Patent Application
20200407401
Not applicable.
The Sequence Listing, which is a part of the present disclosure, includes a computer readable form comprising nucleotide and/or amino acid sequences of the present invention. The subject matter of the Sequence Listing is incorporated herein by reference in its entirety.
The present disclosure generally relates to compositions and methods for inhibition of arthritogenic alphavirus infections.
Among the various aspects of the present disclosure is the provision of compositions and methods of treating arthritogenic alphavirus infection and screening methods.
An aspect of the present disclosure provides for a fusion protein comprising an Fc region and at least one Mxra8 region or functional fragment or functional variant thereof, wherein the Mxra8 region has Mxra8 activity or Mxra8 receptor activity.
In some embodiments, the Mxra8 region comprises a polypeptide, wherein the polypeptide is encoded by a polynucleotide comprising SEQ ID NO: 1 or functional fragment or functional variant thereof; or the polypeptide comprises SEQ ID NO: 2 or a functional fragment or functional variant thereof.
In some embodiments, the functional fragment or functional variant thereof has a nucleotide sequence or an amino acid sequence of at least 90-95% identity to SEQ ID NO: 1 or SEQ ID NO: 2; and Mxra8 activity or Mxra8 receptor activity.
In some embodiments, the Mxra8 region is a Mxra8 ectodomain region or functional fragment or variant thereof. In some embodiments, the Fc region is selected from human IgG1, human IgG1 with N297Q mutation, or mouse IgG2b.
In some embodiments, the fusion protein comprises a human rhinovirus 3C (HRV) protease site between the Mxra8 C-terminus and an Fc domain.
Another aspect of the present disclosure provides for a method of treating a Mxra8-associated alphavirus comprising: administering a therapeutically effective amount of a Mxra8 inhibiting agent.
In some embodiments, the Mxra8-associated alphavirus is an arthritogenic alphavirus having a Mxra8 entry receptor.
In some embodiments, the Mxra8 inhibiting agent comprises an anti-Mxra8 antibody or Mxra8 fusion protein.
In some embodiments, the Mxra8 inhibiting agent is selected from one or more of the group consisting of anti-Mxra8, Mxra8-Fc, MXRA8-2-Fc, or a fusion protein comprising an Fc antibody domain fused to an ectodomain of Mxra8 or ectodomain of MXRA8-2 or a functional fragment or variant thereof having Mxra8 or Mxra8 receptor activity.
In some embodiments, the Mxra8 inhibiting agent comprises a small molecule Mxra8 inhibitor, Mxra8 siRNA, Mxra8 sgRNA, or Mxra8 shRNA.
In some embodiments, the therapeutically effective amount of a Mxra8 inhibiting agent reduces or prevents infection by an arthritogenic alphavirus.
In some embodiments, the arthritogenic alphavirus is selected from the group consisting of: Chikungunya Virus (CHIKV), Mayaro Virus (MAYV), Ross River Virus (RRV), O'nyong nyong (ONNV), Barmah Forest Virus (BFV), Semliki Forest virus, and Getah virus.
In some embodiments, the arthritogenic alphavirus is selected from the group consisting of: Semliki Forest and Getah viruses.
Yet another aspect of the present disclosure provides for a method of treating an arthritogenic alphavirus infection comprising: genetically modifying Mxra8 in a subject or genetically modifying a subject to reduce or prevent expression of the Mxra8 gene, such as through the use of CRISPR-Cas9 or analogous technologies, wherein, such modification reduces or prevents infection of a host by an arthritogenic alphavirus.
In some embodiments, the subject is a mammal.
In some embodiments, the subject is selected from the group consisting of: a human, a fish (e.g., salmon, trout), a mouse, or a mosquito.
In some embodiments, the subject is selected from the group consisting of: a mammal, such as a horse, dog, cat, sheep, pig, mouse, rat, monkey, hamster, guinea pig, chicken, and human.
Yet another aspect of the present disclosure provides for a pharmaceutical composition comprising a Mxra8 inhibiting agent.
In some embodiments, the Mxra8 inhibiting agent reduces inflammation and infection by an arthritogenic alphavirus.
In some embodiments, the Mxra8 inhibiting agent is an anti-Mxra8 antibody.
In some embodiments, the Mxra8 inhibiting agent is selected from one or more of the group consisting of anti-Mxra8, Mxra8-Fc, MXRA8-2-Fc, or a fusion protein comprising an Fc antibody domain fused to an ectodomain of Mxra8 or an ectodomain of MXRA8-2.
In some embodiments, the Mxra8 inhibiting agent comprises a small molecule Mxra8 inhibitor, Mxra8 siRNA, Mxra8 sgRNA, or Mxra8 shRNA.
Yet another aspect of the present disclosure provides for a method of generating antibodies against Mxra8, the method comprising: (a) immunizing a subject (e.g., a mammal) with Mxra8 protein or analog; and/or (b) isolating Mxra8-specific antibodies.
