Patent Application
20210214413
Rhinoviruses (RV) are a large group of non-enveloped, single-stranded, positive-sense RNA viruses in the Enterovirus genus of the Picornaviridae family. More than 160 types of rhinoviruses (RVs) are known. RVs are currently classified into three species (A, B and C) of Enteroviruses in the family Picornaviridae. RV-A and RV-B have been known for many years, but the discovery of RV-C in 2006 surprised the molecular and clinical virology communities.
The RV-C are clearly rhinoviruses, but unlike RV-A or RV-B, they are not readily propagated in typical cell culture systems. For example, conventional cell lines such as NCI-H358, WI-38, WisL, HEK293T, BEAS-2B, A549 and HeLa, do not support any detectable RV-C replication. The RV-C are not “new” in terms of evolution, but rather they were physically undetected by typical characterization methods that required cultured virus growth and induction of cytopathic effects, such as endpoint dilution (TCID50) or plaque assays. Initially, virus propagation was restricted to sinus mucosa organ tissues or to airway epithelial air-liquid-interface cultures (ALI) because the isolates proved refractive to standard cell culture [11,12]. These labor-intensive, low virus-titer systems did allow the determination that RV-C growth required a cellular receptor component distinct from intercellular adhesion molecule 1 (ICAM-1) or the low-density lipoprotein receptor (LDLR) used by the RV-A/B species [10,13,14].
Collectively, RV are the primary etiological agents of upper respiratory tract common colds, but many also induce more severe lower respiratory tract illnesses, including bronchitis and pneumonia [1-3]. Rhinovirus (RV)-C viruses (55 types), together with RV-A and RV-B viruses (˜100 types), are the leading cause of common colds. However, the RV-C lead to more severe respiratory infections among children than any other known rhinoviruses and it is now recognized these strains are associated with up to half of rhinovirus illnesses in young children.
In contrast to other RV, the RV-C utilize cadherin related family member 3 (CDHR3) as a cellular receptor. This childhood asthma susceptibility gene product is expressed in the human lower respiratory tract. In line with this etiology, RV-Cs cause a significantly higher rate of lower respiratory tract infections in children than in adults and are directly associated with childhood asthma exacerbations. Similar to influenza, RV-C infections peak in winter months. Currently, there are no vaccines or effective antiviral treatments available.
CDHR3 belongs to the cadherin superfamily of transmembrane calcium-dependent adhesion proteins. The better-described classical cadherins are expressed in a variety of tissues, where they mediate cell-cell interactions, usually through homologous protein contacts, or where they participate in cell signaling, epithelial polarity, and tissue development and organization [16-19]. CDHR3 expression, in contrast, is generally restricted to airway tissues, with protein display primarily on the apical surfaces of ciliated epithelial cells [20,21]. The biological role of CDHR3 in lung development or function is unknown. Cadherins are Ca++-dependent cell adhesion proteins whose primary job is holding cells together through homologous contacts on or between cell surfaces. The sequence of human CDHR3 (885 amino acids) predicts a linear arrangement of 6 extracellular (EC) repeat domains (7 β-strands each) preceded by a signal sequence and tailed with a transmembrane domain (TM) linked to cytoplasmic recognition units. The role of these domains in viral entry and infectivity is not known.
Therefore, there is a need for understanding the virus receptor interactions in order to produce antivirals, including soluble receptors and in vitro assays for screening potential antiviral therapies.
The present invention overcomes the aforementioned drawbacks by providing soluble truncated CDHR3 peptides and methods of making and using for both therapies and in in vitro screening assays to detect antiviral agents.
In one aspect, the invention provides a soluble truncated CDHR3 peptide comprising (A) (a) domain 1 of CDHR3 (domain 1 of SEQ ID NO:1, e.g., SEQ ID NO:2), (b) domain 1 and domain 2 of CDHR3 (domain 1 and 2 of SEQ ID NO:1); (c) domain 1 and domain 3 of CDHR3 (domain 1 and 3 of SEQ ID NO:1) or (d) domains 1, 2 and 3 of CDHR3 (domain 1, 2 and 3 of SEQ ID NO:1), and (B) at least one linker, wherein the at least one linker is selected from linker 1, linker 2, linker 3 and linker 4.
In some aspects, the soluble truncated peptide further comprises at least one of the following: (i) linker 1 (SEQ ID NO:15) before domain 1, (ii) linker 2 (SEQ ID NO:16) between domain 1 and 2, (iii) linker 3 (SEQ ID NO:17) between domain 2 and 3 and (iv) linker 4 (SEQ ID NO:18) after domain 3.
In another aspect, the disclosure provides a soluble truncated CDHR3 peptide comprising: (a) domain 1 (SEQ ID NO:2) of CDHR3 or (b) domain 1 of CDHR3 with a mutation at position 76 relative to SEQ ID NO1 (SEQ ID NO:31), wherein X is selected from the amino acids consisting of A, G, V, L, I, S, and T, preferably A.
In some embodiments, the soluble truncated CDHR3 peptide further comprises at least one linker, wherein the at least one linker is linker 1 (SEQ ID NO:15) before domain 1, linker 2 (SEQ ID NO:16 after domain 1, or both.
In another aspect, the present disclosure provides a vector comprising the nucleic acids encoding a soluble truncated CDHR3 peptide described herein.
In yet another aspect, the disclosure provides a method of making the soluble truncated recombinant peptides, the method comprising: (a) transforming bacteria cells with the vector encoding a soluble truncated CDHR3 peptide described herein; (b) inducing recombinant protein expression in the bacteria cells; (c) lysing bacterial cells and collecting the inclusion bodies comprising the soluble truncated peptide by centrifugation; (d) solubilizing the protein within the inclusion body; and (e) dialyzing and refolding the protein in buffer supplemented with Ca++ to produce soluble truncated recombinant peptides of CDHR3.
In yet another aspect, the disclosure provides a therapeutic composition for reducing or preventing Rhinovirus C entry into cells, the composition comprising a soluble truncated peptide described herein and Ca++ in a pharmaceutically acceptable carrier.
In yet another aspect, the disclosure provides a method for reducing the infection by human rhinovirus (HRV) C of a host cell susceptible to infection by HRV-C, comprising: contacting the virus with the soluble truncated peptide described herein in an amount effective to reduce the infectivity of the virus.
In a further aspect, the present disclosure provides an in vitro assay for testing an agent for anti-viral activity against rhinovirus C, the assay comprising the steps of: (a) contacting the agent with a soluble truncated peptide described herein and rhinovirus C; and (b) assaying the ability of the agent to disrupt binding of the soluble truncated peptide to the virus. In some aspect, the assay further comprises (c) incubating the rhinovirus pre-incubated with the peptide and the agent or with the agent alone with host cells, and measuring the infectivity of the rhinovirus in the host cell, wherein the truncated peptide is used as a positive control.
The foregoing and other aspects and advantages of the invention will appear from the following description. In the description, reference is made to the accompanying drawings which form a part hereof, and in which there are shown, by way of illustration, preferred embodiments of the invention. Such embodiments do not necessarily represent the full scope of the invention, however, and reference is made therefore to the claims and herein for interpreting the scope of the invention.
The invention provides soluble truncated CDHR3 peptides which inhibit rhinovirus C binding to cells and inhibit viral infectivity. The present invention comprises specific regions (specifically the 1st domain of CDHR3) that are required to provide both the binding to RV-C cells and ability to block viral infectivity. These soluble truncated receptors provide the basis for a new in vitro assay for anti-RV-C drug development therapy.
Full-length gene expression of CDHR3 in transfected Hela cells has been described (Bochkov, 2015, PNAS 112:5485). The present invention, using the CDHR3 gene as starting material, produced a series of mammalian and bacterial expression vectors that express different recombinant proteins (see:
The panel of deleted transfected versions of the CDHR3 gene was tested for virus binding in HeLa cell lysates. As demonstrated in the examples, truncated proteins synthesized in HeLa cells and containing EC domain 1 (SEQ ID NO:2), including constructs with EC domains 1-2 and 1-3 were uniquely necessary and sufficient for effective virus binding in IP pulldown assays.
The EC1 (SEQ ID NO:20 or 22 (with W76A mutation), EC1-2 (SEQ ID NO:23 or 25 (with W76A mutation)), EC1-3 (SEQ ID NO:24), and EC1+3(Δ2) (SEQ ID NO:26) sequences were engineered into bacterial expression vectors, including point mutations of W76 and C345 relative to SEQ ID NO:1. The rEC1 segment encoded residues 20-130, rEC1-2 encoded residues 20-237, rEC1-3 encoded residues 20-345, rEC2+3(Δ1) encoded residues 20-345 (Δ27-124), rEC1+3(Δ2) encoded residues 20-345 (Δ129-231), rEC1-3(+10aa) encoded residues 20-355, and rEC1-3(−5aa) encoded residues 20-340 relative to SEQ ID NO:1 amino acid sequence. These units were amplified by PCR from the pHL-FLAG-CDHR3-His cDNAs described above and then ligated into pET11a vectors between the NheI and BamHI restriction sites.
To prevent spurious disulfide formation, most plasmids encoding EC3 segments had a point mutation converting Cys345 to Ala345 (C345A). Additional point mutations were engineered by standard, primer-directed two-step PCR. After much experimentation, a functional protocol for soluble protein isolation was achieved that produces biologically active recombinant proteins capable of binding RV-C15, in a manner similar to the transfection-derived materials previously tested as described in detail in Example 1 in Exhibit A. These recombinant CDHR3 proteins are refolded in the presence of Ca++ which is critical to proper refolding and activity of the proteins, and these E. coli derived proteins are not glycosylated as in the naturally occurring CDHR3 receptor. The protein fragment-binding to virus was independent of Asn-186 glycosylation. These peptides were tested and shown that all the constructs containing domain 1 were able to block viral infectivity and replication for at least 4 genotypes of RVC virus.
