1.351 from South Africa, B.1.617 from India or variant B.1.1.28.1 from Brazil, by administering or applying an effective amount of SRFs according to the present invention or a pharmaceutical composition according to the present invention to a subject, in particular to a human or animal.

In a further aspect the present invention provides a method for capturing viral particles, the method comprises: a) providing SRFs according to the present invention, wherein the SRFs are immobilised, bound, coupled or linked on beads, on a column or its column material, on membranes, or other surfaces, and b) contacting a liquid sample or fluid with the SRFs of step a) under conditions for allowing the SRFs to bind the viral particles.

The viral particles are preferably viral particles or complete viruses preferably Coronaviridae, more particularly selected from the group consisting of SARS coronavirus, SARS coronavirus- 2, human coronavirus NL63 or SARS-CoV-2 including any mutation thereof such as variants from British lineage B.1.1.7, B.1.351 from South Africa, B.1.617 from India or variant B.1.1.28.1 from Brazil.

In a preferred embodiment the method for capturing viral particles is a method of detecting the captured viral particles. The detection method comprises additionally a step of detecting the captured viral particles. The detection may be performed by detecting the captured viral particles. Alternatively, the method may comprise a step of eluting the virus particle and detecting the eluted virus particle. In addition, the method for capturing viral particles and/or the method of detecting the captured viral particles may comprises further steps such as one or more washing steps.

In a further preferred embodiment, the method for capturing viral particles is a method of washing a liquid sample or fluid, wherein the virus load in the liquid sample or fluid is reduced due to the capturing of the viral particles. The method of washing may be suitable for neutralising the viral receptors and preferably allowing washing out of the neutralised viruses. This method may for example be used in dialysis for reducing the virus load in the subject’s body fluid. In particular, the washing may result in neutralising the viral receptors and preferably allowing washing out of the neutralised viruses from the subject’s body fluid. Alternatively, or in addition this might prevent the uptake of viruses by cells of the subject or at least to massively reduce the viral load entering the human body. For doing this, the SRFs according to the present invention are preferably immobilized, bound, coupled or linked on a dialysis membrane and the fluid is a body fluid such as whole blood, blood plasma or a blood fraction.

Some particularly preferred embodiments of the invention are summarized in the following items 1 to 24:

1. A soluble receptor fragment (SRF) of the ACE-2 receptor, wherein the SFR comprises the peptidase domain (PD) of ACE-2 or a fragment and/or derivates thereof.

2. The SRF according to item 1, wherein the SRF binds to the receptor-binding cleft of a virus spike protein, in particular to the receptor-binding cleft of a virus spike protein S of Coronaviridae, more particularly of the SARS coronavirus, the SARS coronavirus-2, the human coronavirus NL63 or SARS-CoV-2 including any mutation thereof such as variants fromBritish lineage B.1.1.7, B.1.351 from South Africa, B.1.617 fromlndia or variant B.1.1.28.1 from Brazil.

3. The SRF according to item 1 or 2, wherein the fragment and/or derivatives thereof comprise one, two, or more fragments of the PD of ACE-2 and/or derivatives of one, two or more fragments of the PD of ACE-2. The SRF according any one of the preceding items, wherein the fragment and/or derivative thereof comprise 3, 4, 5, 6, 7, 8, 9 or 10 of the alpha-helical structures of the PD of ACE-2 as shown in SEQ ID NO: 34 - 43 or a derivative thereof, in particular wherein said derivative comprises 1, 2, 3, 4 or 5 deletions or mutations under the provision that the derivative it is still able to form an alpha-helical structure. The SRF according any one of the preceding items, wherein the fragment and/or derivatives thereof comprise at least 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 of the Sars-CoV- 2 contacting residues Q24, T27, F28, D30, K31, H34, E35, E37, D38, Y41, Q42, L79, M82, Y83 of SEQ ID NO: 3. The SRF according any one of the preceding items, wherein the fragment and/or derivatives thereof comprise at least 5, 6, 7, 8, 9, 10, 11, 12 or 13 of the Sars-CoV contacting residues Q24, T27, F28, K31, H34, E37, D38, Y41, Q42, L45, L79, M82, Y83 of SEQ ID NO: 3. The SRF according any one of the preceding items, wherein the fragment and/or derivatives thereof comprise 1, 2, 3, 4, 5 or 6 of the Sars-CoV-2 contacting residues N330, K353, G354, D355, R357, R393 of SEQ ID: 3. The SRF according any one of the preceding items, wherein the fragment and/or derivatives thereof comprise 1, 2, 3, 4, 5, 6 or 7 of the Sars-CoV contacting residues Q325, E329, N330, K353, G354, D355, R357 of SEQ ID NO: 3. The SRF according any one of the preceding items, wherein the fragment and/or derivatives thereof comprise at least the alpha-helical structures of

