GB2593742A - Coronavirus - Google Patents

Abstract

Surfactant protein D (SP-D) or a derivative thereof, is disclosed, but not exemplified, for use in preventing and/or treating a coronavirus infection such as SARS-CoV-2 which has caused one or more of pneumonia, bronchiolitis, bronchitis and severe acute respiratory syndrome (SARS). Preferably, the SP-D fragment has carbohydrate binding activity and comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO:2 or SEQ ID NO:4 and may be a fusion protein comprising a carbohydrate-binding domain from SP-D fused an anti-inflammatory polypeptide. A composition of SP-D may further comprise Surfactant Protein A (SP-A) or Mannan Binding Lectin (MBL) or an additional anti-viral agent. The composition is further disclosed for use in the treatment of inflammation, testicular dysfunction and pain in a subject infected with a coronavirus. A nucleic acid encoding SP-D for use in treatment and/or prevention of a coronavirus infection in a subject is also disclosed wherein the nucleic acid has at least 70% homology to SEQ ID: NO.1 or NO:3. The use of Surfactant Protein D in an in vitro method of detecting a coronavirus infection is also disclosed but not exemplified.

Description

Intellectual Property Office Application No. GB2004755.1 RTM Date:29 September 2020 The following terms are registered trade marks and should be read as such wherever they occur in this document: Sepharose Triton Superose Polymixin B Intellectual Property Office is an operating name of the Patent Office www.gov.uk/ipo

CORONAVIRUS

Field of the Invention

The present invention relates to a composition comprising surfactant protein D (SP-D), or a fragment, homologue or variant thereof, for use in preventing and/or treating a coronavirus infection, including coronavirus-associated disease and co-morbidities, in a subject, and a composition as above for use in preventing and/or treating inflammation and other co-morbidities in a subject with a coronavims infection. The present invention further relates to a composition comprising a nucleic acid encoding SP-D, or a fragment, homologue, variant or derivative thereof, for use in preventing and/or treating a coronavirus infection, including coronavirus-related disease and co-morbidities in a subject, and use of SP-D, or a fragment, homologue, variant or derivative thereof in an in vitro method for diagnosing a coronavims infection..

Background of the Invention Coronavims family

Coronaviruses are enveloped, positive-sense RNA viruses that infect a broad range of vertebrates, and cause disease of medical and veterinary significance. The genome size of coronaviruses ranges from approximately 27 to 34 kilobases, the largest among known RNA viruses. In humans, infections are generally localised to the respiratory system. Respiratory symptoms may range from the common cold to high morbidity outcomes such as pneumonia, and Severe Acute Respiratory Syndrome (SARS).

Some coronaviruses may also affect the enteric system (for example, in certain coronavirus infections, diarrhoea has been reported as a common symptom), and reproductive and nervous systems, in both human and in non-human subjects. Coronaviruses may also cause systemic disease. Additionally, after recovery from the acute phase of disease, physical and psychological dysfUnctions following SARS have been documented, These include both neurocognitive impairment such as memory deficit, impaired attention and concentration, and psychological impairment such as depression and post-traurnatic: stress (ESC:1111ff. 1.

The name "coronavirus" is derived from the Latin term corona-, meaning "crown" or "wreath". The name refers to the fringe of large, bulbous projections on the surface of the viral particles, reminiscent of a crown, as observed by electron microscopy. This morphology is created by viral spike ("S") peplomers or glycoproteins on the surface of the viral particles.

Human coronaviruses were first discovered in the late 1960s. The earliest ones were discovered in human patients with the common cold (HCoV-229E and HCoV-0C43). Other members of this family have since been identified, including SARS-Coy in 2003, HCoV 1N1L63 in 2004, HKU1 in 2005, MERS-CoV in 2012, and SARS-CoV-2 (formerly known as 2019-nCoV) in 2019. Most of these have involved serious respiratory tract infections The coronaviral genome encodes four major structural proteins: the spike (S) protein, nucleocapsid (N) protein, membrane (M) protein, and the envelope (E) protein. Individually, each protein primarily plays a role in the structure of the virus particle, but they are also involved in other aspects of the replication cycle.

Coronavirus S-protein is a significant determinant of virus entry into host cells. It has a distinct trimeric structure. The S-protein of SARS-CoV binds to ACE2 receptors and CD209L on host cells, the S-protein of SARS-CoV-2 binds to ACE2 receptors on host cells, and the S-protein of MERS-CoV binds to DPP4. There are distinct differences between the S-protein of the human coronaviruses which cause the common cold and those which cause more severe symptoms such as SARS. S-protein trimers of the coronavirus types that cause the common cold exist in a "closed state" compared to the more severe strains which are "partially open" (Walls et al., Cell. 2020, March 6, pii: S0092-8674(20)30262-2; doi: 10.1016/j.ce11.2020.02.058. 2020).

The entry of coronaviruses into host cells is thought to be accomplished accomplished by direct membrane fusion between the virus and plasma membrane, and by endocytosis. After the virus enters the cells, the viral RNA gen om e is released into the cytoplasm, translated, and replicated, ultimately resulting the formation of new viral particles which are released from the infected cells. The main targets of coronavirus are pneumocytes, macrophages, and dendritic cells.

In some coronaviruses, expression of S-protein at the cell membrane can also mediate cell-cell fusion between infected and adjacent, uninfected cells. The resultant formation of giant, multinucleated cells, or syncytia, has been proposed as a strategy to allow direct spreading of the virus between cells, subverting virus-neutralising antibodies. Also, it is possible that the virus sheds its trimeric S-protein into the systemic space, and this can activate cells such as macrophages directly, in the absence of virus, causing them to release pro-inflammatory cytokines.

SARS emerged as a regional and global health threat in 2002-2003 caused bt SARS COV. SARS-CoV-2, the most recent coronavirus to have been discovered in Wuhan, Hubei Province, China, has resulted in widespread disease across the world. Declared by the World Health Organisation as a pandemic and Public Health Emergency of International Concern, as of 29 March 2020, there have been over 634,800 confirmed cases of SARS-CoV-2 infection and over 29,900 confirmed deaths associated with SARS-CoV-2 infection across 203 different countries. Although those infected with the virus may be asymptomatic, many develop flu-like symptoms, including fever, cough, shortness of breath and fatigue. In some, particularly in the elderly and in those with underlying health conditions, the disease may progress to pneumonia, bronchiolitis, bronchitis, severe acute respiratory syndrome (SARS), multi-organ failure, and death. SARS is the major cause of death resulting from SARS-CoV-2 infection.

One of the main mechanisms for SARS in SARS-CoV-2 infection is an uncontrolled systemic inflammatory response resulting from the release of large amounts of pro-inflammatory cytokines and chemokines by immune effector cells. The high levels of pro-inflammatory cytokines trigger a violent attack by the immune system on body, resulting in alveolar oedema, decreased lung compliance and, ultimately, hypoxaemia, multiple organ failure, and eventual death in severe cases of SARS-CoV-2 infection. Infection appears to be largely via the respiratory route so protection of that surface is absolutely key to prevention against the sequelae of the disease.

There is currently no clinically proven specific anti-viral agent available for preventing or treating SARS-CoV-2 infection, or indeed SARS-CoV and MERS-CoV infections. Supportive treatment, including oxygen therapy, conservation fluid management, and the use of broad-spectrum antibiotics to cover secondary bacterial infection, remains the most important management strategy. There is also no vaccine available for humans even though the severe disease-causing coronavirus types have been in the population since 2002. Strategies to prevent coronavirus-associated diseases in other species (for example, feline infectious peritonitis) have largely proven unsuccessful, and in some cases, have made the disease worse. Attempts are being made to produce polyclonal and monoclonal neutralizing antibodies to SARS -COV 2 but the in vivo activity is unknown, and they are not guaranteed to be effective.

It would therefore be extremely desirable to urgently develop effective interventions against coronavirus infection.