Yet another aspect of the present disclosure provides for a method of screening for a Mxra8 inhibitor, which method comprises: (a) contacting a receptor having a Mxra8 function with a test compound; (b) measuring the ability of Mxra8 to bind a viral protein or to allow viral infection; and/or (c) determining whether the test compound is a Mxra8 inhibitor from the measurements taken in step (b).
Yet another aspect of the present disclosure provides for a method of binding a surface-displayed E2 protein in alphavirus-infected cells comprising: administering to an alphavirus infected cell a therapeutically effective amount of a fusion protein comprising SEQ ID: 2 and/or SEQ ID NO: 8 or a functional variant or functional fragment of SEQ ID: 2 and/or SEQ ID NO: 8.
Yet another aspect of the present disclosure provides for an in silico method of identifying a compound that binds to Mxra8 or Mxra8 entry receptor comprising: (i) providing or receiving an estimated three-dimensional structure of atomic coordinates of Mxra8 or atomic coordinates Mxra8 entry receptor, their complexes, or a portion thereof, the three-dimensional structure comprising a plurality of amino acids; (ii) selecting a chemical probe; or (iii) determining if the chemical probe binds to Mxra8, Mxra8 entry receptor, or a portion of Mxra8, or a portion of a Mxra8 entry receptor (e.g., E2).
Yet another aspect of the present disclosure provides for an in silico method comprising: identifying a set(s) of one or more protein conformations to which the chemical probe binds; testing the compound or chemical probe in an in vitro or in vivo assay to determine its efficacy; determining a toxicity of the compound or chemical probe; determining if the compound or chemical probe has an off-target effect; determining toxicity or the off-target effect in an in vitro, an in vivo, or an in silico assay; optimizing the compound or chemical probe; or optimizing the compound or chemical probe to reduce toxicity of the compound to reduce an off-target effect; to increase or decrease a binding affinity of the compound or chemical probe; or to increase or decrease an activity of an off-target protein.
Yet another aspect of the present disclosure provides for a the three-dimensional structure comprising cryo-EM atomic coordinates according to PDB code 6NK3, PDB code 6NK5, EMDB code EMD-9393, PDB code 6NK6, EMDB code EMD-9394, PDB code 6NK7, EMDB code EMD-9395, TABLE 5,
Yet another aspect of the present disclosure provides for a method of treating a Mxra8-associated alphavirus comprising: administering a therapeutically effective amount of a Mxra8 inhibiting agent identified using the in silico method.
Yet another aspect of the present disclosure provides for a method of measuring integrity of an alphavirus vaccine or alphavirus antigen comprising: capturing Mxra8, Mxra8 fusion protein, or functional fragment or variant thereof, on a substrate; incubating the alphavirus vaccine or alphavirus antigen and the substrate; and detecting with a monoclonal or polyclonal anti-vaccine/antigen antibody or Mxra8-Fc; capturing the alphavirus vaccine or alphavirus antigen in a solid phase (e.g., directly or via a bridging antibody); and/or incubating the captured alphavirus vaccine or alphavirus antigen with Mxra8, Mxra8 fusion protein, or functional fragment or functional variant thereof.
Yet another aspect of the present disclosure provides for a fusion protein selected from Mxra8-Fc; the substrate is selected from a microtiter well plate, chip, or pin; or a method comprising an assay selected from ELISA, biolayerinterferometry, or surface plasmon resonance.
Yet another aspect of the present disclosure provides for an alphavirus vaccine or antigen selected from a live-attenuated virus, a virus-like particle, a viral structural protein, or a nucleic acid or vector producing viral proteins or particles.
Yet another aspect of the present disclosure provides for a method of reducing or preventing alphavirus infection comprising gene editing of Mxra8.
Yet another aspect of the present disclosure provides for a gene editing selected from CRISPR-Cas9-based gene editing.
Yet another aspect of the present disclosure provides for the gene editing comprises insertion of at least one GEQRL/V in Domain 1 or the Mxra8 ectodomain; three or more GEQRL/V in Domain 1 or the Mxra8 ectodomain; or GEQRLGEQRVGEQRV in Domain 1 or the Mxra8 ectodomain, wherein the insertion disrupts interactions with the Mxra8 receptor on an alphavirus or wherein the insertion provides for a disordered extension of the Mxra8 C′-C″ loop in Domain 1.
Other objects and features will be in part apparent and in part pointed out hereinafter.
Those of skill in the art will understand that the drawings, described below, are for illustrative purposes only. The drawings are not intended to limit the scope of the present teachings in any way.
The present disclosure is based, at least in part, on the discovery that Mxra8 is a receptor for arthritogenic alphaviruses, including chikungunya (CHIKV), Ross River, Mayaro, and O'nyong nyong (ONNV) viruses.
MXRA8
One aspect of the present disclosure provides for targeting of Mxra8 (also called DICAM, ASP3, or limitrin). The present disclosure provides methods of treating alphaviruses based on the discovery that Mxra8 is critical for an entry step in the virus lifecycle.