The present disclosure provides truncated engineered CDHR3 peptides comprising domain 1 (SEQ ID NO:2) of CDHR3 (SEQ ID NO:1). These soluble truncated peptides can include a heterologous tag and are able to bind to RVC virus and to block viral infectivity of RVC. Suitable soluble truncated peptides include (a) soluble truncated peptide comprising domain 1 (SEQ ID NO:2), (b) soluble truncated peptides comprising domain 1 with a W76X mutation (e.g. SEQ ID NO:31 wherein X is an amino acid selected from A, G, V, L, I, S, or T, preferably A (as demonstrated in SEQ ID NO:30)), (c) soluble truncated peptides comprising sequentially linker 1 (SEQ ID NO:15) and domain 1 (SEQ ID NO:2) (for example, SEQ ID NO:33); (d) soluble truncated peptides comprising linker 1 (SEQ ID NO:15) and domain 1 with a W76X mutation (SEQ ID NO:31) (for example, SEQ ID NO:34); (e) soluble truncated peptides comprising SEQ ID NO:20 or 22; (f) soluble truncated peptides comprising SEQ ID NO:21 and sequences which have at least 75% identity to the associated SEQ ID NOs. described, alternatively at least 80% sequence identity, alternatively at least 90% sequence identity, alternatively at least 95% sequence identity, alternatively at least 98% sequence identity to the associated SEQ ID NOs described. It is contemplated that these soluble truncated peptides further comprise an exogenous tag, for example, an N-terminus, C terminus tag or both. In some embodiments, the peptides comprise a C-terminus tag. In other examples, the peptides comprises an N-terminus tag. In some examples, the peptides comprise both a C- and N-terminus tag. For example, SEQ ID NO:19 and SEQ ID NO:21 show two suitable soluble truncated peptides comprising linker 1, domain 1, and suitable tags, however, the present invention contemplates the use of other suitable tags or agents.
As demonstrated in Example 1, the soluble truncated peptides comprising only domain 1 (e.g., rEC1 (SEQ ID NO:20 or 22) and sufficient linker sequences were found to be monomeric, thus do not require higher order oligomerization to be effective for viral binding or biological activity to prevent viral infection and replication in cells. Further, the single domain truncated peptides were also able once properly folded (after production in E. coli) to be removed from the Ca++ buffer and then reintroduced back into Ca++ containing buffer to allow for proper refolding, which was not observed for the longer truncated peptides containing domains 2 or 3 in addition to domain 1 (See
In one embodiment, the soluble truncated peptides comprises domain 1 (SEQ ID NO:2) and domain 2 (SEQ ID NO:3) and at least one linker (for example, linker 1 (SEQ ID NO:15), linker 2 (SEQ ID NO:16), linker 3 (SEQ ID NO:17), or a combination thereof). Suitable embodiments contemplated include soluble truncated peptides selected from (a) a soluble truncated peptide comprising domain 1 (SEQ ID NO:2), domain 2 (SEQ ID NO:3) and linker 1 (SEQ ID NO:15) before domain 1; (b) a soluble truncated peptide comprising domain 1 (SEQ ID NO:2), domain 2 (SEQ ID NO:3) and linker 1 (SEQ ID NO:15) before domain 1 and linker 2 (SEQ ID NO: 16) between domain 1 and domain 2 (e.g., SEQ ID NO:23); (c) a soluble truncated peptide comprising domain 1 with the W76X mutation (SEQ ID NO:31), domain 2 (SEQ ID NO:3) and linker 1 (SEQ ID NO:15) before domain 1; (d) a soluble truncated peptide comprising domain 1 with the W76X mutation (SEQ ID NO:31), domain 2 (SEQ ID NO:3) and linker 1 (SEQ ID NO:15) before domain 1 and linker 2(SEQ ID NO:16) between domain 1 and domain 2; (e) SEQ ID NO:25; and sequences which have at least 75% identity to the associated SEQ ID Nos., alternatively at least 80% sequence identity, alternatively at least 90% sequence identity, alternatively at least 95% sequence identity, alternatively at least 98% sequence identity to the associated SEQ ID NOs. It is contemplated that these soluble truncated peptides further comprise an exogenous tag, for example, an N-terminus, C terminus tag or both. In some embodiments, the peptides comprise a C-terminus tag. For example, SEQ ID NO: 5 and SEQ ID NO:7 are two contemplated soluble truncated peptides which include both domain 1 and domain 2 and at least one tag, although other iterations are contemplated.
In some embodiments, the soluble truncated CDHR3 peptide consists essentially of (i) linker 1 (SEQ ID NO:15) and domain 1 (SEQ ID NO:2) of CDHR3, (ii) linker 1 (SEQ ID NO:15), domain 1 (SEQ ID NO:2), linker 2 (SEQ ID NO:16), and domain 2 (SEQ ID NO:3) of CDHR3; (iii) linker 1 (SEQ ID NO:15), domain 1 (SEQ ID NO:2), linker 2 (SEQ ID NO:16), domain 2 (SEQ ID NO:3, linker 3 (SEQ ID NO:17), domain 3 (SEQ ID NO:4) and linker 4 (SEQ ID NO:18) of CDHR3 or (iv) linker 1 (SEQ ID NO:15), domain 1, linker 3 (SEQ ID NO:17), domain 3 (SEQ ID NO: 4) and linker 4 (SEQ ID NO:18) of CDHR3 (as referenced in SEQ ID NO:1).
In some further embodiments, the peptides consist essentially of SEQ ID NO: 20, 22, 23-27, 30-31, 33, or 34 and at least one tag sequence. The tag may be at the N- or C-terminus. In some further embodiments, the peptides consists essentially of SEQ ID NO: 20, 22, 23-27, 3031, 33 or 34 and at least two tag sequences.
Experiments demonstrated in Example 1 also showed that mutation of amino acid Cys-345 to Ala (C345A) (as referenced to SEQ ID NO:1, located in linker 4) was important to the process of making biologically active receptor materials, specifically the recombinant bacterially-produced protein. When CDHR3 is truncated (to rEC1-3) in bacterial contexts, Cys-345 forms unwanted exogenous disulfide interactions which decrease the solubility of recombinant protein fragment. Mutation of this residue (C345A) increased virus accessibility and binding efficiency by eliminating spurious disulfide formation, which is demonstrated in the denaturing gel data confirming this finding. The binding of rEC1-3 to C15 virus is very tight (i.e. natural interaction) because it can withstand 500 mM NaCl.
A second mutation of Trp-76 to Ala (W76A) (located in domain 1 in reference sequence SEQ ID NO:1) also resulted in truncated peptides (bacterial or in eukaryotic cells) that bind higher quantities of virus. Not to be bound by any theory, this mutation (W76A) works by interfering with native protein dimerization (most cadherins form self-dimers) opening the protein to better virus interactions.
The combination of W76A and C345A is a preferable combination that works synergistically in the rEC1-3 format to increase virus-binding of the engineered soluble truncated peptides.
In some embodiments, when making a recombinant truncated peptide comprising rEC1-3 (with or without W76A), care must be taken in the exact length of the EC3 domain. If the sequence is extended slightly or partially deleted, the virus binding activity may be significantly diminished. Therefore, the present invention contemplates truncated peptides comprising domain 3 and linker 4 having less than 10 additional amino acids at the terminal end beyond position 345 in SEQ ID NO:1. Further, the truncated peptides contemplated herein include domain 3 sequence that is not truncated shorter than position 345 in SEQ ID NO:1.
The recombinant truncated peptide of rEC1, rEC1-2, rEC1-3 (C345A) or rEC1+3(Δ2) demonstrates inhibitory biological activity that prevents C15 virus from binding to susceptible HeLa cells, and consequently inhibits subsequent virus replication. The degree of inhibition is proportional to the amount of protein used to treat cells, as would be expected if these truncated peptides (a) bound directly to the virus, or (b) formed complexes with native cellular CDHR3, thereby blocking virus binding sites.
As described in more detail below, soluble truncated CDHR3 peptides comprising domain 1, domains 1 and 2, domains 1, 2, and 3, or domains 1 and 3 are able to bind to virus and the binding is independent of the peptide being glycosylated. EC domain 1 is necessary for effective blocking of viral infectivity. The present invention contemplates soluble truncated CDHR3 peptides comprising, consisting essentially of, or consisting of domain 1, domains 1 and 2, domains 1 and 3 or domains 1, 2 and 3 and, in some embodiments, having at least one mutation of the cysteine at position 345 (C345) relative to SEQ ID NO:1. In some preferred embodiments, the peptides further contain at least one tag that does not alter their viral binding or inhibitory properties. In preferred embodiments, the contemplated soluble truncated CDHR3 peptides do not contain domains 4, 5, or 6 of SEQ ID NO:1 as depicted in
The term “truncated” with regard to the specific protein/peptide sequence as used herein describes the soluble truncated CDHR3 peptides including at least domain 1, for example, domains 1 and 2, domains 1, 2 and 3 and domains 1 and 3 as depicted in
In a preferred embodiment, the soluble truncated peptides do not contain amino acids of domains 4, 5, or 6 of CDHR3 of SEQ ID NO:1. In some embodiments, the truncated peptides encompass slight variation in the amino acid sequences that do not alter the functional properties of the peptide (e.g. does not disrupt the ability of the peptide to bind virus and to inhibit infectivity).
The invention encompasses truncated peptides that have more than one mutation, for example, the additional mutation at amino acid position 76 in domain 1 (as referenced in SEQ ID NO:1, e.g., W76X, wherein X is selected from A, G, V, L, I, S or T). For example, but not limited to, suitable sequences of the truncated peptides can be found in SEQ ID NO:22 or 31 which are domain 1 with a W76X mutation, and SEQ ID NO:30 which is domain 1 with W76A mutation. In contemplated soluble truncated peptides that contain domain 3 in addition to domain 1, suitable peptides have the mutation of amino acid position 76 in domain 1 (as referenced in SEQ ID NO:1) in addition to the cysteine mutation at position 345, the combination of which may increase the functionality of the peptides but does not greatly diminish the functionality of the peptide (e.g., does not reduce the functionality of the peptide by more than 20%).
The term “at least one mutation” includes additional amino acid mutations or substitutions within the contemplated soluble truncated peptides that do not greatly diminish (e.g. reduce by more than 20%) the functionality of the peptides in binding of virus or inhibiting infectivity.
As used herein, the term “mutated” or “mutation” refers to both substitutions of amino acids or a deletion of an amino acid within the wild type sequence to produce a modified sequence. In a preferred embodiment, the mutation is a substitution.