Lung Surfactant Proteins Pulmonary surfactant is essential for normal lung mechanics and gaseous exchange in the lung. The main function of the pulmonary surfactant is to ensure minimal surface tension within the lung to avoid collapse during respiration. Pulmonary surfactant is produced by type II epithelial cells and includes a major phospholipid component (90%) and a minor protein component (10%). Pulmonary surfactant contains four different proteins: surfactant proteins A, B, C and D. Hydrophobic surfactant proteins B and C are involved in the reduction of surface tension at the air-water interface. Hydrophilic surfactant proteins A and D are members of the collectin family, and play an important role in protection of the lung against viral, bacterial and fungal pathogens.

Surfactant protein D (SP-D) is secreted by Alveolar Epithelial Type II cells (ATM) cells, submucosal cells and Clara cells. Although the majority of SP-D is expressed in the lung, SP-D has been shown to be expressed in other parts of the body, such as the intestine, thymus, prostrate, brain, testes, salivary gland, lachrymal gland and heart.

SP-D is a C-type (Ca211-dependent) lectin that comprises four domains: a cysteine-containing N-terminal region, a triple-helical collagen region composed of repeating Gly-X-Y triplets, an a-helical coiled coil neck region, and a C-terminal calcium-dependent carbohydrate-recognition domain (CRD) (Crouch E. et al. (1994) J Biol Chem 269:17311-9). Monomers form trimers through folding of the collagenous region into triple helices and the assembly of a coiled-coil bundle of a-helices in the neck region. These trimers are stabilized by two disulfide bonds in the cysteine-rich N-terminal domain. The SP-D trimer has a total molecular weight of 129 kDa which comprises three identical 43 kDa polypeptide chains. SP-D trimers can form higher order oligomerization states which vary by size and conformation. Typically, SP-D is found as a dodecamer formed from the linking of four trimers by disulphide bonds at the N-termini. Higher order oligomerization states may be important for SP-D function (Hakansson K, et al, Protein Sci (2000) 9:1607-17; Crouch E. Respir Res (2000) 1:93-108; Crouch E. e tat (2006) J Biol Chem 281:18008-14).

Native (full length) SP-D has a role in the pulmonary innate immune system by providing anti-inflammatory and antimicrobial activities that are particularly beneficial in chronic pulmonary diseases such as asthma, cystic fibrosis, and smoking-induced emphysema (Clark H, et al, Immunobiology (2002) 205:619-31). Interestingly, SP-D has also been shown to enhance the binding of HIV to immature monocyte-derived dendritic cells and subsequent transfer to T-cell-like cells in vitro, suggesting a potential role for native SP-D facilitating HIV infection (Madsen et al., PLoS One 2013,8(3).059047 The role of SP-D in coronavirus infection is unknown. SP-T) levels are elevated in serum of patients infected with SARS-CoV and suffering from SARS-associated pneumonia, suggesting a possible role for SP-D as a marker of alveolar damage (Hartshorn et al., Frontiers in bioscience (Scholar edition). 2010; 2:527-46 and Wu et al., Scandinavian Journal of Immunology, 69: 508-515. doi:10.1111/j.1365-3083.2009.02245.x). Raised serum SP-D levels are also seen in other respiratory disorders such as COPD. SP-D also recognizes carbohydrate moieties on a recombinantly-produced S-protein of SARS-CoV which comprises only the extra-AMar domain and lacks the transmembrane and internal domains present in the native form of the protein (Leth-Larsen et al., Immunobiology 2007; 212, 201-211). However, the coronaviruses use conformational masking, and glycan shielding to limit recognition by the immune system and therefore, this isolated S-protein does not necessarily predict the binding of SP-D to the whole virus.

Additionally, the S-protein of SARS-CoV is known to exist in trimeric form within the virus envelope, and it is not known whether human SP-D would bind to native, trimeric form of the S-protein. Furthermore, whilst there is structural homology between the S-glycoprotein of SARS-CoV and SARS-CoV-2, there are significant conformational differences between their respective receptor binding domains (RBDs). In this regard, it has been shown that antibodies to the SARS-CoV RBI) do not cross-react with the SARS-CoV-2 PHD (Wrapp et al., Science. 2020;367(6/1841260-3). 'Therefore, it is also not known whether human SP-D would be effective in binding to, and neutralising S.AIRS-C:OV-2, nor is it known if SR-I) from other species would be effective in binding to and. neutralising SA RS-CoV-2 more effectively that human SP-D.

SP-D has also been shown to bind to FR:oV-229E a coronas,drus type responsible for the common cold. Pre-treatment of liCoV-229E with SP-*D inhibits infection of 16HBE. (Human Bronchial Epithelial) cells. However, pre-treatment of Deo-V-229E with SP-D is not effective in reducing infection lveola mcrophages (Funk et aL, The Journal of General Virology, 201 2 93(Pt 3):494-503), WO 03/035679 describes a 6( kDa trimeric recombinant fragment of human SP-D (iihSP-D) which includes the enninal carbohydrate recognition domain and a-helical coiied coil neck region of SP-D. In the absence of the N-termina: eysteine-rich region, renSP-D is unable to form higher order el:isomers. Despite the inability to form higher order oligomers, rthSP-D has been shown to mannthri many of the immune fumti OTIS of full-length, particularly those involved in immune suppression. (Clark FPN, Neonatology 2010; 97(4)380-7). The prophylactic and therapeutic effects of rtISP-D in coronavirus infection have not been determined.

In view of the above, it would be desirable to further cEucidate the role of pulmonary col lectins and recombinant fragments thereof, particularly SP-D and rthSP-,D, in preventing coronavirus infection, at the in mire level, and in reducliw the sequelae of disease both in the lung and nic space. It would further be desirable to provide novel therapies for coronavirus irifection.

Summary of the Invention

Accordingly, in a first aspect, the present invention provides a composition comprising surfactant protein D (SP-D), or a fragment, homologue, variant or derivative thereof, for use in preventing and/or treating a coronavirus infection in a subject, wherein the use comprises administering the composition to the subject.

In a second aspect, the present invention provides a composition comprising surfactant protein D (SP-D), or a fragment, homologue, variant or derivative thereof, for use in preventing and/or treating inflammation in a subject infected with a coronavirus, wherein the use comprises administering the composition to the subject.

In a third aspect, the present invention provides a composition comprising a nucleic acid encoding SP-D, or a fragment, homologue, variant or derivative thereof, for use in treatment and/or prevention of a coronavirus infection in a subject, wherein the use comprises administering the nucleic acid encoding SP-D, or a fragment, homologue, variant or derivative thereof to the subject.

In a fourth aspect, the present invention provides a use of surfactant protein D (SP-D), or a fragment, homologue, variant or derivative thereof, in an in vitro method of detecting a coronavirus infection Preferably, the coronavirus comprises SARS-CoV-2 and the infection is COVID-19.

Further preferred features of all aspects of the present invention are defined in the dependent claims.

The present inventors believe that SP-D, and notably, rfhSP-D, is an effective prophylactic and therapeutic agent in coronavirus infection and coronavinis-associated disease, particularly following SARS-CoV-2 infection.

Detailed Description of the Invention

In one aspect, the present invention provides a composition comprising surfactant protein D (SP-D), or a fragment, homologue, variant or derivative thereof, for use in preventing and/or treating a coronavirus infection in a subject, wherein the use comprises administering the composition to the subject In a further aspect, the present invention provides a composition comprising surfactant protein D (SP-D), or a fragment, homologue, variant or derivative thereof, for use in preventing and/or treating inflammation in a subject with a coronavirus infection, wherein the use comprises administering the composition to the subject. The inflammation is preferably airway inflammation.

The term comprising" or comprises" as used herein denotes the inclusion of at least the features following the term, and does not exclude the inclusion of other features which have not been explicitly mentioned. The term may also denote an entity which consists of features following the term.