As described herein, the mouse protein Mxra8 was identified in a whole genome CRISPR/Cas9 screen looking for host factors that were required for infection of chikungunya virus (CHIKV). Mxra8 was one of the top hits of the screen. Subsequent validation studies revealed that gene editing of mouse Mxra8 via CRISPR/Cas9 resulted in markedly reduced infection of chikungunya, Ross River, Mayaro, and O'nyong nyong viruses but did not directly impact distantly related alphaviruses (Sindbis, EEEV, WEEV) or unrelated flaviviruses. Transcomplementation of KO cells with mouse or human Mxra8 restored infectivity of CHIKV.
As described herein, mechanism of action studies using CHIKV pseudotyped viruses or transfection of genomic RNA revealed that Mxra8 was critical for an entry step in the virus lifecycle. Direct binding and entry assays showed Mxra8 was not an attachment receptor but rather functioned as an entry receptor critical for allowing the virus to gain access to the cytoplasm.
As described herein, using a genome-wide CRISPR/Cas9-based screen, cell adhesion molecule Mxra8 was identified as an entry mediator for multiple emerging arthritogenic alphaviruses including chikungunya (CHIKV), Ross River, Mayaro, and O'nyong nyong (ONNV) viruses.
As described herein, gene editing of mouse Mxra8 or human MXRA8 resulted in reduced viral infection of cells, and reciprocally, ectopic expression resulted in increased infection.
As described herein, Mxra8 bound directly to CHIKV particles and enhanced virus attachment and internalization into cells.
As described herein, Mxra8-Fc protein or anti-Mxra8 monoclonal antibodies blocked CHIKV infection in multiple cell types including primary human synovial fibroblasts, osteoblasts, chondrocytes, and skeletal muscle cells.
As described herein, mutagenesis experiments suggest that Mxra8 binds to a surface-exposed region across the A and B domains of CHIKV E2, a speculated site of attachment.
As described herein, administration of Mxr8a-Fc protein or anti-Mxra8 blocking antibodies reduced CHIKV or ONNV infection and associated foot swelling in mice.
As such, it has been shown that pharmacological targeting of Mxra8 can form a strategy for mitigating infection and disease by multiple arthritogenic alphaviruses.
Consistent with a role in attachment and entry, Mxra8-Fc protein or anti-Mxra8 antibody directly blocked CHIKV infection.
Mxra8 can be an isoform of Mxra8. For example, an isoform of Mxra8 can be selected from one or more of the group selected from MXRA8-1, MXRA8-2, MXRA8-3, and MXRA8-4.
Mxra8-Associated Viruses
As described herein, the Mxra8 receptor was discovered on cells as an entry receptor for viruses (e.g., arthritogenic alphaviruses) that expressed Mxra8 on its surface. A Mxra8-associated virus can be any virus expressing Mxra8 on the surface of the virus and using a Mxra8 receptor on cell as the entry point of infection. As described herein, arthritogenic alphaviruses comprise Mxra8 and cells comprise Mxra8 entry receptors. Inhibition of Mxra8, inhibition of Mxra8 receptors, or knock out of the Mxra8 receptors reduce arthritogenic alphavirus infection.
Arthritogenic alphaviruses comprise a group of enveloped RNA viruses that can be transmitted to humans by mosquitoes and cause debilitating acute and chronic musculoskeletal disease1. The host factors required for alphavirus entry remain poorly characterized2.
As described herein, Mxra8 was discovered as an entry receptor for several arthritogenic alphaviruses. For example, a Mxra8-associated alphavirus can be any arthritogenic alphavirus. As another example, the Mxra8-associated alphavirus can be Chikungunya (e.g., CHIKV-181/25, 181/25-mKate2, CHIKV-AF15561, CHIKV-LR 2006, CHIKV-37997 (West African lineage)), Ross River (e.g., RRV (T48)), Mayaro (e.g., MAYV (BeH407)), Barmah Forest, or O'nyong nyong (e.g., ONNV (MP30)) viruses. Other Mxra8-associated alphaviruses can be Semliki Forest virus (SFV) (e.g., SFV (Kumba)) or Getah virus. Mxra8 can also be associated with or play an entry role in additional viruses or alphaviruses that have not yet been tested or discovered.
Alphaviruses that were discovered to not be Mxra8-associated alphaviruses were distantly related alphaviruses (e.g., Sindbis, Bebaru, Una, Middleburg, Eastern equine encephalitis, Western equine encephalitis or Venezuelan equine encephalitis viruses) or unrelated flaviviruses (e.g., West Nile virus), bunyaviruses (Rift Valley fever virus), rhabdoviruses (e.g., Vesicular stomatitis virus), or picornaviruses (encephalomyocarditis virus). It was also discovered that Mxra8 showed no effect for unrelated positive- or negative-sense RNA viruses.