The soluble truncated peptides may be covalently or non-covalently attached to a heterologous tag which does not disrupt the functionality of the peptide (e.g., does not disrupt the ability of the peptide to bind virus and to inhibit infectivity). The heterologous tag is a sequence that is not naturally occurring in the CDHR3 protein or peptide.
A “peptide” is used interchangeably with the term “protein” and “polypeptide” and refers to a chain-type polymer formed by amino acid residues which are linked to each other via peptide bonds.
In one embodiment, the present invention provides a soluble truncated CDHR3 peptide comprising, consisting essentially of, or consisting of domain 1 and domain 3 of CDHR3 (SEQ ID NO:1) or sequence that is at least 90% identical to domains 1 and domains 3 of CDHR3, or domain 1, 2 and 3 of CDHR3 or sequence that is at least 90% identical to domains 1, 2 and 3, and comprising at least one amino acid mutation comprising mutation of the cysteine at position 345 (C345) relative to SEQ ID NO:1 mutated to another amino acid, for example, an amino acid selected from the group consisting of alanine (A), glycine (G), valine (V), leucine (L), isoleucine (I), serine (S), and threonine (T). In a preferred embodiment, the cysteine is mutated to an alanine (e.g. C345A).
For clarity of the invention described herein, the domains of CDHR3 are domains of the CDHR3 protein found in SEQ ID NO:1 and all numbers refers to this full protein sequence that corresponds to Genbank #Locus:aic58018. As described herein, domain 1 (SEQ ID NO:2) is amino acids 26-128 of SEQ ID NO:1, domain 2 (SEQ ID NO:3) is amino acids 141-231 of SEQ ID NO:1; and domain 3 (SEQ ID NO:4) is amino acids 242-341 of SEQ ID NO:1. There are linker/spacer regions associated before/after each domain which are believed to be important for proper folding of the proteins. For use herein, the linker/spacers are numbered from N-terminus to C terminus as follows: linker 1 before domain 1 (SEQ ID NO:15, aa 20-25 of SEQ ID NO:1), linker 2 between domain 1 and 2 (SEQ ID NO:16, aa 129-140 of SEQ ID NO:1), linker 3 (SEQ ID NO:17, aa 232-241 of SEQ ID NO:1) located between domain 2 and domain 3, and linker 4 (SEQ ID NO:18, aa 342-345) located after domain 3 (C-terminus).
In one embodiment, a truncated peptide comprising or consisting essentially of domain 1, domain 2 and domain 3 and a sufficient amount of the linkers to allow proper folding of the domains, for example, linker 1, 2, 3 and 4.
In one embodiment, a truncated peptide comprising or consisting essentially of SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4 and linkers 1, 2, 3 and 4, and containing at least one mutation at the C-terminal cysteine in linker 4 (SEQ ID NO:4) to an amino acid selected from the group consisting of A, G, V, L, I, S, and T is contemplated. In some embodiments, linkers 1, 2, 3 and 4 are further included in the truncated peptide. In another embodiment, a truncated peptide comprises or consists essentially of SEQ ID NO:2 and SEQ ID NO:4 consecutive in the soluble truncated peptide and including linker 1, 3 and 4 is contemplated (e.g., consecutive SEQ ID NO:15 (linker 1)-SEQ ID NO:2 (domain 1)-SEQ ID NO:17 (linker 3)-SEQ ID NO:4 (domain 3)-SEQ ID NO:18 (linker 4), for example: SEQ ID NO:14).
In some embodiments, the truncated peptides comprise at least one mutation at the terminal cysteine (e.g. amino acid position C345 of SEQ ID NO:1) and in further embodiments, also include at least one mutation in amino acid at position 76 (W76) relative to SEQ ID NO:1, wherein each of the positions are mutated to an amino acid selected from the group consisting of alanine, glycine, valine, leucine, isoleucine, serine and threonine. In a preferred embodiment the two amino acids are mutated to alanine.
In one embodiment, the soluble truncated CDHR3 peptide comprises or consists essentially of domain 1, domain 2 and domain 3 of CDHR3 or a consecutive sequence at least 90% similar to domains 1, 2 and 3 wherein the terminal cysteine at position 345 relative to SEQ ID NO:1 is mutated to another amino acid, for example, an amino acid selected from the group consisting of alanine, glycine, valine, leucine and isoleucine, serine and threonine. In one embodiment, the soluble truncated peptide is amino acids 20-345 of SEQ ID NO:1 containing the mutation of C345A. In a further embodiment, the soluble truncated peptide is amino acids 20-345 of SEQ ID NO:1 containing the C345A and W76A mutations. In a further embodiment, the soluble truncated peptide comprises or consists essentially of amino acids 20-345 of SEQ ID NO:1 with the C345A mutation and a sequence comprising a tag, for example a HIS tag or a FLAG tag located at the N-terminus, C-terminus or both of the truncated peptide. In another embodiment, the truncated peptide sequence comprises the amino acids of an N-terminal FLAG tag, a C-terminal HIS tag or a combination thereof.
In some embodiments, the soluble truncated CDHR3 peptides containing domains 1, 2 and 3 or domains 1 and 3 have substantial identity of those domains to the domains identified in SEQ ID NO:1 but contain at least one amino acid mutation altered from the peptide sequence of SEQ ID NO:1 (preferably mutation of the W at position 76 in domain 1 or the terminal cysteine, e.g. C345 mutation relative to SEQ ID NO:1 wherein the mutation is to an amino acid selected from the group consisting of alanine, glycine, valine, leucine and isoleucine, serine and threonine). In some embodiments, the domains have at least 75% identity to the CDHR3 domains 1 2 and 3, alternatively at least 80% sequence identity, alternatively at least 90% sequence identity, alternatively at least 95% sequence identity. It is contemplated in some of these embodiments, there are at least two mutations within the soluble truncated CDHR3 peptides from the wild-type sequence of the truncated domains.
Protein and nucleic acid sequence identities are evaluated using the Basic Local Alignment Search Tool (“BLAST”) which is well known in the art (Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87: 2267-2268; Altschul et al., 1997, Nucl. Acids Res. 25: 3389-3402). The BLAST programs identify homologous sequences by identifying similar segments, which are referred to herein as “high-scoring segment pairs,” between a query amino or nucleic acid sequence and a test sequence which is preferably obtained from a protein or nucleic acid sequence database. Preferably, the statistical significance of a high-scoring segment pair is evaluated using the statistical significance formula (Karlin and Altschul, 1990), the disclosure of which is incorporated by reference in its entirety. The BLAST programs can be used with the default parameters or with modified parameters provided by the user.
“Percentage of sequence identity” or “percent similarity” is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or peptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.
The term “substantial identity” or “substantial similarity” of polynucleotide or peptide sequences means that a polynucleotide or peptide comprises a sequence that has at least 75% sequence identity. Alternatively, percent identity can be any integer from 75% to 100%. More preferred embodiments include at least: 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% compared to a reference sequence using the programs described herein; preferably BLAST using standard parameters, as described. These values can be appropriately adjusted to determine corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning and the like.
“Substantial identity” of amino acid sequences for purposes of this invention normally means polypeptide sequence identity of at least 75%. Preferred percent identity of polypeptides can be any integer from 75% to 100%. More preferred embodiments include at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.7%, or 99%.
In another embodiment, the soluble truncated CDHR3 peptide consists essentially of (i) amino acids 20-345 of SEQ ID NO:1 with C345A mutation; (ii) amino acids 20-345 of SEQ ID NO:1 with C345A mutation and W76A mutation, (iii) amino acids 20-128 and 232-345 of SEQ ID NO:1 consecutive in the peptide and wherein the peptide contains C345A mutation; or (iv) amino acids 20-128 and 232-345 of SEQ ID NO:1 with C345A and W76A mutations, and optionally wherein any of the truncated peptides (i)-(iv) further comprise the amino acid sequence of a tag at the C-terminus, N terminus or both of the peptide sequence.
In one embodiment, the soluble truncated CDHR3 peptide consists essentially of the peptide of SEQ ID NO:19 (FLAG-CDHR3 EC1-HIS), SEQ ID NO:22 (FLAG-CDHR3-EC1-His W76A); SEQ ID NO:5 (FLAG_CDHR3 EC1-2), SEQ ID NO:6 (FLAG-CDHR3 EC1-3 HIS), SEQ ID NO:7 (FLAG-CDHR3 EC1-2 W76A), SEQ ID NO:8 (FLAG-CDHR3-W76A EC1-3-HIS) or SEQ ID NO:9 (FLAG-CDHR3 EC1+3 (Δ2)).
Suitable soluble truncated CDHR3 peptides of the present disclosure are truncated peptides that contain at least domain 1 (SEQ ID NO:2) or domain 1 with a W76X mutation (SEQ ID NO:31). In some embodiments, the soluble truncated CDHR3 peptides comprise or consist essentially of (a) domain 1 (SEQ ID NO:2) of CDHR3 or (b) domain 1 of CDHR3 with a mutation at position 76 relative to SEQ ID NO1 (e.g., SEQ ID NO:31), wherein X is selected from the amino acids consisting of A, G, V, L, I, S, and T, preferably in one embodiment, A).
In some embodiments, the soluble truncated CDHR3 peptide containing domain 1 (or domain 1 with W76X mutation) further comprises at least one linker, for example, linker 1 (SEQ ID NO:15) before domain 1, linker 2 (SEQ ID NO:16) after domain 1, or both. For example, suitable soluble truncated CDHR3 peptides of the present disclosure are SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:30, SEQ ID NO:33, or SEQ ID NO:34. In some embodiments, the soluble truncated CDHR3 peptide further comprises a heterologous tag. Suitable peptides with a heterologous tag include, but are not limited to, for example, SEQ ID NO:19 (FLAG-EC1-his) or SEQ ID NO: 21 (FLAG-EC1-his with W76A).