SEQ ID NO: 34 or a derivative thereof,

SEQ ID NO: 35 or a derivative thereof, and SEQ ID NO: 36 or a derivative thereof. The SRF according to any one of the preceding items, wherein the fragment and/or derivatives thereof comprise at least an amino acid sequence according to SEQ ID NO: 8 or a derivative thereof having at least 60, 70, 80, 90, 95 or 98% sequence identity with SEQ ID NO: 8, in particular wherein said derivative comprises at least 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 of residues Q6, T9, F10, D12, K13, H16, E17, E19, D20, Y23, Q24, L61, M64, Y65 of SEQ ID NO: 8 and preferably at least the alpha- helical structures of SEQ ID NO: 34 or a derivative thereof,

SEQ ID NO: 35 or a derivative thereof, and SEQ ID NO: 36 or a derivative thereof. The SRF according to any one of the preceding items, wherein the SRF comprises a fragment combination of two fragments of the PD of ACE-2 or derivatives thereof, in particular wherein the first fragment is selected from the amino acid sequences as shown in SEQ ID NO: 8 and 9 or a derivative thereof and the second fragment is selected from the amino acid sequences as shown in SEQ ID NO: 15, 23, 28 and 32 or each a derivative thereof. The SRF according to any one of the preceding items, wherein the SRF comprises an inactivated PD of ACE-2 or a derivative thereof or an inactivated fragment, or fragment combination of the PD of ACE-2, in particular wherein the inactivated PD, derivative, fragment or fragment combination comprises a mutation such as an insertion, addition, deletion or substitution at one or more of the following positions: The SRF according to any one of the preceding items, wherein the SRF comprises an amino acid sequence according to SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6 or a derivative and/or fragment thereof, in particular wherein the SRF comprises an amino acid according to SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14,

SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21,

SEQ ID NO: 22, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 29,

SEQ ID NO: 30, SEQ ID NO: 31, or a derivative having at least 60, 70, 80, 90, 95 or

98% sequence identity with SEQ ID NO: 3-14, 17-22, 25-27, or 29-31 under the provision that said derivative comprises at least 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 of Q24, T27, F28, D30, K31, H34, E35, E37, D38, Y41, Q42, L79, M82, Y83 of SEQ ID NO: 3 and at least the alpha-helical structures of