Mxra8 Inhibiting Agent
As described herein, a Mxra8 inhibiting agent reduces, prevents, or aborts infection of many different alphaviruses (e.g., Mxra8-associated alphaviruses). For example, a Mxra8 inhibiting agent can be an agent that interrupts the interaction of Mxra8 expressed on the virus and the Mxra8 entry receptor on the cell surface. As another example, the Mxra8 inhibiting agent can bind to Mxra8 on the virus or can bind to the Mxra8 receptor on the cell. The Mxra8 inhibiting agent can be an agent that binds to the viral structural protein (e.g., E2) of the viron on the cell surface. As another example, a Mxra8 inhibiting agent can be an inhibitor of Mxra8 or an inhibitor of Mxra8 receptors (e.g., antibodies, fusion proteins, small molecules).
A Mxra8 inhibiting agent can be any agent that can inhibit Mxra8, inhibit a Mxra8 receptor (e.g., Mxra8 fusion proteins, Mxra8, Mxra8 dimers, and functional variants thereof), downregulate Mxra8, or knockdown Mxra8.
As another example, a Mxra8 inhibiting agent can be a mutated or genetically edited Mxra8 to render it unable to bind to the Mxra8 receptor on an alphavirus.
As another example, the Mxra8 inhibiting agent can be a fusion protein. For example, the Mxra8 fusion protein can comprise a fragment or variant of Mxra8 (e.g., Mxra8-Fc, MXRA8-2-Fc). As another example, the fusion protein can comprise a mouse or human Fc antibody domain fused to the ectodomain of Mxra8 or MXRA8-2. Fusion proteins as described herein can comprise a Mxra8 or functional variant thereof.
Mxra8 can comprise a Mxra8 ectodomain (see e.g., SEQ ID NOs: 1-4) or a Mxra8 isoform (see e.g., (SEQ ID NOs: 25-31)) or a functional variant or functional fragment thereof.
As an example, the following fusion proteins were developed: human Mxra8 on mouse Fc (see e.g., SEQ ID NOs: 17-18); mouse Mxra8 on mouse Fc (see e.g., SEQ ID NOs: 19-20); mouse Mxra8 on human Fc (see e.g., SEQ ID NOs: 21-22); and mouse Mxra8 on human Fc N297Q (see e.g., SEQ ID NOs: 23-24). As another example, the fusion proteins can comprise a signal peptide (e.g., an IL-2 signal peptide), a Mxra isoform fragment, or functional variant thereof (e.g., fragment of Mxra8 comprising the ectodomain of Mxra8); a linker (optionally) (e.g., GS linker), or an Fc region (e.g., mlgG2b Fc region).
STYRVVSVLTVLHQDWLNGKEYKCKVSNAKLPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLT
STYRVVSV
indicates data missing or illegible when filed
As another example, the Mxra8 inhibiting agent can comprise an ectodomain of Mxra8 or any of Mxra8 isoforms SEQ ID NOs: 25-31 (e.g., XP_016857006.1; XP_016857005.1 NP_001269511.1, NP_001269512.1, NP_001269513.1; NP_001269514.1; NP_115724.1) or functional fragments or variants thereof with Mxra8 activity or Mxra8 receptor activity.
As described herein, the Mxra8 inhibiting agent can comprise an Fc region. The Fc region can be chosen from any Fc region known in the art. For example, the Fc region can comprise human IgG1, human IgG1 Fc with N297Q mutation, or mouse mlgG2b (see e.g., Saxena et al. 2016 Front Immunol. 7: 580).
As an example, a Mxra8 inhibiting agent can be an anti-Mxra8 antibody. The anti-Mxra8 antibody can be an anti-Mxra8 antibody that has Mxra8 inhibitory activity. As an example, the anti-Mxra8 antibody can be anti-Mxra8 (mouse) armenian hamster serum, anti-Mxra8 (mouse) hamster monoclonal antibodies, or anti-MXRA8 (human) monoclonal antibodies. As another example, the anti-Mxra8 purified hamster monoclonal antibody clones can be 1G11.E6, 1H1.F5, 3G2.F5, 4E7.D10, 7F1.D8, 8F7.E1, 9G2.D6 or any combination of the aforementioned clones (e.g., 1G11.E6+7F1.D8, or 4E7.D10+8F7.E1).
As another example, Mxra8 inhibiting agent can be SYKi, which can be used to downregulate Mxra8.A Mxra8 inhibiting agent can be a short hairpin RNA (shRNA) or a short interfering RNA (siRNA). A Mxra8 inhibiting agent can be an sgRNA targeting Mxra8 (e.g., SEQ ID NOs: 34-40).
Measuring Vaccine or Diagnostic Antigen Integrity
As described herein, Mxra8 can be used to assess an alphavirus vaccine or a diagnostic antigen integrity.