In some embodiments, the truncated CDHR3 peptides of the present disclosure are truncated peptides that contain at least domain 1 (SEQ ID NO:2) or domain 1 with a W76X mutation (SEQ ID NO:31) and further comprise additional subsequent sequence selected from the group consisting of (i) domain 2 of CDHR3 (SEQ ID NO:3); (ii) domain 3 of CDHR3 (SEQ ID NO:4); (iii) domain 2 (SEQ ID NO:3) and domain 3 (SEQ ID NO:4) of CDHR3; (d) at least one linker, wherein the at least one linker is before domain 1 (linker 1), between domain 1 and 2 (linker 2), between domain 2 and 3 (linker 3), or after domain 3 (linker 4), or (e) a combination of any one of (i)-(iii) and (iv). For example, the suitable linkers can be selected from (a) linker 1 (SEQ ID NO:15) before domain 1, (b) linker 2 (SEQ ID NO: 16) between domain 1 and 2; (c) linker 3 (SEQ ID NO:17) positioned between domain 2 and 3; (d) linker 4 (SEQ ID NO:18) positioned after domain 3, wherein X is, and (e) a combination of any of (a)-(d).
One suitable example of a soluble truncated protein comprises domain 1 (SEQ ID NO:2) and domain 3 of CDHR3 (SEQ ID NO:4) and at least linker 4 (SEQ ID NO:18). In another example, the soluble truncated protein comprises linker 1 (SEQ ID NO:15), domain 1 (SEQ ID NO:2), linker 3 (SEQ ID NO:17), domain 3 of CDHR3 (SEQ ID NO:4) and at least linker 4 (SEQ ID NO:18)
Other suitable truncated CDHR3 peptides comprise or consist essentially of sequentially: (a) linker 1 (SEQ ID NO:15), domain 1 (SEQ ID NO:2), linker 2 (SEQ ID NO:16), and domain 2 (SEQ ID NO:3); (b) linker 1(SEQ ID NO:15), domain 1 with W76X mutation (SEQ ID NO:30), linker 2 (SEQ ID NO:16), and domain 2 (SEQ ID NO:3); (c) linker 1(SEQ ID NO:15), domain 1 with W76X mutation (SEQ ID NO:30), linker 2 (SEQ ID NO:16), domain 2 (SEQ ID NO:3), and linker 3 (SEQ ID NO:17); (d) linker 1 (SEQ ID NO:15), domain 1 (SEQ ID NO:2), linker 2 (SEQ ID NO:16), domain 3 (SEQ ID NO:4) and linker 4 (SEQ ID NO:18); (e) linker 1 (SEQ ID NO:15), domain 1 with W76X mutation (SEQ ID NO:30), linker 2 (SEQ ID NO:16), domain 3 (SEQ ID NO:4) and linker 4 (SEQ ID NO:18); and (f) linker 1(SEQ ID NO:15), domain 1 with W76X mutation (SEQ ID NO:30), linker 2 (SEQ ID NO:16), domain 2 (SEQ ID NO:3), and linker 3 (SEQ ID NO:17), domain 3 (SEQ ID NO:4) and linker 4 (SEQ ID NO:18). For example, suitable truncated CDHR3 peptides may be the amino acid sequence of any one of SEQ ID NO: 23-27.
In other examples, the soluble truncated CDHR3 peptide consists essentially of an amino acid sequence selected from the group consisting of: SEQ ID NO:2, 5-9, 19-27, 30-31, 33-34, and a sequence at least 90% identity to SEQ ID NO:2, 5-8, 19-27, 30-31 and 33-34. In a further example, the soluble truncated CDHR3 peptides is an amino acid sequence selected from the group consisting of SEQ ID NO, 5-9, 19 and 21, or an amino acid sequence having at least 90% identity to SEQ ID NO, 5-9, 19 and 21.
In another set of examples, the soluble truncated CDHR3 peptide comprises or consists essentially of (i) amino acids 20-345 of SEQ ID NO:1 with C345A mutation; (ii) amino acids 20-345 of SEQ ID NO:1 with C345A mutation and W76A mutation, (iii) amino acids 20-128 and 232-345 of SEQ ID NO:1 with C345A mutation; (iv) amino acids 20-128 and 232-345 of SEQ ID NO:1 with C345A and W76A mutations, (v) amino acids 20-237 of SEQ ID NO:1; (vi) amino acids 20-130 of SEQ ID NO:1; or (vi) amino acids 20-130 of SEQ ID NO:1 with W76A mutation. Some suitable examples of the truncated CDHR3 peptide are SEQ ID NO:6 (FLAG-CDHR3 EC1-3 HIS), SEQ ID NO:8 (FLAG-CDHR3-W76A EC1-3-HIS), SEQ ID NO:9 (FLAG-CDHR3 EC1+3 (Δ2)), SEQ ID NO:19 (FLAG-CDHR3-EC1) or SEQ ID NO:21.
All soluble truncated peptides described herein can be made in a suitable host cells line. Suitable host cells include prokaryotic or eukaryotic cells. Suitable eukaryotic cells include mammalian cells, for example, animal or human cells (including, but not limited to, for example, HeLa cells, HEK293S cells or CHO cells) by transfection or transduction of the cells with a vector encoding and capable of expressing the CDHR3 peptides. In some embodiments, the soluble truncated peptides are fully or partially glycosylated. In other embodiments, the proteins are not glycosylated. In another embodiment, a suitable host cell is a yeast cell.
In some embodiments, as demonstrated by the Examples, the soluble truncated peptides described herein may be completely unglycosylated as glycosylation is not necessary for viral binding or inhibiting viral infectivity of the peptides. In one embodiment, the peptides are made as recombinant proteins in bacterial cells by transfection of bacterial cells with a suitable vector encoding and able to express the recombinant truncated CDHR3 peptides. Suitable bacteria for producing recombinant protein are known in the art and include, but are not limited to E. coli. The recombinant truncated peptides are expressed in bacteria and contained within inclusion bodies within the bacteria. The inclusion bodies can be isolated and the proteins solubilized and refolded by the methods described herein, requiring refolding and preparing in a suitable carrier containing Ca++.
The term “soluble peptide” as used herein refers to the peptide that does not include the membrane-spanning and cytoplasmic domains present in the naturally occurring, non-soluble form.
In some embodiments, the truncated peptide further contains an exogenous or heterologous tag or agent. In some embodiments, the truncated peptide is directly or indirectly linked to an exogenous or heterologous tag or agent. The suitable tag or agent does not interfere with the functionality of the soluble truncated CDHR3 peptides' function in viral binding or inhibiting viral infectivity. Further, the exogenous or heterologous tag is not native to the CDHR3 protein nor derived from the human CDHR3 protein.
In some embodiments, the heterologous tag or agent is a polypeptide, wherein the polypeptide is translated concurrently with the soluble truncated CDHR3 peptide's nucleic acid sequence.
The term “tag” or “agent” as used herein includes any useful exogenous moiety that allows for the purification, identification, detection, diagnosing, imaging, or therapeutic use of the peptides of the present invention. The terms tag or agent includes epitope tags or detection markers, including, for example, enzymatic markers, fluorescence markers, radioactive markers, among others. The tags or agents are not naturally found in the peptide sequence. Additionally, the term agent includes therapeutic agents, small molecules, and drugs, among others. Suitable tags are known in the art and include, but are not limited to, affinity or epitope tags (non-limiting examples include, e.g., cMyc, HIS, FLAG, VS-tag, HA-tag, NE-tag), florescence tags (RFP, GFP, etc.). Suitable agents include agents that help with the bioavailability or targeting of the peptide. In some embodiments, the peptide is encoded in a nucleic acid sequence that encodes both the peptide and the tag (for example a FLAG, HIS or HA tag).
The present invention also contemplates purified and isolated nucleic acid sequences (e.g. DNA sequences) encoding the soluble truncated CDHR3 peptides, vectors comprising the DNA sequences able to express the soluble truncated CDHR3 peptide in host cells and host cells comprising the DNA or vectors capable of expressing the soluble truncated CDHR3 peptides. In one embodiment, the present invention provides nucleic acid sequences encoding the truncated peptides comprising domain 1 domain 1 with a W76 mutation; domain 1 and 2; domain 1 and 2 with a W76 mutation; domain 1, 2, and 3 of CDHR3 with a C345 mutation; domain 1, 2 and 3 with a W76 and C345 mutation; domain 1 and 3 of CDHR3 with a W76 and C345 mutation; and domain 1 and 3 of CDHR3 with a C345 mutation as described herein. In one example, the vector comprises the nucleic acid sequence selected from the group consisting of SEQ ID NO:10 (EC1-2), SEQ ID NO:11 (EC1-2 W76A), SEQ ID NO:12 (EC1-3), SEQ ID NO:13 (EC1-3W76A), SEQ ID NO:14 (EC1+3(Δ2)), SEQ ID NO:28 (EC1), or SEQ ID NO:29 (EC1 W76A).
In suitable exemplary embodiments, the DNA sequences contemplated herein include, but are not limited to, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO: 12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:28 or SEQ ID NO:29.
The term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked and expressing the gene encoded within the nucleic acid sequence. One type of vector is a “plasmid”, which refers to a circular double stranded DNA loop into which additional DNA segments may be ligated, specifically exogenous DNA segments of the targeted protein. Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of exogenous genes to which they are operatively linked. Such vectors are referred to herein as “recombinant expression vectors” (or simply, “expression vectors”). In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, “plasmid” and “vector” may be used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.
In some embodiments, the heterologous sequence of the vector is a viral sequence. Suitable viral sequences include, but are not limited to, an adeno-associated viral sequence or a retroviral sequence. In a preferred embodiment, the heterologous nucleic acid sequence is a recombinant adeno-associated virus. In some embodiments, the virus is an adeno-associated virus (rAAV), a lentivirus, an adenovirus, a herpes simplex virus, a baculovirus, among others. Further embodiments include viruses made using the viral vectors described herein. Suitable methods for making the viruses are known in the art.
Vectors can also include additional selectable marker genes and other genetic elements known in the art.
A vector can preferably transduce, transform or infect a cell, thereby causing the cell to express the proteins encoded by the vector.
In one embodiment, the vectors can comprise a nucleic acid sequence selected from the group consisting of SEQ ID NO:10 (EC1-2), SEQ ID NO:11 (EC1-2 W76A), SEQ ID NO:12 (EC1-3), SEQ ID NO:13 (EC1-3W76A), SEQ ID NO:14 (EC1+3(Δ2)), SEQ ID NO:28 (EC1), and SEQ ID NO:29 (EC1 W76A).