SEQ ID NO: 34 or a derivative thereof,

SEQ ID NO: 35 or a derivative thereof, and SEQ ID NO: 36 or a derivative thereof. The SRF according to any one of the preceding items, wherein the SRF is immobilised, bound, coupled, linked or fused to proteins, antibodies, antibody fragments, as e.g. the Fc part of IgGl, or other compounds or molecules. The SRF according to any one of the preceding items, wherein the SRF is immobilised, bound, coupled or linked on beads, membranes such as dialysis membranes, a column or column material or other surfaces. The SRF according to any one of the preceding items for use in a method for treatment of the human or animal body by surgery or therapy, as a vaccine or in diagnostic methods practiced on the human or animal body or with fluids or other material from the human or animal body. The SRF according to any one of items 1 to 14 for use in a method of treating and/or preventing a virus infection, in particular a virus infection caused by Coronaviridae, more particularly caused by SARS coronavirus, SARS coronavirus-2, human coronavirus NL63 or SARS-CoV-2 including any mutation thereof such as variants from British lineage B.1.1.7, B.1.351 from South Africa, B.1.617 from India or variant B.1.1.28.1 from Brazil. The SRF for use according to item 17, wherein preventing a virus infection comprises inactivating or neutralizing the virus, in particular by blocking the binding pockets of the spike proteins, more particularly by binding the SRFs to the binding proteins of the virus, more particularly by blocking the binding pockets of the spike proteins (protein S) of Coronaviridae, more particularly by binding the SRFs to the binding proteins S of the virus. The SRF for use according to item 17 or 18, wherein the SRFs are administered in an amount sufficient to reduce the virus load of viruses which are capable of infecting cells of the subject and/or to inactivate the viral particles at the place of infection in the subject’s body. The SRF for use according to any one of items 17 to 19, wherein the SRF is formulated for nasal, oral, intraocular, topical, systemic, intravenous or wound flushing administration or for administration by inhalation or injection. A pharmaceutical composition or medical product comprising SRF according to any one of items 1 to 14 and a pharmaceutical or physiological acceptable excipient and/or carrier, in particular wherein the pharmaceutical composition or medical product is formulated as a tablet, a lozenge, a bonbon, a drop, a chewing gum, a lollipop, a spray, in particular a nasal, oral, mouth, throat or wound spray, a rinsing solution, in particular a nasal, oral, wound or eye rinsing solution, an injection solution, a balm, an ointment, as eyedrops, or as mouth or throat washes. A method for capturing viral particles, the method comprises: a) providing SRFs according to item 14 or 15, and b) contacting a liquid sample or fluid with the SRFs of step a) under conditions for allowing the SRFs to bind the viral particles. The method according to item 22, wherein the method is a method of detecting the captured viral particles and wherein the method comprises additionally a step of detecting the captured viral particles. 24. The method according to 22, wherein the method is a method of washing a liquid sample or fluid and wherein the virus load in the liquid sample or fluid is reduced due to the capturing of the viral particles, in particular wherein the SFRs are immobilized, bound, coupled or linked on a dialysis membrane and the fluid is a body fluid such as whole blood, blood plasma or a blood fraction.

The following examples explain the present invention but are not considered to be limiting. It should be understood that the detailed description and specific examples disclosed herein, indicating particular embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this description.

Example 1 - Expression and purification of SRFs and spike proteins in Baculo virus cells

SRF (according to SEQ ID NO: 4 with or without Strep-tag) was expressed in the Baculo virus system (Sf9) according to the manufacturer's protocols (BD Biosciences). In brief, 2pg SRF (according to SEQ ID NO: 4) encoding-plasmid was mixed with 0.5pg BD BaculoGold linearized Baculovirus DNA which sat for 5 min at room temperature before adding 1 ml BD BaculoGoldTransfection Buffer B. The mixture was added dropwisely to Sf9 insect cells (ATCC) pre-co vered with 1 ml Transfection Buffer A, followed by incubation of the cells at 28 °C for 4 h, and further culture of the transfected cells in fresh SF-900 II SFM (Invitrogen) for an additional 4 days. Supernatant was collected 4 days later, which was used to infect cells with three more 3-day culture cycles for virus amplification. Cell culture supernatant from the 4th cycle was collected for purification of SRF (according to SEQ ID NO: 4) protein.

SRF (according to SEQ ID NO: 5 with or without Strep-tag) was expressed in the Baculovirus system (Sf9) according to the manufacturer's protocols (BD Biosciences). In brief, 2pg SRF (according to SEQ ID NO: 5) encoding-plasmid was mixed with 0.5pg BD BaculoGold linearized Baculovirus DNA which sat for 5 min at room temperature before adding 1 ml BD BaculoGoldTransfection Buffer B. The mixture was added dropwisely to Sf9 insect cells (ATCC) pre-co vered with 1 ml Transfection Buffer A, followed by incubation of the cells at 28 °C for 4 h, and further culture of the transfected cells in fresh SF-900 II SFM (Invitrogen) for an additional 4 days. Supernatant was collected 4 days later, which was used to infect cells with three more 3-day culture cycles for virus amplification. Cell culture supernatant from the 4th cycle was collected for purification of SRF (SEQ ID NO:5) protein. Spike protein S with His-tag was obtained from Sino Biological 40592-V08B-1. A Strep-tag variant was cloned into an expression vectors and expressed soluble in Baculovirus SF9 cells to allow correct glycosylation. Purification was performed via Strep-Tag or other standard purification techniques.