The present inventors believe that SP-D is capable of binding to, and neutralising coronavirus particles, thereby preventing or reducing entry into target cells. Cells include, but are not limited to, cells expressing ACE2 receptors such as dendritic cells, alveolar macrophages, alveolar Type I epithelial cells, and alveolar Type II epithelial cells, oral mucosal cells including those of the tongue, epithelial cells of the ileum and colon, choloangiocytes, myocardial cells, kidney proximal cells, bladder urothelial cells, gonadal cells, neutrophils, lymphocytes and peripheral macrophages. The inventors believe that consequently, SP-D is effective in preventing or treating coronavirus infection, and/or treating inflammation associated with coronavirus infection. It is unexpected that SP-D recognizes the S-protein of coronaviruses in its native (natural) multimeric form and inhibits or reduces infection of target cells.

Types of coronavirus In the present invention, the coronavirus infection may be caused by one or more coronaviruses selected from: HCoV-229E (alpha coronavirus), HCoV-NL63 (alpha coronavirus), HCoV0C43 (beta coronavirus), HCoV-HKU1 (beta coronavirus), MERS-CoV (a beta coronavirus that causes Middle East Respiratory Syndrome, or MERS), SARS-CoV (a beta coronavirus that causes severe acute respiratory syndrome, or SARS) and SARS-CoV-2 (the novel coronavirus that causes coronavirus disease 2019, or COV1D-19).

In preferred embodiments, the coronavirus infection is a COVID-19 infection caused by SARSCoV-2. SARS-CoV-2 may include the L and/or S subtypes. In other embodiments, the coronavirus infection is not caused by SARS-CoV. In further embodiment, the coronavirus infection may include co-infection with other respiratory viruses including, but not limited to, Influenza and RSV.

SP-D polypepti des and nucleotides As used herein, "surfactant protein D (SP-D)" refers to an SP-D polypeptide or nucleic acid encoding an SP-D polypeptide (unless otherwise specified and as the context allows).

The SP-D, or fragment, homologue, variant or derivative thereof, may originate from any of a human, primate, cat, dog, cow, sheep, pig, rabbit, rat, mouse or bat. Preferably, the SP-D for use according to the present invention is of human origin. The SP-D may have any of the sequences of GenBank accession numbers NM 003019.1, X65018.1 and AH005286.2.

In a preferred embodiment, the SP-D is a human SP-D comprising a sequence according to GenBank accession number NNI 003019.1. The nucleic acid and amino acid sequences of such a human SP-D are shown in SEQ ID NO: I and SEQ ID NO: 2 respectively.

SEQ ID NO: 1 (Nucleotide sequence of human SP-D cDNA clone (accession number NM 003019.1)) AT GCT GOT OTT COT CCT CT CT GCACTGGTCCTGCTCACACAGCCOCTGGGCTACCTGGAAGCAGAAATG AAGAC C:TAC:T C: C:CACAGAACAATGCCCAGT GOT GCAC C: C: T GGT CAT GT GTAGCT CAGT GGAGAGTGGC GT GC CT GGT C GCGAT GGACGGGAT GGGAGAGAGGGCCCT CGGGGCGAGAAGGGGGAGC CAGGTTT GCCA GGAGCT GCAGGGOAAGGAGGGATGCCT GGACAAGCT GGC2 CAGTT GGGCCAAAAGGGGACAAT GGCT CT GT T GGAGAAC CT GGACCAAAGGGAGP,CACT GGGCCAAGT GGACCT CCAGGACCT CCC GGT GT GCCT GGT CAGCT GGAAGAGAAGGT GG C CTGGGGAAGCAGGGGARCATAGGACCT CAGGGCAAGC CAGGCCCAPLAA GGAGAAGOTGGGOCIAAAGGAGAAGTAGGTGCCCCAGGOATGCAGGGCTCGGCAGGGGCAAGAGGCCTC GCAGGC C CTAAGGGAGAGCGAGGT GT CCCT GGT GAGCGT GGAGT CCCT GGAAACACAGGGGCAGCAGGG T CT GCT GGAGC CAT GGGT CC C CAGGGAAGT CCAGGT GC CAGGGGACCCCCGGGAT T GAAGGGGGACAAA GGCATT C CT GGAGACAAAGGAGCAAAGGGAGAAAGT GGGCTT CCAGAT GT T GCT I CT CT GAGGCAGCAG Gil GAGGC CTTACAGGGAGAAGTACAGCAGGT GGAGGCT GOTTT CT CT GAGTATAAGAAAGTT GA= G T T CC CAAAT GGCCAAAGT GT GGGGGAGAAGATTITCAAGACAGCAGGCTITGTAAAACCATTTACGGAG GCACAGCT GCT GT GCACACAGGCT GGT GGACAGT T GGC CT CT CCACGCT CT GCCGCT GAGAATGCCGCC TT GCAACAGCT GGT GGTAGGTAAGAACGAGGGT GGT T T C CT GAGCAT GAGT GAT T GGAAGACAGAGGGG AAGTT CAC CTACCCCACAGGAGAGT CCCT GGT CTAT T C CAACT GGGCGCCAGGGGAGC C CAACGAT GAT GGC GGGT CAGAGGACT GT GT GGAGAT CTT CACCAAT GGC2AAGT GGAAT GACAGGGCT T GT GGAGAAAAG C GT OTT GT GGT CT GC GAGT T CT GA SEQ ID NO: 2 (Amino acid sequence of human SP-D (translated from SEQ NO: 1)) ML L FL L SALVL LT Q P LGYLEAEMKTY SHRTMP SACT LYME.: S SVES GL P GRDGRDGRE GP RGEKGDPGLP GAAGQAGMP GQAGPVGP KGDNGSVGEP GP KGDT GP SGP P GP P GVP GPAGREGALGKQ GNI GPOGK P GP K GRACE' KGEVGAP GMQGSAGARGLAG P KGERGVP GERGVP GMT GAAG SAGAMGP QGS P GARGPPGLKGDK GI P GDKGAKGE S GLPDVASLRQQVEALQGQVQHLQAAFSQYKKVELFPNGQSVGEKI FKTAGFVKP FT E AQ L L CTQAGGQ LAS P RSAAENAALQQLVVAKNEAAFL SMT DS KT EGK FT YP T GESLVYSNWAP GEPNDD GGSEDCVEI FTNGKWNDRAGGEKELVVCEF In some embodiments, the SP-D comprises an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 98% identity with SEQ ID. NO: 2. In other embodiments, the SP-D comprises a nucleotide sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 98% identity with SEQ ID. NO: 1.

The "percentage identity" of two amino acid or nucleic acid sequences as provided herein may be determined using the algorithm of Karlin and Altschul Proc. Natl. Acad. Sci. USA 87:226468, 1990, modified as in Karlin and Altschul Proc. Natl. Acad, Sci. USA 90:5873-77, 1993. Such an algorithm is incorporated into the NBLAST and XBLAST programs (version 2.0) of Altschul, eta]. J. Mol. Biol. 215:403-10, 1990. BLAST protein searches can be performed with the)(BLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to the protein molecules of interest. Where gaps exist between two sequences, Gapped BLAST can be utilized as described in Altschul et al, Nucleic Acids Res. 2507):3389-3402, 1997. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs can be used. Another preferred program for carrying out a sequence alignment to determine the percentage identity between two sequences is the GCG Wisconsin Bestfit package (University of Wisconsin, U.S.A, Devereux et al., 1984, Nucleic Acids Research 12:387). Examples of other software than can perform sequence comparisons include, but are not limited to, FASTA (Atschul et al., 1990, J. Mol, Biol., 403-410), DNAsis, ESEE, and the GENEWORKS suite of comparison tools.

SP-D polypeptides for use according to the present invention include a fragment, homologue, variant or derivative of SP-D. Preferred fragments, homologues and variants have one or more biological activities of full-length, native (natural) or endogenous SP-D. A preferred biological activity is carbohydrate-binding activity. Other biological activities include, but are not limited to, oligomerization (for example, trimerization), pathogen (particularly coronavirus) binding and neutralisation, and immune cell activation.