In another embodiment, the invention provides a method of making the soluble truncated recombinant peptide described herein in bacterial cells. The method comprises the steps of: (a) transforming bacterial cells with the vector encoding and able to express the soluble truncated recombinant peptide; (b) lysing bacterial cells and collecting the inclusion bodies comprising the soluble peptide by centrifugation; (c) solubilizing the peptide within the inclusion body; and (d) dialyzing and refolding the peptide in buffer supplemented with Ca++(e.g., CaCl2) to produce soluble truncated recombinant peptides of CDHR3 In some embodiments, the method further comprises before step (b) inducing recombinant peptide expression in the bacteria cells. Methods of inducing bacterial cells are known in the art, and include, but are not limited to, Isopropyl β-D-1-thiogalactopyranoside (IPTG) for use in bacterial vectors containing a lac operator. Methods of solubilizing the protein and refolding the protein are known in the art and described in the examples. Suitably, the soluble truncated CDHR3 peptides described herein require the presence of Ca++(e.g. CaCl2) during the refolding and dialysis. The final soluble truncated CDHR3 peptides must be stored in a buffer containing Ca++(e.g. CaCl2). Suitable ranges of CaCl2) in the buffer solution include from about 2 mM to 10 mM, preferably from 2 mM to 5 mM, for example, about 3 mM.
A specific protocol for making the rEC, rEC1-2, rEC1-3 and rEC1+3(Δ2) is described here. Bacterial plasmids for the expression of various CDHR3 rEC domains, linked to amino-terminal FLAG-tags and carboxyl-terminal 6× His tags, were constructed. The rEC1 segment encoded residues 20-130, rEC1-2 encoded residues 20-237, rEC1-3 encoded residues 20-345, rEC2+3(Δ1) encoded residues 20-345 (Δ27-124), rEC1+3(Δ2) encoded residues 20-345 (Δ129-231), rEC1-3(+10aa) encoded residues 20-355, and rEC1-3(−5aa) encoded residues 20-340. These units were amplified by PCR from the pHL-FLAG-CDHR3-His cDNAs described above and then ligated into pET11a vectors between the NheI and BamHI restriction sites. To prevent spurious disulfide formation, most plasmids encoding EC3 segments had a point mutation converting Cys345 to Ala345 (C345A). Additional point mutations were engineered by standard, primer-directed two-step PCR. Escherichia coli BL21(DE3) pLysS cells, transformed with each plasmid were induced with IPTG (isopropyl-β-d-thiogalactopyranoside) for recombinant protein expression. The cells were collected by centrifugation, resuspended in lysis buffer (20 mM Tris, pH 8.0, 137 mM NaCl, 1% Triton-X100), and sonicated. The majority of recombinant material was insoluble and collected by centrifugation (20,000×g for 45 min at 4° C.). The pellets were washed (1 M NaCl, then 1 M urea, then water) and solubilized (6 M urea, 20 mM Tris, pH 8.0, 137 mM NaCl for 1 h at 25° C. or O/N at 4° C.). After clarification (20,000×g for 45 min at 4° C.), the supernatants of rEC1 and rEC1-2 proteins were purified under denaturing conditions (6 M urea, 20 mM Tris, pH 8.0, 137 mM NaCl) on HisTrap FF columns (GE Healthcare). After elution with 200 mM Imidazole, the proteins were diluted (to 0.1-0.2 mg/mL) in the solubilization buffer (above) supplemented with 3 mM CaCl2) and then dialyzed (4 times, 8-12 hrs each, against 20 mM Tris, pH 8.0, 137 mM NaCl, 3 mM CaCl2), 2 mM β-mercaptoethanol). Proteins containing the EC3 domain [rEC1-3, rEC2+3(Δ1), and rEC1+3(Δ2)] bound poorly to the HisTrap FF columns even under denaturing conditions so the clarified supernatants were diluted and refolded as described above. Refolded proteins were concentrated using Amicon Ultra centrifugal filters. The soluble receptors bind virus in an in vitro assay. We note that the bacterial constructs can be made without one of the tags, for example, without the FLAG tag and the C-terminal tag (for example the C terminal HIS tag) may be used for purification of the peptide. Other suitable tags are known in the art and contemplated to be able to be switched out the FLAG or HIS tag described above.
The present invention also contemplates therapeutic compositions for reducing or preventing rhinovirus C entry into cells. The therapeutic compositions comprise the soluble truncated peptides of CDHR3, Ca++(e.g. CaCl2) and a pharmaceutically acceptable carrier.
A “pharmaceutically acceptable carrier” means any conventional pharmaceutically acceptable carrier, vehicle, or excipient that is used in the art for production and administration of compositions to a subject. Pharmaceutically acceptable carriers are typically non-toxic, inert, solid or liquid carriers which are physiologically balanced. Typically phosphate buffered saline or other saline solutions are physiologically acceptable carriers. Water is not contemplated as a suitable physiologically acceptable carrier. In some embodiments, additional components may be added to preserve the structure and function of the peptides of the present invention, but are physiologically acceptable for administration to a subject. Ca++(for example, but not limited to, CaCl2) is a necessary addition to the pharmaceutically acceptable carrier in order to preserve the function of the peptides' ability to bind to virus and inhibit viral entry.
Methods for reducing infection by human rhinovirus C in a host cell susceptible to infection by HRV-C are also contemplated. The methods comprise contacting the virus with an effective amount of the soluble truncated peptide or a pharmaceutical composition comprising the soluble truncated peptide described herein in an amount effective to reduce the infectivity of the virus. In some embodiments, the method is performed in vivo. Methods of performing in vivo include administering to a subject the soluble truncated peptide or pharmaceutical composition in an amount effective to reduce HRV-C infection within the subject.
Methods for treating a subject having human rhinovirus C infection are also contemplated in the present invention. The methods comprise administering to the subject an effective amount of the soluble truncated peptide or pharmaceutical composition comprising the soluble truncated peptide, wherein the effective amount is able to treat one or more symptoms of human rhinovirus C infection.
Routes of administering the soluble truncated peptides and therapeutic compositions in vivo are by appropriate contact with those areas of the body susceptible for infection by HRV, e.g., by any intranasal spray.
The term “treating” or “treatment” includes, but is not limited to, reducing, inhibiting or preventing one or more signs or symptoms associated with rhinovirus C infection. Treating also includes the ability to inhibit or reduce rhinovirus infection within the subject, including the ability to reduce the number of infective rhinovirus particles within the subject, for example, by reducing the number of rhinovirus particles in respiratory droplets or mucus. Symptoms of rhinovirus C infection include, but are not limited to, sore throat, runny nose, nasal congestion, sneezing and cough; muscle aches, fatigue, malaise, headache, muscle weakness, or loss of appetite.
The terms “subject” and “patient” are used interchangeably and refer to any animal (e.g., a mammal), including, but not limited to, humans, non-human primates, rodents, and the like, which is to be the recipient of a particular treatment. Typically, the terms “subject” and “patient” are used interchangeably herein in reference to a human subject.
The terms “effective amount” or “therapeutically effective amount” refer to an amount sufficient to effect beneficial or desirable biological and/or clinical results. In some embodiments, the “effective amount” is an amount able to inhibit viral infectivity by at least 50% or more, preferably by at least 75% or more, more preferably by at least 90% or more.
Suitable amounts of CaCl2) or other soluble Ca++ salt for preparing compositions comprising the soluble truncated peptide include from about 2 mM to about 10 mM, preferably about 3 mM. Other suitable ranges in between are contemplated, including for example, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM and 10 mM.
The soluble truncated peptides of CDHR3 can be used in assays for testing agents for anti-viral activity. Suitable in vitro assay for testing an agent for anti-viral activity against rhinovirus C are contemplated herein. In one embodiment, the assay comprising the steps of: (a) contacting the agent with the soluble truncated peptide of CDHR3; and (b) assaying the ability of the agent to disrupt binding of the soluble peptide to the virus. Some embodiments, further include the step of (c) incubating rhinovirus pre-incubated with the truncated peptide and the agent with host cells, and measuring the infectivity of the rhinovirus in the host cell. Suitable methods for testing inhibition of binding in a pull-down assay include, but are not limited to, for example,
The soluble truncated peptides described herein are contemplated to be used in high-throughput screening methods for screening compounds and agents for anti-viral activity. Suitable methods are known in the art and can incorporate the novel soluble truncated peptides described herein.
In some embodiments, the truncated peptide is attached to a solid support, for example, tissue culture plates (including, but not limited to, 6-well, 12-well, 24-well, 48-well, 96-well, 384-well, etc.), membranes, glass slides or plates, among others.
Drugs or agent can be added to screen for inhibitory effects that disrupt this binding.
In some assays, the ability of the virus to bind to cells and infect cells in the presence of the soluble truncated peptide and test agent are assayed.
In some assays, the ability of the virus to bind cells in the presence of the test agent alone are assayed with soluble truncated peptide as a positive control for infection inhibition.
The present invention has been described in terms of one or more preferred embodiments, and it should be appreciated that many equivalents, alternatives, variations, and modifications, aside from those expressly stated, are possible and within the scope of the invention.
It should be apparent to those skilled in the art that many additional modifications beside those already described are possible without departing from the inventive concepts. In interpreting this disclosure, all terms should be interpreted in the broadest possible manner consistent with the context. Variations of the term “comprising” should be interpreted as referring to elements, components, or steps in a non-exclusive manner, so the referenced elements, components, or steps may be combined with other elements, components, or steps that are not expressly referenced. Embodiments referenced as “comprising” certain elements are also contemplated as “consisting essentially of” and “consisting of” those elements. In places where ranges of values are given, this disclosure explicitly contemplates other combinations of the lower and upper limits of those ranges that are not explicitly recited. For example, recitation of a value between 1 and 10 or between 2 and 9 also contemplates a value between 1 and 9 or between 2 and 10. Ranges identified as being “between” two values are inclusive of the end-point values. For example, recitation of a value between 1 and 10 includes the values 1 and 10.