Example 2 -Measurement of binding of the SRFs to spike protein S in an Elisa assay

An SRF (according to SEQ ID NO: 4) coated microtiter plate was incubated with soluble spike protein S (Strep-tagged). Unbound spike protein was washed off by three subsequent washing steps with PBS-buffer. Protein S bound to immobilised SRF (according to SEQ ID NO: 4) was then quantified using Streptavidin-coupled horseradish peroxidase. Binding of protein S to immobilised SRF (according to SEQ ID NO: 4) was successfully measured.

A protein S coated microtiter plate was incubated with SRF (according to SEQ ID NO: 5) and additionally comprising a Strep-tag. Unbound SRF (according to SEQ ID NO: 5) was washed off by three subsequent washing steps with PBS-buffer. SRF (according to SEQ ID NO: 5) bound to immobilised protein S was then quantified using Streptavidin-coupled horseradish peroxidase. Binding of SRF (according to SEQ ID NO: 5) to immobilised protein S was successfully measured.

Example 3 - Affinity measurement on a Biacore chip - Part 1

A sensor chip SA with pre-immobilized Streptavidin was used to capture of Strep-tagged SRFs. Then spike protein S was injected and binding to immobilised SRFs (according to SEQ ID NO: 4) was measured. Blank flow cells were used for correction of the binding response. The surface plasmon resonance (SPR) method was used with a BIAcore system to measure the binding affinities of the SRFs. Binding of protein S to immobilised SRF (according to SEQ ID NO: 4) was successfully measured, whereas when protein S was incubated with soluble SRF (according to SEQ ID NO: 4) and injected to the Biacore system clearly reduced binding to immobilised SRF (according to SEQ ID NO: 4) was detected. Incubating protein S at higher concentrations with soluble SRF (according to SEQ ID NO: 4) before injecting to a Biacore chip with immobilised SRF (according to SEQ ID NO: 4) resulted in that no binding was detected. Example 4 - Virus inactivation assay: Treatments of Vero E6 cells with SRFs

Vero E6 cells were seeded in 48-well plates in DMEM containing 10% FBS. 24 hours post- seeding, SRFs were mixed with different concentration of virus ( 1 : 1 ) in a final volume of 100ml per well in DMEM (0% FBS) at 37°C. After 30 minutes, Vero-E6 were infected either with mixes containing SRFs or SRF/SARS-CoV-2 for 1 hour followed by washing or for 15 hours without washing, cells were washed 3 times with PBS and 500ml of new complete medium supplemented with SRF were added. 15 hours post- infection, supernatants were removed, cells were washed 3 times with PBS and then lysed using Trizol before analysis by qRT-PCR for viral RNA detection. Infecting Vero E6 cells with virus was used as a positive control. Infecting Vero E6 cells with SRFs only was used as negative control. Mixing SRF & SARS-CoV-2 before infection of Vero E6 cells showed a clear reduction in infectivity. Applying SRF (according to SEQ ID NO: 4) or SRF (according to SEQ ID NO: 5) did not result in a significant difference.

Example 5 - Expression and purification of SRFs and spike proteins in HEK293 cells

Genes encoding SRFs comprising amino acid sequences according to SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 4, SEQ ID NO: 8 and SEQ ID NO: 9 were cloned into a standard expression vector pTZ (Trenzyme) suitable for transient expression in the supernatant of mammalian HEK293 cells. Due to translation and purification reasons the above listed amino acid sequences were complemented each with a His-tag and a methionine as translation start signal. Thus, the encoded sequences including the methionine as translation start signal, a secretion signal and the His-tag which were actually used for cloning are as follows:

(3)_ACE2_19-615_E375Q as shown in SEQ ID NO: 10 (comprising an amino acid sequence as shown in SEQ ID NO: 5)

(4)_ACE_2_19-103;301-365 as shown in SEQ ID NO: 11 (comprising an amino acid sequence as shown in SEQ ID NO: 7)

(9)_ACE2_19-615 as shown in SEQ ID NO: 12 (comprising an amino acid sequence as shown in SEQ ID NO: 4)

(1)_ACE2_19-103 as shown in SEQ ID NO: 13 (comprising an amino acid sequence as shown in SEQ ID NO: 8), and

(8)_ACE2_19-132 as shown in SEQ ID NO: 14 (comprising an amino acid sequence as shown in SEQ ID NO: 9). Protein expression was performed in shake flasks, with the target proteins being in the cell culture supernatant. After expression the supernatant was collected, and the target proteins were purified via His-tag affinity chromatography.