Because Mxra8 is a receptor for multiple arthritogenic alphaviruses, it can be used to physically assess the integrity of alphavirus vaccine or diagnostic antigens. As such, Mxra8 can be used to define lot-to-lot integrity or variation, for example, in the context of commercial production. Alphavirus vaccine or diagnostic antigens can include, but are not limited to, live-attenuated viruses, virus-like particles, viral structural proteins, or nucleic acids or vectors producing these viral proteins or particles.
The integrity assay can include capturing Mxra8 or Mxra8 fusion proteins (e.g., Mxra8-Fc) on a substrate (e.g., microtiter well plate, chip, or pin) and then incubating the alphavirus vaccine or diagnostic antigen and detection with a monoclonal or polyclonal anti-vaccine/diagnostic antigen antibody or even Mxra8-Fc itself. Alternatively, the alphavirus vaccine or diagnostic antigen can be captured in the solid phase (directly or via a bridging antibody) and then incubated with Mxra8 or Mxra8-Fc fusion proteins for binding. The assay could be performed by ELISA, biolayerinterferometry, surface plasmon resonance, or any other detection based system for binding.
Molecular Engineering
The following definitions and methods are provided to better define the present invention and to guide those of ordinary skill in the art in the practice of the present invention. Unless otherwise noted, terms are to be understood according to conventional usage by those of ordinary skill in the relevant art.
The terms “heterologous DNA sequence”, “exogenous DNA segment” or “heterologous nucleic acid,” as used herein, each refer to a sequence that originates from a source foreign to the particular host cell or, if from the same source, is modified from its original form. Thus, a heterologous gene in a host cell includes a gene that is endogenous to the particular host cell but has been modified through, for example, the use of DNA shuffling. The terms also include non-naturally occurring multiple copies of a naturally occurring DNA sequence. Thus, the terms refer to a DNA segment that is foreign or heterologous to the cell, or homologous to the cell but in a position within the host cell nucleic acid in which the element is not ordinarily found. Exogenous DNA segments are expressed to yield exogenous polypeptides. A “homologous” DNA sequence is a DNA sequence that is naturally associated with a host cell into which it is introduced.
Expression vector, expression construct, plasmid, or recombinant DNA construct is generally understood to refer to a nucleic acid that has been generated via human intervention, including by recombinant means or direct chemical synthesis, with a series of specified nucleic acid elements that permit transcription or translation of a particular nucleic acid in, for example, a host cell. The expression vector can be part of a plasmid, virus, or nucleic acid fragment. Typically, the expression vector can include a nucleic acid to be transcribed operably linked to a promoter.
A “promoter” is generally understood as a nucleic acid control sequence that directs transcription of a nucleic acid. An inducible promoter is generally understood as a promoter that mediates transcription of an operably linked gene in response to a particular stimulus. A promoter can include necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element. A promoter can optionally include distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription.
A “transcribable nucleic acid molecule” as used herein refers to any nucleic acid molecule capable of being transcribed into a RNA molecule. Methods are known for introducing constructs into a cell in such a manner that the transcribable nucleic acid molecule is transcribed into a functional mRNA molecule that is translated and therefore expressed as a protein product. Constructs may also be constructed to be capable of expressing antisense RNA molecules, in order to inhibit translation of a specific RNA molecule of interest. For the practice of the present disclosure, conventional compositions and methods for preparing and using constructs and host cells are well known to one skilled in the art (see e.g., Sambrook and Russel (2006) Condensed Protocols from Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ISBN-10: 0879697717; Ausubel et al. (2002) Short Protocols in Molecular Biology, 5th ed., Current Protocols, ISBN-10: 0471250929; Sambrook and Russel (2001) Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, ISBN-10: 0879695773; Elhai, J. and Wolk, C. P. 1988. Methods in Enzymology 167, 747-754).
The “transcription start site” or “initiation site” is the position surrounding the first nucleotide that is part of the transcribed sequence, which is also defined as position+1. With respect to this site all other sequences of the gene and its controlling regions can be numbered. Downstream sequences (i.e., further protein encoding sequences in the 3′ direction) can be denominated positive, while upstream sequences (mostly of the controlling regions in the 5′ direction) are denominated negative.
“Operably-linked” or “functionally linked” refers preferably to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is affected by the other. For example, a regulatory DNA sequence is said to be “operably linked to” or “associated with” a DNA sequence that codes for an RNA or a polypeptide if the two sequences are situated such that the regulatory DNA sequence affects expression of the coding DNA sequence (i.e., that the coding sequence or functional RNA is under the transcriptional control of the promoter). Coding sequences can be operably-linked to regulatory sequences in sense or antisense orientation. The two nucleic acid molecules may be part of a single contiguous nucleic acid molecule and may be adjacent. For example, a promoter is operably linked to a gene of interest if the promoter regulates or mediates transcription of the gene of interest in a cell.
A “construct” is generally understood as any recombinant nucleic acid molecule such as a plasmid, cosmid, virus, autonomously replicating nucleic acid molecule, phage, or linear or circular single-stranded or double-stranded DNA or RNA nucleic acid molecule, derived from any source, capable of genomic integration or autonomous replication, comprising a nucleic acid molecule where one or more nucleic acid molecule has been operably linked.