SP-D fragments generally include any recombinant fragment of SP-D, preferably human SP-D, which lacks the N-terminal domain. At least a portion of the collagen domain is preferably retained to stabilise lower order multimers (for example, trimers). The SP-D polypeptide may be a recombinant fragment of SP-D, preferably human SP-D depicted in SEQ ID NO: 2, which lacks substantially residues 1-178. The SP-D polypeptide may thus be a recombinant fragment of SP-D, preferably human SP-D, having a sequence shown in SEQ ID NO: 2, comprising substantially residues 179-355.

The nucleic acid sequence and amino acid sequence of a 60kDa trimeric human SP-D fragment (rfhSP-D) have been disclosed previously in WO 03/035679, which is incorporated herein by reference, and further provided below as SEQ ID NO:3 and SEQ ID NO:4, respectively.

SEQ ID NO: 3 (Nucleic acid encoding ifliSP-D)

GGAAGCCCGGGATTGAAGGGGGACAAAGGCATICCTGGAGACAAAGGAGCAAAGGGAGAAAGTGGGCTICCAGA T GT T GCTT CT CT GAGGCAGCAGGTT GAGGCCT TACAGGGACAAGTACAGCACCT CCAGGCT GCTTT CT CT CAGTAT AAGAPAGTT GAGOT CT T CCCAAAT GGCCAAAGT CT GGGGGAGAAGATT T T CAAGACAGCAGGCTTT GTAAAACCA ITTACGGAGGOACAGCTGCT GT GCACACAGGGIGGIGGACAGTT GGCCTCT CCACGCT CT GC CGCT GAGAAT GCC GC OTT GCAACAGCT GGI CGTAGCTAAGAACGAGGCT GCTTT COT GAGCAT GACT GAT T CCAAGACAGAGGGCAAG TT CAC CTAC C C CACAGGAGAGT CCCT GGT CTAT T CCAACT GGGCCCCAGGGGAGCCCAAC GAT GAT GGCGGGT CA GAGGACT GT GT GGAGAT CT T CACCAATGGCAAG

T GGAAT GACAGGGCT T GT GGAGAAAAGC GT CT T GT GGT CT GCGAGTT CT GA

SEQ ID NO: 4 (Amino acid sequence of rfliSP-D) GS P GLKGDKGI PGDKGAKGES GLPDVASLRQQDTEALQGQVQHLQAAFS QYKKVEL FPNGQ SVGEK I FKTAGFVKP FT EAQ I, CTQAGGQIJAS P RSAAENAALQQLVVAKNEAAFL SMT D S KT EGKFT YP T GE S LVYSNWAP GE PNPDGGS ED CVEI FTNGKWNDRACGEKRLWCEF In a preferred embodiment, the SP-D fragment is rfhSP-D and comprises an amino acid sequence according to SEQ ID NO:4. In some embodiments, the SP-D fragment comprises an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 98% identity with SEQ ID. NO: 4. In other embodiments, the SP-D fragment comprises a nucleotide sequence according to SEQ ID NO:3. In further embodiments, the SP-D fragment comprises a nucleotide sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 98% identity with SEQ ID. NO: 3.

In yet further embodiments, the SP-D polypeptide for use according to the present invention comprises a carbohydrate recognition domain (CRD), comprising substantially the following residues: SEQ ID NO: 5 (Amino acid sequence of the CRD of SP-D)

VELFPNGQSVGEKI FKTAGFVKP FT EAQLLCT QAGGQLAS PRSAAENAALQQLWAKNEAAFLSMTDSKTEGKFIY PT GESLVYSNWAPGEPNDDGGSEDCVEI ETNGKKINDRACGEKRLWCE

The SP-D polypeptide may further comprise a "neck" region comprising substantially the following residues: SEQ ID NO: 6 (Amino acid sequence of the neck region of SP-D)

DVASLRQQVEALQGQVQHLQAAFSQYKK

Preferably, such a neck region is N-terminal to the carbohydrate recognition domain CRD.

The SP-D polypeptide may additionally comprise at least two Gly-Xaa-Yaa repeating units, and preferably, a plurality of Gly-Xaa-Yaa repeating units to facilitate multimeri sati on. In one embodiment, the SP-D polypeptide additionally comprises an N-terminal sequence comprising a sequence according to SEQ ID NO:7 provided below: SEQ ID NO: 7 (Gly-Xaa-Yaa repeat sequence)

GS P GL KGDKGI PGDKGAKGESGL

Without wishing to be bound by theory, the absence of the cysteine-rich N-terminal domain in rfhSP-D and other comparable SP-D fragments may render rfhSP-D and other fragments advantageous in preventing and treating a coronavirus infection, and in reducing inflammation in a subject with coronavirus infection.

Gardai et al. proposed a model by which SP-D might simultaneously mediae anti-and pro-inflammatory processes in the lung through the opposing actions of signal regulating protein a CSIRP0 and calretictilin/CD91 (Gardal, S. J et al., (2003) Cell 115:13-23). Their model indicates that in the unbound state, the C RD ot'sP-D inhibits macrophage acti'vation by binding to SIRPa which inhibits P38 mediated activation of NiFKB In comrast, ilthe eRn of SP-0 is occupied by a microbial 1,g,and, binding to SIRPa is inhibited and the col ecun binds to the macrophage activating receptor, calreticulin/CD9 I via the N-terminal domain. Calreticulin/CD9 t subseottently stimulates P38 mediated activation of NFKB which induces pro-inflammatory mediators and activates alveolar macrophages. Therefore, depending on the presence or absence of infectious particles in the CRD and type of receptor bound, SPA) can either enhance or suppress inflammation.

The present inventors believe that by utilising an SPA) fragment such as riliSP-D which lacks the N-terminal domain, the capacity to induce inflammation is no longer present. Consequently, in coronavirus infection, where uncontrolled inflammation in the lung can often be fatal, SP-D fragments may have a therapeutic advantage Over their full-length counterparts.

Additionally, the N-terminal domain of SPA) is known to influence multimerization via the formation of disulphide bridges between monomers. The multimerization may in turn facilitate the binding of SP-D to, and opsonization of, pathogens, thereby enhancing their uptake into immune cells such as macrophages and dendritic cells, and promoting-viral propagation. In the case of coronavirus it may be desirable to restrict viral uptake into immune cells to prevent the immune dystegulati Oil that is observed with severe in:fediOIL By using a fragment of SP-D that lacks the higher order multimeri radon properties of the full-length molecule, neutralization of the virus (i.e. inhibition of binding of the virus to host cells) may be achieved through binding of the fragment to viral particles, without any facilitation of uptake of the virus into inuntme In some ernnodiriienis., an SPA) homologue, variant or derivative is used to prevent or treat coronavirus infection, or inflammation associated with coronavirus infection. As discussed above, variants, homologues or derivatives of SP-D for use according to the present invention may encompass related polypeptides which provide one or more of the biological activities of endogenous or native SPA). A preferred biological activity is carbohydrate-binding activity. Other biological activities include, but are not limited to, oligomerization (for example, trimerizati on), pathogen (particularly coronavirus) binding and neutralisation, and immune cell activation.

The SP-D homologue, variant or derivative may comprise a sequence which has homology to any of the sequences described above, and ftnther, may be obtained from any source such as a human, primate, domestic species such as cat, dog, cow, sheep, pig,rabbit, rat, mouse, civet cat or bat. The SP-*D homologue may also comprise a synthetic peptide.

As used herein; a. el ogixis sequence is taken to include an amino acid sequence which has at least 40%; 50%, 60%, 70%, 80% or 90% identity, preferably at least 95 or 9817iii identity at the amino acid level, with SEQ ID NO:2. The sequence identity is preferably over at least 50, 100, 200."00; 400 or 500 amino acids.