Constructs (1) and (8) were expressed as MBP-fusions by fusing the respective construct via a short linker to MBP. The encoded sequences included the methionine as translation start signal, a secretion signal, the MBP, a short linker and the His-tag. Protein expression was performed in shake flasks, with the target proteins being in the cell culture supernatant. After expression the supernatant was collected, and the target proteins were purified via His-tag affinity chromatography.

Example 6 - Affinity measurement on a Biacore chip - Part 2

The measurement of SARS-CoV-2 Spike Protein SI (aal4-683), His-Avi-Tag recombinant protein (Invitrogen, Catalog # RP-87681) to three variants of SRF ((3)_ACE2_19-615_E375Q, 1.24 mg/ml; (4)_ ACE_2_19-103;301-365, 1 mg/ml; (9)_ACE2_19-615, 0.74mg/ml) was conducted at a Biacore X100 from Cytiva using the Biotin CAPture kit on a sensor chip CAP. Buffer was PBS pH 7.4.

After equilibration and conditioning of the Biacore system, the CAP chip was coated with Biotin CAPture reagent.

Immobilisation of Spike protein SI on flow cell one yealded -100 RU (Ligand concentration 0.43mM; Contact time: 420s; Stabilization period: 300s)

The relative response of the binding was measured with the highest concentrations of ACE-2 variants, without referencing flow cell two due to unspecific reference binding (Contact time: 120s; Dissociation time: 600s; no regeneration needed). Three startup cycles with buffer were made.

Results are: (3)_ACE2_19-615_E375Q 443.6 RU

(4)_ ACE_2_19-103;301-365, 1 mg/ml 352.0 RU

(9)_ACE2_19-615 55.1 RU Results:

Binding to the spike protein of Sars-CoV-2 immobilised on a biocore chip was observed for all three proteins tested, with variant (3)_ACE2_19-615_E375Q showing the highest binding response. Variant (4)_ ACE_2_19-103;301-365 was binding less efficiently and variant (9)_ACE2_19-615 showed the lowest binding. Even if (9)_ACE2_19-615 showed the lowest binding affinity in comparison with to other constructs, binding to SARS-CoV-2 Spike Protein S 1 was clearly confirmed.

Example 7 - Measuring binding affinity by MicroScale Thermophoresis

MicroScale Thermophoresis was used to measure the affinity between the spike protein of Sars- CoV-2 and the respective target proteins. In principle the binding affinity of two molecules was measured by detecting variations in fluorescence signal as a result of an IR-laser induced temperature change. The range of the variation in the fluorescence signal correlates with the binding of a ligand to the fluorescent target. By that it allows for quantitative analysis of molecular interactions in solution on the microliter scale with high sensitivity.

Fluorescence labelling to the spike protein:

SARS CoV 2 Spike Protein SI (aal4683), His tag, Avi tag; Invitrogen, Cat. Number: RP 87681 at a stock concentration of 10.0 mM in lx PBS buffer pH 7.4

The target protein was diluted into the labelling buffer 1 x PBS pH 7.4, 0.005% Tween to reach the labelling concentration 9.7 pM. DMSO was introduced into the labelling buffer, because the dye stock solution is prepared in DMSO. Labelling concentration of the dye was 20.1 pM (3:1 dye:protein molar ratio). As fluorescent dye Red NHS 650 ^{2nd gen} was used. Labelling was performed at 25 °C for 30 minutes. After labelling, unbound dye was removed via a gel filtration step and the target protein was brought into the final buffer. 0.005% Tween 20 were added in order to reduce protein sticking/adhesion and enhance protein recovery. The concentration of the labelled target protein was determined by measurement of the dye fluorescence. Total protein concentration and protein recovery were determined by comparative fluorescence measurements using a Tycho NT.6 instrument. Labelling was performed to introduce roughly 2 fluorescence labels per protein.