A constructs of the present disclosure can contain a promoter operably linked to a transcribable nucleic acid molecule operably linked to a 3′ transcription termination nucleic acid molecule. In addition, constructs can include but are not limited to additional regulatory nucleic acid molecules from, e.g., the 3′-untranslated region (3′ UTR). Constructs can include but are not limited to the 5′ untranslated regions (5′ UTR) of an mRNA nucleic acid molecule which can play an important role in translation initiation and can also be a genetic component in an expression construct. These additional upstream and downstream regulatory nucleic acid molecules may be derived from a source that is native or heterologous with respect to the other elements present on the promoter construct.
The term “transformation” refers to the transfer of a nucleic acid fragment into the genome of a host cell, resulting in genetically stable inheritance. Host cells containing the transformed nucleic acid fragments are referred to as “transgenic” cells, and organisms comprising transgenic cells are referred to as “transgenic organisms”.
“Transformed,” “transgenic,” and “recombinant” refer to a host cell or organism such as a bacterium, cyanobacterium, animal or a plant into which a heterologous nucleic acid molecule has been introduced. The nucleic acid molecule can be stably integrated into the genome as generally known in the art and disclosed (Sambrook 1989; Innis 1995; Gelfand 1995; Innis & Gelfand 1999). Known methods of PCR include, but are not limited to, methods using paired primers, nested primers, single specific primers, degenerate primers, gene-specific primers, vector-specific primers, partially mismatched primers, and the like. The term “untransformed” refers to normal cells that have not been through the transformation process.
“Wild-type” refers to a virus or organism found in nature without any known mutation.
Design, generation, and testing of the variant nucleotides, and their encoded polypeptides, having the above required percent identities and retaining a required activity of the expressed protein is within the skill of the art. For example, directed evolution and rapid isolation of mutants can be according to methods described in references including, but not limited to, Link et al. (2007) Nature Reviews 5(9), 680-688; Sanger et al. (1991) Gene 97(1), 119-123; Ghadessy et al. (2001) Proc Natl Acad Sci USA 98(8) 4552-4557. Thus, one skilled in the art could generate a large number of nucleotide and/or polypeptide variants having, for example, at least 95-99% identity to the reference sequence described herein and screen such for desired phenotypes according to methods routine in the art.
Nucleotide and/or amino acid sequence identity percent (%) is understood as the percentage of nucleotide or amino acid residues that are identical with nucleotide or amino acid residues in a candidate sequence in comparison to a reference sequence when the two sequences are aligned. To determine percent identity, sequences are aligned and if necessary, gaps are introduced to achieve the maximum percent sequence identity. Sequence alignment procedures to determine percent identity are well known to those of skill in the art. Often publicly available computer software such as BLAST, BLAST2, ALIGN2 or Megalign (DNASTAR) software is used to align sequences. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared. When sequences are aligned, the percent sequence identity of a given sequence A to, with, or against a given sequence B (which can alternatively be phrased as a given sequence A that has or comprises a certain percent sequence identity to, with, or against a given sequence B) can be calculated as: percent sequence identity=X/Y100, where X is the number of residues scored as identical matches by the sequence alignment program's or algorithm's alignment of A and B and Y is the total number of residues in B. If the length of sequence A is not equal to the length of sequence B, the percent sequence identity of A to B will not equal the percent sequence identity of B to A.
Generally, conservative substitutions can be made at any position so long as the required activity is retained. So-called conservative exchanges can be carried out in which the amino acid which is replaced has a similar property as the original amino acid, for example the exchange of Glu by Asp, Gln by Asn, Val by Ile, Leu by Ile, and Ser by Thr. For example, amino acids with similar properties can be Aliphatic amino acids (e.g., Glycine, Alanine, Valine, Leucine, Isoleucine); Hydroxyl or sulfur/selenium-containing amino acids (e.g., Serine, Cysteine, Selenocysteine, Threonine, Methionine); Cyclic amino acids (e.g., Proline); Aromatic amino acids (e.g., Phenylalanine, Tyrosine, Tryptophan); Basic amino acids (e.g., Histidine, Lysine, Arginine); or Acidic and their Amide (e.g., Aspartate, Glutamate, Asparagine, Glutamine). Deletion is the replacement of an amino acid by a direct bond. Positions for deletions include the termini of a polypeptide and linkages between individual protein domains. Insertions are introductions of amino acids into the polypeptide chain, a direct bond formally being replaced by one or more amino acids. Amino acid sequence can be modulated with the help of art-known computer simulation programs that can produce a polypeptide with, for example, improved activity or altered regulation. On the basis of this artificially generated polypeptide sequences, a corresponding nucleic acid molecule coding for such a modulated polypeptide can be synthesized in-vitro using the specific codon-usage of the desired host cell.