Aspects of the present disclosure that are described with respect to methods can be utilized in the context of the compositions of matter or kits discussed in this disclosure. Similarly, aspects of the present disclosure that are described with respect to compositions of matter can be utilized in the context of the methods and kits, and aspects of the present disclosure that are described with respect to kits can be utilized in the context of the methods and compositions of matter.
The invention will be more fully understood upon consideration of the following non-limiting examples.
This Example demonstrates that identification and production of soluble truncated recombinant CDHR3 peptides which inhibit rhinovirus C binding to cells and inhibit viral infectivity. As detailed below, specific regions (specifically any construct comprising the 1st domain of CDHR3) are required to provide both the binding to RV-C cells and ability to block viral infectivity. These soluble truncated receptors provide the basis for a new in vitro assay for anti-RV-C drug development therapy.
This Example in part demonstrates that soluble recombinant materials comprising domain 1 (e.g., rEC1), domain 1 and 2 (e.g., rEC1-2), domain 1 and 3(Δ2) (e.g., rEC1+3(Δ2)) or domains 1-3 (e.g., rEC1-3) when properly folded in the presence of Ca++, can recapitulate virus:receptor binding interactions and can inhibit RV-C infection of susceptible cells for at least 4 different virus genotypes.
This Example in part further shows that the peptides described herein can be used to reduce viral infection. When pre-incubated with virus before inoculation of cell monolayers, all recombinant proteins containing EC1 inhibited infection in a dose dependent manner (
Introduction for Example 1: A defining feature of all cadherins is their distinctive rod-like arrangement of linear tandem-repeat extracellular domains (EC). Collectively or individually, these units mediate the various cis and trans contacts needed for adhesion specificity [26]. Each domain of about 110 amino acids is distinct in sequence, but they typically configure into similar 7-stranded, anti-parallel “Greek key” motifs. The linked repeat units (usually 5) are preceded by a signal sequence and followed with a short transmembrane segment and a cytoplasmic tail. Calcium ions (2 or 3) chelated by multiple acidic residues, are set into each EC junction, and required for relative domain orientation as well as the overall rigidity of the long, slightly curved, rod-like conformations. Homotypic and heterotypic cadherins interact in cis (parallel orientation, same cell) and/or trans (anti-parallel orientation, opposing cell) by reciprocal ionic contacts on their various EC surfaces to provide adhesion functions [27]. The CDHR3 sequence (885 amino acids) encodes six EC repeats with the usual, easily defined amino and carboxyl extensions (
This Example demonstrates the development of relevant biochemical pull-down assays, leading to determination of the minimum forms of CHDH3 capable of direct virus interactions. As soluble recombinant materials, the required protein units, rEC1, rEC1-2, rEC1+3(Δ2) or rEC1-3, when properly folded in the presence of Ca++, could recapitulate virus:receptor binding interactions and could inhibit RV-C infection of susceptible cells for at least 4 different virus genotypes.
RV-C will bind and infect HeLa cells that are transfected or transduced for surface expression of full-length CDHR3 sequences [15]. During transfections however, the exterior protein presentation and therefore formal virus access is dependent on each individual cell's cDNA uptake as well as that sequence's innate display potential (e.g. C529 vs Y529). Transduced cells introduce additional surface variabilities by the very nature of clonal selection. A reproducible assay for virus binding, dependent only on the introduced CDHR3 sequence, was achieved by reacting C15 virus with whole-cell lysates, after transfection or transduction of preferred cDNAs. Except for the CDHR3 glycosylation status (see below) there was no indication in any experiment of virus preference for intra- or extracellular protein pool locales. Indeed, even the predominantly non-surface C529 materials readily interacted with virus, if given the chance as lysate extracts (e.g. see
The initial application of this assay tested the overall EC domain requirements (
The N186 mutation data fit the binding model's putative expectations that glycosylation at this site might contribute to virus interactions. Almost all classical cadherins are post-translation-ally modified with glycans [31,33-35]. The sequence of CDHR3 predicts 6 possible N-linked sites (
CDHR3 human protein variants Y529 and C529 from transfected HeLa cells migrate identically during gel fractionation (
Unlike transfected cells, transduced cells expressing CDHR3 Y529 (fCR3Y) show 2 forms of protein upon Western analyses (
The lysate binding assays mapped the segments and sequences of CDHR3 required for virus pull-down. In a formal sense though, those experiments could not preclude contributory partner interactions from other lysate components. Full-length CDHR3 proved insoluble when expressed in bacteria and not readily refolded. Accordingly, the next experiments focused on the EC1-3 domains. Like the successful lysate materials, the recombinant proteins were designed with amino-terminal FLAG-tags and carboxyl-terminal His-tags. The first constructions expressed native CDHR3 residues 20-345 (rEC1-3#). The amino-terminal signal sequence (residues 1-19) was not included because it prevented high-level protein expression in bacteria. Produced this way, >90% of the bacterial inclusion body material was the desired protein, which upon denaturation (urea) and refolding (see Methods) was capable of binding virus (
The next plasmid series assayed domain requirements, successively eliminating EC1, EC2 and EC3 (
The virus captured in these assays was not by weak interactions, because for EC1 containing proteins, it resisted disruption with 500 mM salt (
To resolve why prokaryotic (bacteria) and eukaryotic (transfections) expressed proteins showed apparently different virus interaction requirements for the EC2 domain and for N186, transfection plasmids encoding EC1-3 (N186 or N186A), and EC1+3(Δ2), were introduce into HeLa cells (
The rod-like arrangement of EC domains in classical cadherins is dependent upon calcium binding to multiple acidic clusters at the inter-domain junctions. Removal of calcium causes the proteins to collapse into more condensed structures [31,39,40] and can also affect the oligomerization status when the domains no longer orient properly [41,42]. The CDHR3 structure model predicts analogous acidic clusters at each EC junction [23], but modeling cannot accurately anticipate the exact ion placement or count. In the lysate assay with full-length HeLa-produced CDHR3, the addition of EDTA or EGTA reduced the virus binding to background levels (
Soluble ICAM-1 and LDLR materials can bind their appropriate RV-A and RV—B isolates to inhibit virus infection of susceptible cells [43-45]. The soluble recombinant CDHR3 protein panel was tested for its ability to inhibit C15 infection of stably transduced fCR3Y cells. When pre-incubated with virus before inoculation of cell monolayers, all recombinant proteins containing EC1 inhibited infection in a dose dependent manner (
The binding of a virus to accessible external receptor(s) is an initiating step in host cell entry. CDHR3 cell surface display, mediated by the Y529 variant SNP of this gene, is required for optimal RV-C entry into cells [15]. The dominant human allele, encoding C529, shows much lower protein surface expression in transfected cells and consequently poorer cell-binding interactions with virus. For homozygous or heterozygous Y529 human carriers, especially children, there is a correlate higher rate of virus-induced asthma exacerbations [25]. The current study examined three important questions concerning these observations. First, we asked if there might be measurable discrepancies between the Y529 and C529 proteins, in addition to surface display, that could influence virus interactions? Second, if RV-C did bind directly with either or both proteins, could we devise reproducible assays to map the elements of CDHR3 or its glycosylation format that might be required for this interaction? Third, assuming CDHR3 like the ICAM-1 and LDLR receptors of the RV-A and RV-B could be isolated in a cell-free format, would such materials independently react with virus and potentially inhibit infections?
Cadherin proteins share a common architecture in that the tandem repeat EC domains (EC1-6 for CDHR3) assume a rigid, slightly curved elongated structure, anchored like a waving stalk in the cell membrane. The C-proximal cytoplasmic domain does the anchoring. The N-proximal distal domains (e.g. EC1-3) usually confer adhesion properties by mediating dimer formation or higher order arrangements [26]. The linked EC orientations and even the folding of individual EC units depend on multiple calcium ions bound at various Kd, between the EC junctions. The first challenge in examining CDHR3 was to devise a virus-binding assay that was not cell surface dependent. In transfected or stably transduced cells, the intracellular protein pools are frequently much larger than that which is membrane anchored [15]. Clarified cell lysates proved a ready source of assay materials, and we found no CDHR3 sequences, fragments or conditions that required cell anchoring for demonstrable reactivity with virus. Tested this way, C529, Y529 and H529 proteins were equivalently capable of virus IP (
The basic N-linked glycosylation sites of human C529 and Y529, mapped to N186 (EC2), N384 (EC4), and N624 (EC6). These proteins migrated equivalently on gels by molecular weight, indicating both must undergo similar Golgi transport and modification pathways on the way to the cell surface. But once there, the Y529 abides, and can be labeled with biotin, while the C529 somehow withdraws, or undergoes a faster surface cycling pattern and is not labeled with biotin. In mature, plated stably transduced cells (fCR3Y), the presumed longer surface “hang time” of constitutively expressed protein apparently then permits Y529 to undergo additional multiple sialyations with α2-6 linkages. Transfected cells, even for Y529 do not have detectable amounts of these modifications, perhaps because the signal strength is masked by the much larger cytoplasmic pool created by overexpression, or because Y529 does not have time to fully surface-mature within 24 hrs post-transfection.
Surprisingly, none of these parameters proved relevant to virus binding. Whether the materials were from transfections, transductions, or bacterially produced, virus could be extracted with almost any CDHR3 format, including after de-glycosylation, as long as a properly reconstituted EC1 domain was present. In fact, EC1 alone was sufficient to bind virus (
The R166A mutation, also in EC2, models immediately adjacent to the EC1-EC2 junction, a region predicted to have 11 Asp and Glu calcium-chelating residues within 5-9 Å of its location (
An innate property of all cadherins on cell surfaces or in solution is to find dimeric and/or oligomeric partners. Most certainly the EC3 domain of our proteins promotes self-association because, although soluble upon refolding, proteins containing this domain flow directly through a Sephacryl S200 column and therefore are of dimer or higher order (
Consistent with what is known for other cadherins, we presumed the W76A mutation acts positively by reducing (trans) oligomer states, thereby freeing more EC1 units for productive virus binding. However, the rEC1 protein appears to be monomeric with or without this mutation. While W76 may not mediate EC1-EC1 dimerization like the tryptophans of other cadherins, it could still be involved in inter-protein interactions with other CDHR3 EC domains.