Although homology can also be considered in terms of similarity of residues amino acid residues haying similar chemical propertiesifunctions), in the context of the present invention, horirologi is preferably synonymous with sequence identity. Sequence identity may be determined as described above.

In some embodiments, the homologue, variant or derivative in relation to native or endogenous SP-D sequences for use according to the present invention may include any substitution of, variation of modification of, replacement of, deletion of or addition of one (or more) amino acids within the endogenous or native SP-D sequence providing the resultant amino acid sequence retains substantially the same biological activity as, or at least one biological activity of, the unmodified seq tence h ulogical activities of May be as described above.

IIn other embodiments, the.homologue, variant or derivatiye comprises surfactant (SP-A), marman-binding lec,tin (MBL,), or fragments thereof SW-A and MBL, like SRD are multimeric col it-xi:tins and have carbohydrate-binding activity. The subunits of SP-A. and Mill.. are structurally similar to those of SP-D. SP-A and NIBL" have been shown to play a role in the host innate immune defence against pathogens.

In another aspect the present invention provides a composition comprising a nucleic acid encoding a SP-D polypepti de, or fragment, homologue, variant or derivative thereof, for use in preventing and/or treating a coronavirus infection in a subject, or for use in preventing and/or treating inflammation in a subject infected with coronavirus The prevention or treatment of infection or inflammation comprises administering the composition to the subject.

The nucleic acid sequence may comprise a sequence according to SEQ ID NO: 1. In other embodiments, the nucleic acid sequence has at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 98% identity with SEQ ID. NO: 1. In a preferred embodiment, the SP-D nucleic acid is a recombinant fragment of a native or endogenous SP-D nucleic acid sequence, preferably human SP-D depicted in SEQ ID NO: 1 which lacks substantially residues 1-594, or any homologue, variant or derivative thereof Most preferably, the nucleic acid comprises a sequence according SEQ ID NO: 3. In other embodiments, the nucleic acid sequence has at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 98% identity with SEQ. ID. NO: 3, The SP-D homologue, derivative or variant, in relation to a nucleotide sequence includes any substitution of, variation of, modification of replacement of, deletion of or addition of one (or more) nucleotides in relation to the sequence of a native or endogenous SP-D. Preferably the homologue, variant or derivative encodes a polypeptide retaining at least one or improving upon at least one biological activity of a native or endogenous SP-D, wherein the biological activity is as described above.

The nucleic acid for use according to the present invention may be provided within a vector which is capable of introducing the nucleic acid (for example, by transfection or transduction) into a cell in the subject such that the nucleic acid is expressed within the cell and the peptide encoded by the nucleotide is produced. The vector may, for example, be a plasmid or a viral vector, such as a retroviral vector or a lentiviral vector. A preferred vector is an adenoviral vector. Vectors are commonly known in the field of recombinant DNA technology. The host cell which receives the vector may be a lung cell, for example, alveolar Type I or Type II cell, a submucosal cell, a Clara cell, or a macrophage.

Prevention and treatment of disease condition in a subject In the present invention, compositions comprising SP-D, or a fragment, homologue, variant or derivative thereof, may be used to prevent or treat a coronavirus infection in a subject. When used for the prevention of infection, the invention relates to the prophylactic use of SP-D. In this aspect SP-D may be administered to a subject who has not yet contracted the infection and/or who is not showing any symptoms of disease or onset of disease associated with the infection, to prevent the development of at least one symptom or disease condition associated with the infection. Respiratory symptoms or disease associated with coronavirus infection include but are not limited to fever (temperature of above 38°C in humans), dry cough, shortness of breath, headache, body aches, pain, sore throat, pneumonia, bronchitis, bronchiolitis, and severe acute respiratory syndrome (SARS). Systemic effects of coronavirus infection or diseases associated with coronavirus infection (or shed virus S-proteins) include, but are not limited to, peripheral pain, hypogonadism, diarrhoea, myocardial disease and kidney disease.

When used for the treatment of infection, the invention relates to the therapeutic use of SP-D. The treatment of infection may comprise the amelioration of one or more symptoms and/or diseases associated with the infection. SP-D may be administered to a subject having an existing coronavirus infection or coronavirus-associated disease condition in order to lessen, reduce or improve at least one symptom, and/or at least one disease associated with the infection and/or to slow down, reduce or inhibit the progression of the infection or disease. The symptom and disease may be as defined above.

Preferably, the prevention or treatment of a coronavirus infection comprises preventing or treating inflammation associated with coronavirus infection in the subject. Thus, in one aspect of the invention, a composition comprising SP-D, or a fragment, homologue, variant or derivative thereof, is used to prevent (for example, reduce the likelihood of developing and/or reducing the severity of) inflammation associated with a coronavirus infection in a subject who has not yet acquired a coronavirus infection and/or who is not exhibiting any symptoms of a coronavirus infection. In another aspect, a composition comprising SP-D, or a fragment, homologue, variant or derivative thereof, is used to treat (for example, reduce) inflammation in a subject with coronavirus infection and/or exhibiting at least one symptom associated with a coronavinis infection. As described above, use of a fragment of SP-D which lacks the N-terminal domain such as rfhSP-D, and therefore, may have reduced pro-inflammatory potential, may be particularly useful in this regard.

The subject may be pre-disposed to, or have increased susceptibility to, acquiring a coronavirus infection. For example, the subject may have had an organ transplant, may be undergoing cancer treatment, may be pregnant, and/or may have one or more of the following: blood or bone marrow cancer, such as leukaemia, a lung condition, such as cystic fibrosis or severe asthma, a weakened immune system, diabetes, hypertension, and/or a heart condition. In other embodiments, the subject may otherwise have no existing health conditions and/or have no pre-disposition to acquiring a coronavirus infection. Preferred subjects are human subjects. However, other mammalian subjects including primates, cats, dogs, cows, sheep, and pigs are also envisaged in the present invention.

Ansi otensin-converting-enzyme I (ACEI) inhibitors are a class of medication used primarily for the treatment of hypertension and heart failure. They are also used in the treatment of renal is disease, particularly in patients with diabetes, and may be prescribed following a heart attack. They function by promoting vasodilation as well as a decrease in blood volume, which in turn results in reduced blood pressure and decreased oxygen demand from the heart. Specifically, ACE1 inhibitors inhibit the activity of angiotensin-converting enzyme which converts angiotensin I to angiotensin II, and hydrolyses bradykinin. The result is a reduced level of angiotensin II, which is a vasopressin and promotes vasoconstriction, and an increased level of bradykinin, a peptide vasodilator. Angiotensin II Receptor Blockers (ARBs) are another class of medication used for the treatment of hypertension and heart failure. ARBs function by blocking angiotensin II receptors which mediate the vasoconstricting effect of angiotensin II.

It has been reported that both ACE1 inhibitors and ARBs increase levels of Angiotensin converting enzyme 2 (ACE2) which is an enzyme found in cell membranes of cells in lungs, arteries, heart, kidney, and intestines (Nicholls et al., Nature Medicine, 2005; 11(8): 821-2. doi:10.1038/nm0805-821). ACE2 lowers blood pressure by catalysing the conversion of angiotensin 11 to angiotensin I. It has also been suggested that at least SARS-CoV and SARSCoV-2 gain entry into target cells via binding of their respective S-proteins to ACE2 receptors on the cells (Wan et al., Journal of Virology, 2020; DOT: 10.1128/W1.00127-20). Therefore, subjects undergoing treatment for hypertension, heart problems and renal failure who are taking ACE I inhibitors or ARBs may have increased susceptibility to coronavirus (particularly, SARS-CoV and SARS-CoV-2 infections) and/or may have more severe symptoms by virtue of the raised levels of ACE2. In light of this, there is speculation as to whether such patients who are infected with coronavirus should stop treatment with ACE1 inhibitors and/or ARBs in order to reduce infection.