Binding measurements:

Measurement setup for variant: (9) ACE 2 19-615 Target: SARS CoV 2 spike SI, used at constant 10 nM

Ligand: (9) ACE 2 19-615, titrated from 4.73 mM down in 16 1:1 dilution steps

Instrument: Monolith NT.115 Pico

Buffer: 1 x PBS pH 7.4, 0.005% Tween 20

Measurement setup (3) ACE 2 19-615 E375Q Target: SARS CoV 2 spike SI, used at constant 10 nM

Ligand: (3) ACE 2 19-615 E375Q, titrated from 7.92 pM down in 16 1:1 dilution steps Instrument: Monolith NT.115 Pico Buffer: 1 x PBS pH 7.4, 0.005% Tween 20

Measurement setup (4) ACE 2 19-103:301-365 Target: SARS CoV 2 spike SI, used at constant 10 nM

Ligand: (4)_ACE_2_19-103;301-365, titrated from 22.9 pM down in 16 1:1 dilution steps Instrument: Monolith NT.115 Pico Buffer: 1 x PBS pH 7.4, 0.005% Tween 20

Measurement setup (1) ACE2 19-103 fused to MBP Target: SARS CoV 2 spike SI, used at constant 10 nM

Ligand: (1) ACE2 19-103 fused to MBP, titrated from 1.26 pM down in 16 1:1 dilution steps Instrument: Monolith NT.115 Pico Buffer: 1 x PBS pH 7.4, 0.005% Tween 20

Measurement setup (8) ACE2 19-132 fused to MBP Target: SARS CoV 2 spike SI, used at constant 10 nM

Ligand: (8) ACE2 19-132 fused to MBP, titrated from 1.29 mM down in 16 1:1 dilution steps Instrument: Monolith NT.115 Pico Buffer: 1 x PBS pH 7.4, 0.005% Tween 20

Results:

Variants (8) ACE2 19-132 fused to MBP and (1) ACE2 19-103 fused to MBP showed the best binding affinity. The other three variants were binding less efficiently with variant (3)_ACE2_19-615_E375Q binding stronger than (4)_ ACE_2_19-103;301-365 (ex ACE2_19- 103) and variant (9)_ACE2_19-615 showing the lowest binding affinity. Even if (9)_ACE2_19- 615 showed the lowest binding affinity in comparison to other constructs, binding to SARS CoV 2 spike S 1 was clearly confirmed.

Example 8 - SARS-CoV-2 (B.3 lineage) Neutralisation Plaque Assay with recombinant SRF variants

Materials:

DMEM media: Thermo Fisher 41965039 FBS: Pan Biotech #P30-3702 Penicilin/streptomycin: Sigma #P4333 OptiPro-SFM: Thermo Fisher # 12309019 DPBS (w/o Ca/Mg): Thermo Fisher 14190144 Carboxymethylcellulose: Sigma #C5013 MEM: Pan Biotech # P03-0710 NaHC03: Roth # HN01.1 Paraformaldehyde: AppliChem #141328.1212 Crystal violet (C.I. 42555): Merck # 1159400025

On Day 0, 1.25x10 ^s VeroE6 cells/well were plated in 24- well plates and incubated for 24 hours at 37 °C and 5 % CO2 _. On the day 1, serial dilutions of recombinant SRF variants were made as follows:

Stock concentrations: variant_19-615_E375Q: Mw=70,6 kDa, c=l,24 mg/ml ® 17,563 mM variant_19-615: Mw=70,6 kDa, c=0,74 mg/ml ® 10,481 pM

SRF variants were thawed on ice. A round-bottom 96- well plate was prepared with triplicates of 120 pi SRF variant dilutions in OptiPro-SFM (serum-free media). An additional 12 wells were filled with 120 pi OptiPro-SFM for the controls and several 120 pi vehicle control samples were prepared with PBS in OptiPro-SFM. The plate was sealed tightly and without air bubbles.

Virus dilution

In the BSF-3 lab one aliquot of in vitro-propagated SARS-CoV-2 (B.3 lineage, isolated in February 2020, 1.8xl0 ⁶ PFU/ml) was thawed and prediluted in 1:2250 in OptiPro-SFM (1:4500 final dilution, 80 PFU/200 pi). 120 pi of this dilution are needed for each well of protein dilution and of the positive control.