Alternatively, W76 may lie within or near the virus-contact interface, and elimination of the bulky aromatic side chain may allow tighter virus binding. Ongoing (NMR and cryoEM) structural studies with rCDHR3 proteins should provide some insight on the positive effects we observe for the W76A mutant.
When the panel of recombinant proteins was tested in infectivity inhibition assays, all inhibited virus replication in fCR3Y cells in a dose-dependent manner except rEC2+3(Δ1) (
EC2 is not required, nor is its glycosylation, except perhaps to help the protein fold properly. Apparently, soluble recombinant proteins contributed in this format are sufficient to inhibit infection of stably transduced fCR3Y HeLa and differentiated nasal epithelial ALI cultures. The observed inhibition phenomenon was common to 4 tested genotypes of virus, indicating that all RV-C virions likely share the same receptor landing pad and would respond to these soluble recombinant CDHR3 sequences in a similar manner. We believe these new reagents have great potential utility in RV-C research as useful tools for investigating CDHR3 biology and function, possibly with future antiviral applications.
Plasmids for transient eukaryotic expression of FLAG-tag-CDHR3 C529 and Y529 variants (pCDHR3-FLAG-0529 and pCDHR3-FLAG-Y529), which have a FLAG-tag (DYKDDDDK) inserted between the CDHR3 signal sequence (residues 1-19) and its EC1 (residue 20), were previously described [15]. The protein numbering system is from GenBank: AIC58018. Additional plasmids encoding single point mutations (K43A, W76A, F152A, R166A, R182A, N186A, N186Q, and Y529H) were engineered by two-step PCR into the pCDHR3-FLAG-Y529 plasmid. Analogous units for the expression of various FLAG-tag CDHR3 EC deletion mutants, which included additional carboxy-terminal 6× His tags, were engineered within pHLsec vectors (generously provided by Yue Liu, Michael Rossmann) based on pLEXm plasmid backbones [50]. Pilot experiments indicated that when this vector's innate secretory sequence was linked to the native CDHR3 signal sequence, protein expression was inhibited in transfected cells. Therefore, the 5′ regions of the CDHR3 EC deletion sequences, encoded only the native signal sequence and its inserted FLAG-tag, as engineered into pHLsec vector backbones between BamHI and KpnI restriction sites. pHL-FLAG-CDHR3-His constructs included: WT (CDHR3 residues 1-885, Y529), ΔEC1 (1-885, Δ27-124), ΔEC2 (1-885, Δ129-231), ΔEC3 (1-885, Δ230-334), EC1-6 (1-689), EC1-3 (1-345), EC1-2 (1-237). Transfection protocols used 3 μg cDNA reacted with lipofectamine 2000 (3 μL, Invitrogen) in Opti-MEM media (Invitrogen) according to manufacturer's recommendations, and plated HeLa cells (ATCC CRL-1958 in Eagle's medium, 10% NBCS; 6-well dishes; 37° C.) grown to 80% confluence. The cells were incubated 24 h (37° C. under 5% C02) before collection, lysis and immunoprecipitation assays.
Amplicons containing the C529 and Y529 FLAG-tagged variants of CDHR3 were amplified by PCR from pCDHR3-FLAG cDNAs and then ligated into MIGR1-based IRES-neo retroviral plasmids (NG) which express neomycin resistance [51,52]. Viral vector generation required 293T cell transfection with pNG-FLAG-CDHR3 plasmids (4 μg), pMDGag-Pol (4 μg, packaging plasmid), and a vesicular stomatitis virus G protein-encoding (VSV-G) envelope plasmid (2 μg) in 500 μL of Opti-MEM with 20 μL of polyethylenimine. The transfection medium was replaced 12 h post-transfection, then subsequently harvested and filtered (0.45 μm, at 48 h). After infection of HeLa cells (ATCC CRL-1958) with this material by spinoculation [53] and incubation for genome integration, stably transformed cells were selected with G418 (400 μg/mL, Geneticin) and cloned. The cells were maintained in suspension culture [37° C.; Eagle's medium, 10% newborn calf serum (NBCS), 2% fetal bovine serum (FBS) under 5% C02]. The final transduced HeLa cell lines expressing full-length C529 and Y529 FLAG-tagged variants of CDHR3 were designated fCR3C and fCR3Y, respectively.
Cells (2×106) transfected with pCDHR3-FLAG-Y529 were lysed in PBS (100 μL, 0.5% SDS and 40 mM DTT) and then heated (10 min, 95° C.). The denatured lysate was diluted 2-fold into PBS and 1% NP40 and then equivalent samples were incubated with or without PNGaseF (1 U, 1 h, 37° C., Sigma F8435). The proteins (25 μL lysates) were fractionated by SDS-PAGE and visualized by Coomassie Brilliant Blue staining. Bands corresponding to glycosylated (˜100 kDa) and de-glycosylated (˜93 kDa) CDHR3 gel regions were cut out and submitted for analysis to the Mass Spectrometry/Proteomics facility at the University of Wisconsin Biotechnology Center for MS/MS analysis after in-gel trypsin digestion.
Bacterial plasmids for the expression of various CDHR3 rEC domains, linked to amino-terminal FLAG-tags and carboxy-terminal 6× His tags, were constructed. The rEC1 segment encoded residues 20-130, rEC1-2 encoded residues 20-237, rEC1-3 encoded residues 20-345, rEC2+3(Δ1) encoded residues 20-345 (Δ27-124), rEC1+3(Δ2) encoded residues 20-345 (Δ129-231), rEC1-3(+10aa) encoded residues 20-355, and rEC1-3(−5aa) encoded residues 20-340. These units were amplified by PCR from the pHL-FLAG-CDHR3-His cDNAs described above and then ligated into pET11a vectors between the NheI and BamHI restriction sites. To prevent spurious disulfide formation, most plasmids encoding EC3 segments had a point mutation converting Cys345 to Ala345 (C345A). Additional point mutations were engineered by standard, primer-directed two-step PCR. Escherichia coli BL21(DE3) LysS cells, trans-formed with each plasmid were induced with IPTG (isopropyl-β-d-thiogalactopyranoside) for recombinant protein expression. The cells were collected by centrifugation, resuspended in lysis buffer (20 mM Tris, pH 8.0, 137 mM NaCl, 1% Triton-X100), and sonicated. The majority of recombinant material was insoluble and collected by centrifugation (20,000×g for 45 min at 4° C.). The pellets were washed (1 M NaCl, then 1 M urea, then water) and solubilized (6 M urea, 20 mM Tris, pH 8.0, 137 mM NaCl for 1 h at 25° C. or O/N at 4° C.). After clarification (20,000×g for 45 min at 4° C.), the supernatant of rEC1 and rEC1-2 proteins were purified under denaturing conditions (6 M urea, 20 mM Tris, pH 8.0, 137 mM NaCl) on HisTrap FF columns (GE Healthcare). After elution with 200 mM Imidazole, the proteins were diluted (to 0.1-0.2 mg/mL) in the solubilization buffer (above) supplemented with 3 mM CaCl2) and then dialyzed (4 times, 8-12 hrs each, against 20 mM Tris, pH 8.0, 137 mM NaCl, 3 mM CaCl2, 2 mM β-mercaptoethanol). Proteins containing the EC3 domain [rEC1-3, rEC2+3(Δ1), and rEC1+3(Δ2)] bound poorly to the HisTrap FF columns even under denaturing conditions so the clarified supernantants were diluted and refolded as described above. Refolded proteins were concentrated using Amicon Ultra centrifugal filters.
Recombinant RV-C isolates C02, C15, C41, and C45 were produced by transfecting full-length T7 RNA transcripts synthesized in vitro (Ribomax, Promega) from linearized plasmid cDNAs into HeLa cells [54]. The C02 and C45 sequence encoded a D41K substitution in protein 3A, to increase virus replication in these cells. In contrast to the more prolific, HeLa-adapted C15a sequence, these recombinants do not encode a T125K substitution in capsid protein VP1. Therefore, like their parental clinical isolates, they do not bind heparan sulfate and their cell interactions are entirely dependent upon CDHR3 presentation [54]. Virus purification was by centrifugation of cell lysates through 30% sucrose cushions as described [55].
CDHR3 proteins expressed in transfected or stably transformed HeLa cells (˜2×106 cells scraped, collected in PBS, pelleted) were harvested 24 h after transfection or plating. The cells were pelleted (1.5 min at 1500×g), resuspended and then lysed (350 μL, 20 mM Tris, 137 mM NaCl, 2 mM CaCl2), 2 mM PMSF, 1% Triton x-100). The lysates were clarified (16,000×g, 20 min) and then incubated with sucrose purified C15 virus (107 PFUe) and with antibody (0.8 μL, α-CDHR3, HPA011218, Sigma; or 1 μg α-His Tag, HIS.H8, Millipore) overnight at 4° C. before being reacted with protein-G sepharose beads (1 h, 25° C.). When required, glycosylases PNGaseF (1 U, Sigma F8435) or neuraminidase (0.04 U, Sigma 10269611001) were included during the overnight incubations. After reaction and collection, the beads were washed (3×, lysis buffer) and bound proteins eluted with SDS (boiling), before SDS-PAGE fractionation and visualization by Western blot analysis. For experiments with bacterially-expressed materials, the refolded protein samples (100 pmol) were incubated with virus (107 PFUe, 1 h, 25° C.) and with the α-His Tag antibody (350 μL, 20 mM Tris, 137 mM NaCl, 2 mM CaCl2), 1% Triton x-before reactions with protein-G sepharose beads and treatment as above.
The sialylation status of CDHR3 expressed in fCR3Y or fCR3C was tested by incubating (1 h, 25° C.) cell lysates (˜2×106 cells in 300 μL, PBS 1% TritonX-100) with 5 μg biotinylated Sambucus nigra lectin (SNA) or Maackia amurensis lectin II (MAL II, Vector Labs) before addition to streptavidin beads (1 h, 25° C.). Collected beads were washed (3×PBS) before the bound protein was eluted (in 30 μL 2% SDS, with boiling), fractionated by SDS/PAGE and then visualized by Western blot analysis. Extracellular expression of CDHR3 was examined by treating plated cells (˜2×106 per sample) with EZ-Link Sulfo-NHS-Biotin (2 mM, Thermo-Fisher, in PBS for 1 h at 25° C.). The cells were then washed (3×, 50 mM Tris, pH 8.0; 3×PBS), harvested, lysed (300 μL, PBS 1% TritonX-100). The clarified lysates were reacted with streptavidin beads (1 h, 25° C.). The bound samples were processed for protein detection as above.