However, not only does ACE2 increase the production of the vasodilator angiotensin I, ACE2 has also been shown to have a protective effect against virus-induced lung injury by increasing the production of the vasodilator angiotensin 1-7 (Imai et al, Nature, 2005; 436 (7047). 112-6). Furthermore, according to studies conducted on mice, the interaction of the S-protein of SARS-CoV with ACE2 induces a drop in the levels of ACE2 in cells through internalization and degradation of the protein, which may contribute to lung damage in infection (Kuba et al, 2005; Nature Medicine 11 (8): 875-9). Thus, continuing treatment with ACE1 inhibitors and/or ARBs may be desirable in patients with coronavirus infection.

SP-D advantageously binds to, and neutralises coronavirus preventing its attachment to target cells and subsequent entry into the cells. Thus, in the present invention, SP-D, or an active fragment, homologue, variant or derivative thereof (SP-D active agent), is particularly useful in treating a coronavirus infection in subjects who are taking ACE1 inhibitors and/or ARBs, since any raised level of ACE2 would be counteracted by neutralisation of the virus. Preferably, the coronavirus is SARS-CoV or SARS-CoV-2. Treatment with the SP-D active agent would enable these subjects to continue with ACE1 inhibitor and/or A RB treatment and the higher ACE2 levels to be advantageously maintained. A fragment of SP-D lacking the N-terminal domain required for higher order oligomerisation (such as rflISP-D) may be particularly useful in these circumstances since, as described above, it may have a reduced capacity to aggregate and/or opsonize viral particles, and promote their uptake into target cells via the ACE2 receptor. This would result in reduced internalisation/degradation of the receptor and allow ACE2 levels to be maintained.

Binding of SARS-CoV-2 to ACE2 receptors on macrophages may be associated with peripheral pain in subjects who are infected. It is known that neuropathic pain can be caused by inflammatory diseases affecting the somatosensory system via the ACE2 receptor found on local macrophages releasing inflammatory cytokines (Shepard et al 2018 PNAS 115 (34) E8057-E8068). Therefore, in further aspects of the invention, a composition comprising SP-D, or a fragment, homologue, variant or derivative thereof, may be used to prevent or treat pain associated with a coronavirus infection in a subject. Thus, in one aspect of the invention, a composition comprising SP-D, or a fragment, homologue, variant or derivative thereof, is used to prevent (for example, reduce the likelihood of developing and/or reducing the severity of) pain associated with a coronavirus infection in a subject who has not yet acquired a coronavirus infection and/or who is not exhibiting any symptoms of a coronavirus infection. In another aspect, a composition comprising SP-D, or a fragment, homologue, variant or derivative thereof, is used to treat (for example, reduce the severity of) pain in a subject with coronavirus infection and/or exhibiting at least one symptom associated with a coronavirus infection. Preferably, the coronavirus is SARS-CoV or SARS-CoV-2. The pain may be peripheral pain such as skin tingling.

The ACE2 receptor is further expressed in testes, including sperm atogoni a, Leydig and Sertoli cells. It has been reported that male subjects infected with SARS-CoV-2, exhibit raised levels of luteinizing hormone, and a reduced testosterone: luteinizing hormone ratio, which may be associated with testes dysfunction, and which is indicative of hypogonadism (Ma et al., 2020; https://doi * on.:110 / 01/2020 03.21.20037267). Therefore, in further aspects of the invention, a composition comprising SP-D, or a fragment, homologue, variant or derivative thereof, may be used to prevent or treat testicular dysfunction or hypogonadism associated with a coronavirus infection in a subject. Thus, in one aspect of the invention, a composition comprising SP-D, or a fragment, homologue, variant or derivative thereof, is used to prevent (for example, reduce the likelihood of developing and/or reducing the severity of) testicular dysfunction or hypogonadism associated with a coronavirus infection in a subject who has not yet acquired a coronavirus infection and/or who is not exhibiting any symptoms of a coronavirus infection. In another aspect, a composition comprising SP-D, or a fragment, homologue, variant or derivative thereof, is used to treat (for example, reduce the severity of) testicular dysfunction or hypogonadism (such as reduced testosterone levels) in a subject with coronavirus infection and/or exhibiting at least one symptom associated with a coronavirus infection. Preferably, the coronavirus is SARS-CoV or SARS-CoV-2.

Further provided herein is a method of preventing and/or treating a coronavirus infection in a subject, and a method of preventing and/or treating inflammation in subject with a coronavirus infection, wherein the methods comprises administering to the subject a composition comprising SP-D, or a fragment, homologue, variant or derivative thereof The composition, prevention, treatment and subject may be as defined herein.

Fusion Proteins The SP-D, fragments, homologues, variants and derivatives thereof for use in the present invention may be obtained from endogenous sources, or more preferably, generated using standard methods of recombinant DNA technology. In some embodiments, the recombinant protein is a fusion protein which comprises the SP-D polypeptide, fragment, homologue, variant or derivative thereof, fused by means of an optional linker region to a second polypeptide which has beneficial activity in coronavirus infection. Preferably, the carbohydrate recognition domain of SP-D is fused to a second polypeptide which has beneficial activity in coronavirus infection. In a preferred embodiment, rfliSP-D is fused to a second polypeptide which has beneficial activity in coronavirus infection. The second polypeptide may bind to the neonatal Fc receptor in the liver to aid removal of viral particles from the body. In other embodiments, the second polypeptide may have anti-inflammatory activity. By way of example, the second polypeptide may comprise IL-4, IL-6, IL-10, 1L-11 or IL-13 or their respective receptors. In some embodiments, the optional linker region may have a protease binding site such that when administered to the subject, endogenous proteases cleave the fusion protein and separate the SP-D active agent from the second polypeptide. In some embodiments, the linker between recombinant SP _D and the second polypeptide may be a chemical linker such as PEG. In accordance with the invention, the fusion protein described herein may be provided and administered as a nucleotide encoding the protein which is expressed in the subject following administration.

Form of comp° ions and admin strati on The SP-D, fragments, homologues, variants and derivatives thereof for use in the present invention may be provided and administered in a substantially isolated form. The SP-D, fragments, homologues, variants and derivatives may be combined with inert carriers or diluents which do not interfere with the activity of the protein or nucleotide, and may still be regarded as being substantially isolated form. By "substantially isolated" it is meant that the SP-D active agent (protein or nucleotide) is present in the composition in an amount of greater than 90 wt.%, 95 wt.%, 98 wt.% or 99 wt.% by total weight of the composition.

In some embodiments, the composition comprising SP-D or a fragment, homologue, variant or derivative thereof, is a pharmaceutical composition. In addition to the SP-D, or a fragment, homologue, variant or derivative thereof, the pharmaceutical composition may comprise a pharmaceutically acceptable carrier, diluent, excipient or adjuvant. The choice of pharmaceutical carrier, excipient, diluent or adjuvant may depend on the intended route of administration, and falls within the bounds of standard pharmaceutical practice. Other components that may be present in the pharmaceutical compositions include, but are not limited to, buffers, bulking agents, suspending agents, solubilizing agents, sugars, coating agents, and surfactants. In some embodiments, the compositions comprise a lyophile and are in lyophilised form.

It is preferred that compositions for use according to the present invention are delivered to a lung. In some embodiments, compositions for use in the invention are suitable for intranasal, oral, intratracheal, intrabronchial or bronchoalveolar administration. In some embodiments, intranasal, oral, intratracheal, intrabronchial or bronchoalveolar administration can include spraying, lavage, inhalation, flushing or installation, using a physiologically, and preferably, pharmaceutically acceptable carrier fluid in which the composition of the invention has been dissolved. Methods of administration may include the use of continuous positive airway pressure (CPA]?) or direct intubation. In other embodiments, compositions comprising the SP-D active agent may be administered through a ventilation line.