“Highly stringent hybridization conditions” are defined as hybridization at 65° C. in a 6×SSC buffer (i.e., 0.9 M sodium chloride and 0.09 M sodium citrate). Given these conditions, a determination can be made as to whether a given set of sequences will hybridize by calculating the melting temperature (Tm) of a DNA duplex between the two sequences. If a particular duplex has a melting temperature lower than 65° C. in the salt conditions of a 6×SSC, then the two sequences will not hybridize. On the other hand, if the melting temperature is above 65° C. in the same salt conditions, then the sequences will hybridize. In general, the melting temperature for any hybridized DNA:DNA sequence can be determined using the following formula: Tm=81.5° C.+16.6(log10[Na+])+0.41(fraction G/C content)−0.63(% formamide)−(600/I). Furthermore, the Tm of a DNA:DNA hybrid is decreased by 1-1.5° C. for every 1% decrease in nucleotide identity (see e.g., Sambrook and Russel, 2006).
Host cells can be transformed using a variety of standard techniques known to the art (see, e.g., Sambrook and Russel (2006) Condensed Protocols from Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ISBN-10: 0879697717; Ausubel et al. (2002) Short Protocols in Molecular Biology, 5th ed., Current Protocols, ISBN-10: 0471250929; Sambrook and Russel (2001) Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, ISBN-10: 0879695773; Elhai, J. and Wolk, C. P. 1988. Methods in Enzymology 167, 747-754). Such techniques include, but are not limited to, viral infection, calcium phosphate transfection, liposome-mediated transfection, microprojectile-mediated delivery, receptor-mediated uptake, cell fusion, electroporation, and the like. The transfected cells can be selected and propagated to provide recombinant host cells that comprise the expression vector stably integrated in the host cell genome.
Exemplary nucleic acids which may be introduced to a host cell include, for example, DNA sequences or genes from another species, or even genes or sequences which originate with or are present in the same species, but are incorporated into recipient cells by genetic engineering methods. The term “exogenous” is also intended to refer to genes that are not normally present in the cell being transformed, or perhaps simply not present in the form, structure, etc., as found in the transforming DNA segment or gene, or genes which are normally present and that one desires to express in a manner that differs from the natural expression pattern, e.g., to over-express. Thus, the term “exogenous” gene or DNA is intended to refer to any gene or DNA segment that is introduced into a recipient cell, regardless of whether a similar gene may already be present in such a cell. The type of DNA included in the exogenous DNA can include DNA which is already present in the cell, DNA from another individual of the same type of organism, DNA from a different organism, or a DNA generated externally, such as a DNA sequence containing an antisense message of a gene, or a DNA sequence encoding a synthetic or modified version of a gene.
Host strains developed according to the approaches described herein can be evaluated by a number of means known in the art (see e.g., Studier (2005) Protein Expr Purif. 41(1), 207-234; Gellissen, ed. (2005) Production of Recombinant Proteins: Novel Microbial and Eukaryotic Expression Systems, Wiley-VCH, ISBN-10: 3527310363; Baneyx (2004) Protein Expression Technologies, Taylor & Francis, ISBN-10: 0954523253).
Methods of down-regulation or silencing genes are known in the art. For example, expressed protein activity can be down-regulated or eliminated using antisense oligonucleotides, protein aptamers, nucleotide aptamers, and RNA interference (RNAi) (e.g., small interfering RNAs (siRNA), short hairpin RNA (shRNA), and micro RNAs (miRNA) (see e.g., Fanning and Symonds (2006) Handb Exp Pharmacol. 173, 289-303G, describing hammerhead ribozymes and small hairpin RNA; Helene, C., et al. (1992) Ann. N.Y. Acad. Sci. 660, 27-36; Maher (1992) Bioassays 14(12): 807-15, describing targeting deoxyribonucleotide sequences; Lee et al. (2006) Curr Opin Chem Biol. 10, 1-8, describing aptamers; Reynolds et al. (2004) Nature Biotechnology 22(3), 326-330, describing RNAi; Pushparaj and Melendez (2006) Clinical and Experimental Pharmacology and Physiology 33(5-6), 504-510, describing RNAi; Dillon et al. (2005) Annual Review of Physiology 67, 147-173, describing RNAi; Dykxhoorn and Lieberman (2005) Annual Review of Medicine 56, 401-423, describing RNAi). RNAi molecules are commercially available from a variety of sources (e.g., Ambion, Tex.; Sigma Aldrich, Mo.; Invitrogen). Several siRNA molecule design programs using a variety of algorithms are known to the art (see e.g., Cenix algorithm, Ambion; BLOCK-iT™ RNAi Designer, Invitrogen; siRNA Whitehead Institute Design Tools, Bioinformatics & Research Computing). Traits influential in defining optimal siRNA sequences include G/C content at the termini of the siRNAs, Tm of specific internal domains of the siRNA, siRNA length, position of the target sequence within the CDS (coding region), and nucleotide content of the 3′ overhangs.