After SDS-PAGE resolution, proteins were electro-transferred to polyvinylidene difluoride membranes (Immobilon-P, Millipore). The membranes were blocked (1 h, 10% NFD milk in TBST: 20 mM Tris pH 7.6, 150 mM NaCl, 0.5% Tween20) then incubated with a primary antibody (1% NFD milk in TBST, overnight, 4° C.) before washing (3×TBST) and reaction with an appropriate secondary antibody (1 h, 20° C.). Commercial antibodies included: α-CDHR3 (rabbit Ab HPA011218 IgG, Sigma, 1:2000), α-FLAG (rabbit mAb F2555, IgG, Sigma, 1:2000), α-His Tag, (murine mAb HIS.H8 IgG, Millipore, 1:4000), HRP-conjugated α-mouse IgG (goat Ab A1068, Sigma (1:4000), and HRP-conjugated α-rabbit IgG (goat Ab A0545, Sigma, 1:4000). α-C15 (18C4 and 30C12, 1:5000) are custom murine mAbs (1 mg/mL, GeneScript) raised to the VP1 “finger” peptide sequence [28] characteristically exposed on the surface of this RV-C virion structure. For band visualization, the membranes were rinsed (3×, TBST), incubated (1 min) with enhanced chemiluminescence substrate (GE healthcare) and then exposed to film.
Typically, virus (3×106 PFUe) was incubated (1 h, 25° C.) with or without refolded recombinant CDHR3 protein (0.01 to 5 μM) in binding buffer (100 μL, 20 mM Tris pH 8.0, 137 mM NaCl, 2 mM CaCl2) before dilution into Eagle's medium (250 μL). Inoculation was onto plated, stably transformed fCR3Y cells. After attachment (30 min at 25° C., 15 min at 34° C.), the cells were washed (2× with PBS) to remove unattached virus and incubated (24 h at 34° C.) before harvest (lysis in 350 RLT buffer, Qiagen) and assessment of virus replication. Alternatively, the cells were directly incubated with recombinant CDHR3 protein (0-20 μg in 100 μL binding buffer, diluted into 250 μL Eagle's medium, 30 min 25° C., then 15 min 34° C.) and then washed (2×, PBS) before being exposed to virus as above. The cells were washed (2× with PBS) to remove unattached virus, before incubation (24 h at 34° C.), harvest and virus measurements. Viral loads (PFUe) were determined by RT-qPCR according to standardized RNA preparations after total RNA extraction from harvested cells (RNeasy Mini kits, Qiagen). The RT-qPCR reactions used Power SYBR Green PCR mix (Life Technologies) and RV-C specific primers as previously described [10]. For experiments with differentiated primary nasal epithelial cells, cells were obtained from nasal turbinates using ASI Rhino-Pro curette (Arlington Scientific) and cultured at air-liquid interface in collagen-coated Transwell polycarbonante inserts as previously described [10,11]. Fully differentiated cultures (2 months old) were washed with PBS and inoculated with C15 virus (106 PFUe) preincubated (1 h, 25° C.) with or without 1 μM recombinant CDHR3 protein (50 μL 20 mM Tris pH 8.0, 137 mM NaCl, 2 mM CaCl2). After attachment (30 min at 25° C., 15 min at 34° C.), the cells were washed (3× with PBS) to remove unattached input virus and incubated (24 h at 34° C.) before harvest for assessment of virus replication as described above.
Each publication, patent, and patent publication cited in this disclosure (including any listed in Exhibit A) is incorporated in reference herein in its entirety. The present invention is not intended to be limited to the foregoing examples, but encompasses all such modifications and variations as come within the scope of the appended claims.
The application includes the sequence listing that is concurrently filed in computer readable form. This sequence listing is incorporated by reference herein.
Protein Sequences
SEQ ID NO:1 CDHR3 synthetic sequence: Genbank accession AIC58018 (885 aa)-accession KJ900485.1
Bold: domain 1 (SEQ ID NO:2)
Underline: Domain 2 (SEQ ID NO:3)
Bold/italic: Domain 3 (SEQ ID NO:4) with C345 highlighted and underlined
[Domain 1: 26-128, Domain 2: 141-231, Domain 3: 242-341]]
Signal Peptide: aa 1-20
Linker 1 (aa 21-25), Linker 2 (aa 129-140), Linker 3 (aa232-241) and Linker 4 (aa 342-345 with c-to-a-mutation, the c is bold-italic below)
qivnsnplte afrvnwlsgt yfevvttgme qldfetgpni fdlqiyvkde vgvtdlqvlt
vqvtdvnepp qfqgnlaegl hlyiveranp gfiyqveafd pedtsrnipl syflisppks
frmsangtlf stteldfeag hrsfhlivev rdsgglkast elqvnivnln devprftspt
pat
qkftf simvpertak
MASDYKDDDDK
LHLILL
PATGNVAENSPPGTSVHKFSVKLSAS
LSPVIPGFPQIVNSNPLTEAFRVNWLSGTYFEVVTTGMEQLDFET
GPNIFDLQIYVKDEVGVTDLQVLTVQVTDVNEPP
GGTKHHHHH
H
MASDYKDDDDK
LHLILL
PATGNVAENSPPGTSVHKFSVKLSAS
LSPVIPGFPQIVNSNPLTEAFRVN
LSGTYFEVVTTGMEQLDFET
GPNIFDLQIYVKDEVGVTDLQVLTVQVTDVNEPP
GGTKHHHHH
H
MASDYKDDDDK
LHLILL
PATGNVAENSPPGTSVHKFSVKLSAS
LSPVIPGFPQIVNSNPLTEAFRVNWLSGTYFEVVTTG
MEQLDFETGPNIFDLQIYVKDEVGVTDLQVLTVQVTDVNE
PPQF
QGNLAEGL
LYIVERANPGFIYQVEAFDPEDTSRNI
PLSYFLISPPKSFRMSANGTLFSTTELDFEAGHRSFHLIVEVRDSG
GLKASTELQVNIVNLNDEVPRFT
GGTKHHHHHH
MASDYKDDDDK
LHLILL
PATGNVAENSPPGTSVHKFSVKLSAS
LSPVIPGFPQIVNSNPLTEAFRVNWLSGTYFEVVTTG
MEQLDFETGPNIFDLQIYVKDEVGVTDLQVLTVQVTDVNE
PPQF
QGNLAEGL
LYIVERANPGFIYQVEAFDPEDTSRNI
PLSYFLISPPKSFRMSANGTLFSTTELDFEAGHRSFHLIVEVRDSG
GLKASTELQVNIVNLND
EVPRFTSPTR
YTVLEE
LSPGTIVANITAEDPDDEGFPSHLLYSITTVSKYFMINQLTGTIQV
AQRIDRDAGELRQNPTISLEVLVKDRPYGGQENR
IQITFIVEDVNDN
PATA
GTKHHHHHH
MASDYKDDDDK
LHLILL
PATGNVAENSPPGTSVHKFSVKLSAS
LSPVIPGFPQIVNSNPLTEAFRVN
A
LSGTYFEVVTTG
MEQLDFETGPNIFDLQIYVKDEVGVTDLQVLTVQVTDVNE
PPQF
QGNLAEGL
LYIVERANPGFIYQVEAFDPEDTSRNI
PLSYFLISPPKSFRMSANGTLFSTTELDFEAGHRSFHLIVEVRDSG
GLKASTELQVNIVNLNDEVPRFT
GGTKHHHHHH
MASDYKDDDDK
LHLILL
PATGNVAENSPPGTSVHKFSVKLSAS
LSPVIPGFPQIVNSNPLTEAFRVNALSGTYFEVVTTG
MEQLDFETGPNIFDLQIYVKDEVGVTDLQVLTVQVTDVNE
PPQF
QGNLAEGL
LYIVERANPGFIYQVEAFDPEDTSRNI
PLSYFLISPPKSFRMSANGTLFSTTELDFEAGHRSFHLIVEVRDSG
GLKASTELQVNIVNLND
EVPRFTSPTR
YTVLEE
LSPGTIVANITAEDPDDEGFPSHLLYSITTVSKYFMINQLTGTIQV
AQRIDRDAGELRQNPTISLEVLVKDRPYGGQENR
IQITFIVEDVNDNPATA
GTKHHHHHH
MASDYKDDDDK
LHLILL
PATGNVAENSPPGTSVHKFSVKLSAS
LSPVIPGFPQIVNSNPLTEAFRVNWLSGTYFEVVTTG
MEQLDFETGPNIFDLQIYVKDEVGVTDLQVLTVQVTDVNE
EVPR
FTSPTR
YTVLEELSPGTIVANITAEDPDDEGFPSH
LLYSITTVSKYFMINQLTGTIQVAQRIDRDAGELRQNPTISLEVLV
KDRPYGGQENRIQITFIVEDVNDN
PATA
GTKHHHHHH
A
LSGTYFEVVTTGMEQLDFETGPNIFDLQIYVKDEVGVTDLQVLTVQVTD
X
LSGTYFEVVTTGMEQLDFETGPNIFDLQIYVKDEVGVTDLQVLTVQVTD
LHLILLPATGNVAENSPPGTSVHKFSVKLSASLSPVIPGFPQIVNSNPLT
LHLILLPATGNVAENSPPGTSVHKFSVKLSASLSPVIPGFPQIVNSNPLT
DNA Sequences
This application claims priority to U.S. Provisional Application No. 62/678,507 filed on May 31, 2018 and U.S. Provisional Application 62/768,191 filed on Nov. 16, 2018, the contents of which are incorporated by reference in their entireties.
This invention was made with government support under AI104317 awarded by the National Institutes of Health. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2019/034580 | 5/30/2019 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62678507 | May 2018 | US | |
| 62768191 | Nov 2018 | US |