In preferred embodiments, the compositions for use according to the present invention are delivered to the lungs, for example through intranasal administration, and/or by inhaling. The compositions may be administered as a solution, dry powders or aerosols. A wide range of mechanical devices designed for pulmonary delivery of therapeutic products can be employed, including, but not limited to nebulizers, metered dose inhalers, and powder inhalers, all of which are familiar to those skilled in the art of pharmaceuticals. These devices employ formulations suitable for the dispensing of a pharmaceutical composition. Typically, each formulation is specific to the type of device employed and can involve the use of an appropriate propellant material, in addition to diluents, adjuvants, and/or carriers useful in therapy.

Systemic delivery of the compositions for use according to the invention is also envisaged. Systemic delivery may be achieved by oral and parenteral routes of administration, including intranasal, intraperitoneal, intravenous, subcutaneous, and intramuscular routes of administration. Systemic delivery is particularly advantageous where bioavailability of SP-D is required at sites other than the lung and/or at multiple sites. For example, as described above, patients who are undergoing treatment with ACEI inhibitors and/or ARBs have higher expression of ACE2 receptors which may make them more susceptible to coronavirus infection, particularly SARS-CoV and SARS-CoV-2 infection. Since ACE2 receptors are expressed at multiple locations (for example, lungs, arteries, heart, kidneys and intestines), systemic treatment with SP-D, or a fragment, homologue, variant, or derivative thereof would be advantageous in order to inhibit the binding of vials to ACE2 receptors at these multiple extra-pulmonary sites. Moreover, since patients who are undergoing treatment with ACE1 inhibitors and/or ARBs may have a heart condition and/or renal disease, it is important to prevent infection-associated tissue injury at these extra-pulmonary sites. Similarly, systemic administration of SP-D active agents would also be advantageous in preventing or treating testicular dysfunction in male subjects with a coronavirus infection. As SP-D is known to be endogenously expressed in several extra-pulmonary tissues (for example tongue, intestinal tract, thymus, skin, gall bladder, stomach, kidney, liver, prostate and spleen and coronary arteries), and given that in chronic and acute lung conditions lung derived SP-D (including fragments of SP-D may enter the systemic circulation, systemic administration should not pose any adverse effects for the subject.

The SP-D, fragment, homologue, variant or derivative thereof, may be administered in a prophylactically or therapeutically effective dose to achieve the required prevention or treatment of infection or symptoms. Typically, a physician will determine a suitable dosage for an individual subject which will vary with age of subject, weight of subject, severity of infection and route of administration. The dosage may be such that it is sufficient to prevent or treat a coronavirus infection and/or to stabilise or improve at least one symptom associated with a coronavirus infection.

Compositions comprising SP-D, or a fragment, homologue, variant or derivative thereof, may be used in conjunction with one or more other antiviral agents. Antiviral agents include, but are not limited to, interferons, oseltamivir, lopinavir, ritonavir, favilavir, remdesivir, anti-inflammatory JAK inhibitors including banicitnib, fedratinib and ruxditinib, low-dose corticosteroids, and the endosome inhibitor, hydroxychloroquine.

Diagnostic Methods The invention further provides a use of SP-D, or a fragment, homologue, variant or derivative thereof, as described herein, in an in vitro method of diagnosing a coronavirus infection. The method may comprise an enzyme-linked immunosorbent assay (ELISA) to detect coronavirus in a biological sample collected from a subject. The method may comprise coating a well of a microtitre plate or the like with SP-D, or a fragment, homologue, variant or derivative thereof which is capable of binding coronavirus, incubating the coated well with the biological sample, and detecting bound coronavirus with the use of an anti-coronavirus antibody that is labelled with a detectable marker (for example, a chemiluminescent label such as horseradish peroxidase). The compositions described herein may further be provided in a kit for use in a method of diagnosing a coronavirus infection. Such a kit may be suitable for performing an enzyme-linked immunosorbent assay (ELISA) to detect coronavirus in a biological sample collected from a subject as described above.

In other embodiments, the invention provides a use of SP-D, or a fragment, homologue, variant or derivative thereof, as described herein, in an in vitro method of determining levels of SP-D in serum, which in turn may be used as an indicator of a coronavirus infection. For example, a microtitre plate well or the like may be coated with an anti-SP-D antibody. The well may then be incubated with a serum sample potentially comprising SP-D. Bound SP-D may then be detected with another labelled anti-SP-D antibody. Known amounts of SP-D, or a fragment, homologue, variant or derivative thereof, as described herein, would be used as a control/standard for the assay in place of the test serum sample.

The present application provides novel and effective prophylactic and therapeutic applications of SP-D and fragments, homologues, variants or derivatives thereof (SP-D active agents), in coronavirus infection. A significant problem with vaccines and other therapies such as antibody therapies against viral diseases is that viruses evolve/mutate such that newly-emerging strains are no longer susceptible. Consequently, new vaccines and antibody therapies need to be developed with emerging strains. As the anti-coronaviral properties of SP-D active agents of the present invention are believed to be mediated through carbohydrate-binding, treatment of future, emerging strains of coronavirus with SP-D active agents may still be effective despite the presence of mutations in these strains.

Examples

Example 1 -Purification of endogenous SP-D Endogenous SP-D for use in in vitro binding and infectivity assays was purified from bronchoalveolar lavage fluid as previously described (Kingma, P. S. et al., (2006) J Biol Chem 281:24496-24505; Strong, Pet al., (1998) J Immunol Methods 220:139-149, each of which is incorporated herein by reference in its entirety). Lavage fluid was cleared of lipid by centrifugation. The lipid-free supernatant was applied to a 20 ml maltosyl-Sepharose column in 20 mM Tris-HC1 (pH 7.4), 5 mM CaC12. The column was washed with a solution of 20 mIVI Tris-HC1 (pH 7.4), 5 mM CaCl2, and 1NI NaCI, followed by a selective elution of SP-D with manganese chloride. The pooled fractions were diluted 10-fold in a solution of 20 mINI TrisHCI (pH 7.4) and 30 mM CaCl2 and applied to a lml bed volume maltosyl-Sepharose column. The column was stripped of LPS with a solution of 20 mM Tris-HC1 (pH 7.4), 20 mM n-octyld-glucopyranoside, 200 mM NaC1, 2 mM CaC12 and 100 pg/ml polymyxin and washed with a solution of 20 mINITris-HC1 (pH 7.4), 0.5 mM CaC12 and 200 mNINaCl. SP-D was eluted with a solution of 20 mM Tris-HCI (pH 7.4), 200 mM NaC1, and 1 mM EDTA. Under the conditions described, LPS concentration was typically <0.1 endotoxin units/jig protein.

Example 2-Purification of rflISP-D The recombinant fragment of SP-D was expressed in E. coli and purified as described as follows: The cDNA for the neck./CRD, including a short region of the collagen stalk (8 Gly-XY) and representing residues 179-355 was cloned from human lung library DNA and inserted into a pET-21d vector (Novagen, Nottingham). The plasmid was transformed into BL210LIDE3) pLysS and a single colony selected and re-plated to give 100-400 colonies/plate. These were scraped and used to inoculate shake-flasks containing 500 ml LB medium supplemented with 100 jig/ml ampicillin and 25 jig/ml chloramphenicol and grown to an 0D600 of 0.6-0.8 followed by induction with 0.4 mM IPTG for 2-3 hours. Cells were collected by centrifugation and lysed in 20 mM Tris-HC1, 150 mM NaC1, 5 m3.4 EDTA, 0.1% v/v Triton X-100, 0.1 mM PMSF, pH 7.5 and sonicated for 3 minutes. The rfl-ISP-D is expressed in insoluble inclusion bodies and was collected by centrifugation and washed 4 times at 10000 x g. The pellet was solubilized in 100 ml of 8 NI Urea, 100 mINI 2-mercaptoethanol, pH 7.5 and clarified by centrifugation and refolded by overnight dialysis against 10 L of 20 miN1 Tris-HCI, 150 MINI NaC1, 5 mM CaC12 (TCB). Refolded rfhSP-D was separated from denatured rfhSP-D by absorption onto maltose-agarose (Sigma-Aldrich, Poole, UK) and eluted with 20 mNI Tris-HC1, 150 mIVI NaC1, containing 5 mM EDTA after first washing the column with TCB containing 1 M NaC1 to remove impurities. Final purification was by gel filtration column (Superose 12, Amersham Pharmacia, UK) in a running buffer of 20 mM Tris-HC1, 150 mM NaC1, 5 mM EDTA, 0.02% (w/v) sodium azide pH7.4 (TSE). The rfhSP-D eluted as a single peak corresponding to 60 kDa molecular weight. The recombinant preparation was judged to be pure by using SDS-PAGE, immunoblotting, and amino-terminal sequencing. The purified trimeric recombinant protein was assessed for correct folding by disulfide mapping and by its crystallographic structure complexed with maltose in the carbohydrate-binding pockets. Endotoxin levels were reduced by passing the purified rfhSP-D through a 10 ml Polymixin B column (Detoxi-Gel, Pierce & Warriner, UK) and only preparations containing less than 5 pg/jtg were used.