SRF variant-virus coincubation

1:2250 virus dilution was poured into reservoir. With a multichannel pipet 120 pi virus dilution was added to each SRF variant dilution well and positive control well. The plate was sealed and incubated for 1 h at 37 °C and 5 % CO2. Infection of VeroE6 cells

The vacuum pump or a PI 000 was used to remove media from max. 6 wells of the 24 well- plate at a time and 200 mΐ of SRF variant dilution/virus mix, virus (positive control), or OptiProSFM (negative control) was added. After completing the first 24- well plate a timer was started for counting up, and the plate was placed back in the incubator. It was continued accordingly until all samples from your 96- well plates were added to the cells. The time on the timer was noted whenever a plate was finished. The cells were incubated for lh at 37 °C.

Replacing media after infection

A 1:1 mix of 1.5 % (w/v) carboxymethylcellulose (autoclaved) and 2x MEM (sterile-filtered, contained 2x Pen/Strep, 4 % FBS, and NaHCCb for pH) was prepared and prewarmed in water bath. After the timer reached lh after adding the virus, the supernatant of each 24- well plate was aspirated (either by vacuum or P1000) and replaced with 1 ml of carboxymethylcellulose- MEM/well. Plates were returned to incubator and incubated for 72h.

On Day 4, the cells were fixed and stained: A vacuum or 10 ml serological pipet was used to remove part of the volume of the supernatant from each well without touching or coming near the surface of the well bottom. The whole 24- well plate was immersed in 6 % formaldehyde in a fixation/transport container and a lid was added of the 24- well plates. The plates were left for 30 mins at room temperature. The container^ outside was wiped down with a desinfectant pursept wipe and the container/-s were taken out of BSL-3. The container was opened and as much formaldehyde as possible was poured back into the container. Each well was rinsed 3 times with tap water. Then, 1 % crystal violet staining solution was added to each well, the lid was closed, and the plates were incubated 30 min at room temperature. The crystal violet was poured back with a funnel into the bottle, the plate was rinsed with tap water until the draining water was no longer blue and the plates and lids were air dried completely. The plaques in each well were counted.

Results:

(9)_ACE2_19-615 (40 PFU)

(3)_ACE2_19-615_E375Q (40 PFU)

_ untreated 250 nM 500 nM 750 nM 1 mM 1.5 mM 2 mM

(9)_ACE2_19-615 (80 PFU)

(3)_ACE2_19-615_E375Q (80 PFU)

The results as shown in the Tables above and in Fig. 2 and 3 revealed the following:

Neutralisation tested with 80 pfu/ml: Variant 4_ACE_2_19-103;301-365 shows no significant reduction in pfu at all concentrations tested. Variant 9_ACE2_19-615 shows already at InM at minor reduction and a 50% reduction at 250nM. Application of variant 3_ACE2_19- 615_E375Q in the neutralisation assay leads to a measurable reduction at all concentrations tested, with a reduction to about 40%. Neutralisation tested with 40 pfu/ml:

Variant 4_ ACE_2_19-103;301-365 shows no significant reduction in pfu at all concentrations tested. Variant 9_ACE2_19-615, shows a 50% inhibition at 250nM and a full inhibition at 1,5 mM. Application of variant 3_ACE2_19-615_E375Q leads to a significant reduction in the concentration range of 250nM to ImM. At 1,5mM and 2mM an almost complete reduction is observed

Example 9 - SARS-CoV-2 UK-Variant (B.l.1.7) Neutralisation Plaque Assay with recombinant SRF variants

The Neutralisation Plaque Assay with recombinant SRF variants as described in Example 8 was performed with a mutant of SARS-CoV-2, namely SARS-CoV-2 UK-variant (B.l.1.7) as described by Rambaut, A., Loman, N., Pybus, O., Barclay, W., Barrett, J., Carabelli, A., Connor, T., Peacock, T., Robertson, D.L., and Volz, E. (2020). Preliminary genomic characterisation of an emergent SARS-CoV-2 lineage in the UK defined by a novel set of spike mutations.

Results:

(9)_ ACE2_19-615 (80 PFU)

As shown in Fig. 4 and the tables above variant 9_ACE2_19-615 and variant 3_ACE2_19- 615_E375Q show already at 250nM a massive reduction and a complete reduction at ImM. References

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