Other mammalian systems for expressing rfhSP-D may also be used.

Claims (39)

1. Claims A composition comprising surfactant protein D (SP-D), or a fragment, homologue, variant or derivative thereof, for use in preventing and/or treating a coronavirus infection in a subject, wherein the use comprises administering the composition to the subject.
2. 2. A composition for use according to claim 1, wherein the coronavirus comprises SARS-CoV-2.
3. 3. A composition for use according to claim 1 or claim 2, wherein the coronavirus does not comprise SARS-CoV.
4. 4. A composition for use according to any preceding claim, wherein the coronavirus infection comprises an infection of the respiratory tract.
5. 5. A composition for use according to any preceding claim, wherein the use comprises preventing and/or treating one or more of pneumonia, bronchiolitis, bronchitis and severe acute respiratory syndrome (SARS).
6. A composition for use according to any preceding claim, wherein the use further comprises reducing inflammation in the subject.
7. 7. A composition for use according to claim 6, wherein the inflammation comprises airway inflammation.
8. S. A composition for use according to any preceding claim, wherein the prevention and/or treatment comprises binding of the SP-D, or fragment, homologue or variant thereof to the coronavirus, and inhibiting the entry of the coronavirus into a cell of the subject.
9. A composition for use according to claim 8, wherein the cell comprises one or more of a dendritic cell, alveolar macrophage, alveolar Type I epithelial cell, and alveolar Type II epithelial cell, oral mucosal cells including those of the tongue, epitheial cells of the ileum and colon, choloangiocytes, myocardial cells, kidney proximal cells, bladder urothelial cells, gonadal cells, neutrophils, lymphocytes and peripheral macrophages.
10. 10. A composition for use according to any preceding claim, wherein the SP-D, SP-D fragment, homologue, variant or derivative thereof has carbohydrate binding activity.
11. 11. A composition for use according to any preceding claim, wherein the composition comprises SP-D, the SP-D comprising an amino acid sequence according to SEQ ID NO:2, or wherein the composition comprises an SP-D fragment, homologue, variant or derivative, wherein the SP-D fragment, homologue, variant or derivative comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO:2.
12. 12. A composition for use according to any of claims 1 to 10, wherein the composition comprises an SP-D fragment, wherein the SP-D fragment comprises an amino acid sequence according to SEQ ID NO:4, or wherein the SP-D fragment comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO:4.
13. 13. A composition for use according to any of claims 1 to 10, wherein composition comprises an SP-D homologue, variant or derivative, and the SP-D homologue, variant or derivative comprises Surfactant Protein A (SP-A) or Mannan Binding Lectin (MBL).
14. 14. A composition for use according to any preceding claim, wherein the SP-D, or fragment, homologue, variant or derivative thereof is a fusion protein further comprising a polypeptide having anti-inflammatory activity.
15. 15. A composition for use according to claim 14, wherein the fusion protein comprises a carbohydrate-binding domain of SP-D fused to the polypeptide having anti-inflammatory activity.
16. 16. A composition for use according to claim 14 or claim 15, wherein the polypeptide bind the neonatal Fc receptor.
17. 17. A composition for use according to claim 14 or claim 15, wherein the polypeptide having anti-inflammatory activity comprises IL-4, IL-6, IL-10, IL-11 or IL-13 or their respective receptors.
18. 18. A composition for use according to any preceding claim, wherein the subject is a human.
19. 19. A composition for use according to claim 18, wherein the subject is suffering from one or more of hypertension, a heart condition and renal disease.
20. 20. A composition for use according to claim 19, wherein the subject is receiving treatment with an Angiotensin-converting Enzyme 2 (ACE2) inhibitor and/or an angiotensin II receptor inhibitor (ARE).
21. 21. A composition for use according to any preceding claim, wherein the SP-D, or fragment, homologue, variant or derivative thereof is derived from a human.
22. 22. A composition for use according to any of claims Ito 120, wherein the SP-D, or fragment, homologue, variant or derivative thereof is derived from a non-human species selected from mouse, rat, rabbit, pig, sheep, cow and primate.
23. 23. A composition for use according to any preceding claim, wherein the composition is a pharmaceutical composition.
24. 24. A composition for use according to claim 23, wherein the composition comprises a pharmaceutically acceptable excipient and/or carrier.
25. 25. A composition for use according to any preceding claim wherein the composition is delivered to a lung of the subject.
26. 26. A composition for use according to any preceding claim, wherein the composition is aerosolized.
27. 27. A composition for use according to any preceding claim, wherein the composition is administered to the subject intranasally, intratracheaIly, and/or through a ventilation line.
28. 28. A composition for use according to any preceding claim, wherein the composition is administered using one or more of an inhaler and a nebulizer.
29. 29. A composition for use according to any of claims 1 to 24, wherein the composition is administered systemically.
30. 30. A composition for use according to any preceding claim, wherein the composition further comprises an additional anti-viral agent.
31. 31. A composition for use according to claim 30, wherein the anti-viral agent is selected from one or more of interferons, oseltamivir, lopinavir, ritonavir, avilavir, remdesivir, anti-inflammatory JAK inhibitors including banicitnib, fedratinib and ruxditinib, 1 corticosteroids, and the endosome inhibitor, hydroxychloroquine.
32. 32. A composition comprising surfactant protein D (SP-D), or a fragment, homologue, variant or derivative thereof, for use in preventing and/or treating inflammation in a subject infected with a coronavirus, wherein the use comprises administering the composition to the subject.
33. 33. A composition comprising surfactant protein D (SP-D), or a fragment, homologue, variant or derivative thereof, for use in preventing and/or treating testes dysfunction in a subject infected with a coronavirus, wherein the use comprises administering the composition to the subject.
34. 34. A composition comprising surfactant protein D (SP-D), or a fragment, homologue, variant or derivative thereof, for use in preventing and/or treating pain in a subject infected with a coronavirus, wherein the use comprises administering the composition to the subject.
35. 35. A composition for use according to any of claims 32 to 34, wherein the composition, use and/or subject is as defined in any of claims 2 to 31.
36. 36. A composition comprising a nucleic acid encoding SP-D, or a fragment, homologue, variant or derivative thereof, for use in treatment andJor prevention of a coronavirus infection in a subject, wherein the use comprises administering the nucleic acid encoding SP-D, or a fragment, homologue, variant or derivative thereof to the subject.
37. 37. A composition for use according to claim 36, wherein the nucleic acid comprises SEQ ID NO:1, or wherein the nucleic acid has at least 70% homology to SEQ ID: NO.I.
38. 38. A composition for use according to claim 36, wherein the nucleic acid comprises SEQ ID NO:3, or wherein the nucleic acid 70% homology to SEQ ID: NO.3.
39. 39. Use of Surfactant Protein D (SP-D), or a fragment, homologue, variant or derivative thereof in an in vitro method of detecting a coronavirus infection.