Formulation
The agents and compositions described herein can be formulated by any conventional manner using one or more pharmaceutically acceptable carriers or excipients as described in, for example, Remington's Pharmaceutical Sciences (A. R. Gennaro, Ed.), 21st edition, ISBN: 0781746736 (2005), incorporated herein by reference in its entirety. Such formulations will contain a therapeutically effective amount of a biologically active agent described herein, which can be in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the subject.
The term “formulation” refers to preparing a drug in a form suitable for administration to a subject, such as a human. Thus, a “formulation” can include pharmaceutically acceptable excipients, including diluents or carriers.
The term “pharmaceutically acceptable” as used herein can describe substances or components that do not cause unacceptable losses of pharmacological activity or unacceptable adverse side effects. Examples of pharmaceutically acceptable ingredients can be those having monographs in United States Pharmacopeia (USP 29) and National Formulary (NF 24), United States Pharmacopeial Convention, Inc, Rockville, Md., 2005 (“USP/NF”), or a more recent edition, and the components listed in the continuously updated Inactive Ingredient Search online database of the FDA. Other useful components that are not described in the USP/NF, etc. may also be used.
The term “pharmaceutically acceptable excipient,” as used herein, can include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic, or absorption delaying agents. The use of such media and agents for pharmaceutical active substances is well known in the art (see generally Remington's Pharmaceutical Sciences (A. R. Gennaro, Ed.), 21st edition, ISBN: 0781746736 (2005)). Except insofar as any conventional media or agent is incompatible with an active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.
A “stable” formulation or composition can refer to a composition having sufficient stability to allow storage at a convenient temperature, such as between about 0° C. and about 60° C., for a commercially reasonable period of time, such as at least about one day, at least about one week, at least about one month, at least about three months, at least about six months, at least about one year, or at least about two years.
The formulation should suit the mode of administration. The agents of use with the current disclosure can be formulated by known methods for administration to a subject using several routes which include, but are not limited to, parenteral, pulmonary, oral, topical, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, ophthalmic, buccal, and rectal. The individual agents may also be administered in combination with one or more additional agents or together with other biologically active or biologically inert agents. Such biologically active or inert agents may be in fluid or mechanical communication with the agent(s) or attached to the agent(s) by ionic, covalent, Van der Waals, hydrophobic, hydrophilic or other physical forces.
Controlled-release (or sustained-release) preparations may be formulated to extend the activity of the agent(s) and reduce dosage frequency. Controlled-release preparations can also be used to effect the time of onset of action or other characteristics, such as blood levels of the agent, and consequently affect the occurrence of side effects. Controlled-release preparations may be designed to initially release an amount of an agent(s) that produces the desired therapeutic effect, and gradually and continually release other amounts of the agent to maintain the level of therapeutic effect over an extended period of time. In order to maintain a near-constant level of an agent in the body, the agent can be released from the dosage form at a rate that will replace the amount of agent being metabolized or excreted from the body. The controlled-release of an agent may be stimulated by various inducers, e.g., change in pH, change in temperature, enzymes, water, or other physiological conditions or molecules.
Agents or compositions described herein can also be used in combination with other therapeutic modalities, as described further below. Thus, in addition to the therapies described herein, one may also provide to the subject other therapies known to be efficacious for treatment of the disease, disorder, or condition.
Therapeutic Methods
Also provided is a process of treating a Mxra8-associated alphavirus in a subject in need administration of a therapeutically effective amount a Mxra8 inhibiting agent, so as to reduce infection, prevent or block infection, reduce viral load, shorten the duration of the infection, or reduce the symptoms of the infection, including joint or tissue swelling or joint or tissue inflammation.
Methods described herein are generally performed on a subject in need thereof. A subject in need of the therapeutic methods described herein can be a subject having, diagnosed with, suspected of having, or at risk for developing a Mxra8-associated alphavirus. A determination of the need for treatment will typically be assessed by a history and physical exam consistent with the disease or condition at issue. Diagnosis of the various conditions treatable by the methods described herein is within the skill of the art. The subject can be an animal subject, including a mammal, such as horses, cows, dogs, cats, sheep, pigs, mice, rats, monkeys, hamsters, guinea pigs, and chickens, and humans. For example, the subject can be a human subject. The subject can be an insect subject, such as a mosquito.
Generally, a safe and effective amount of a Mxra8 inhibiting agent is, for example, that amount that would cause the desired therapeutic effect in a subject while minimizing undesired side effects. In various embodiments, an effective amount of a Mxra8 inhibiting agent described herein can substantially inhibit a Mxra8-associated alphavirus infection, slow the progress of a Mxra8-associated alphavirus infection, limit the development of a Mxra8-associated alphavirus infection, reduce infection, prevent or block infection, reduce viral load, shorten the duration of the infection, or reduce the symptoms of the infection, including joint or tissue swelling or joint or tissue inflammation.
According to the methods described herein, administration can be parenteral, pulmonary, oral, topical, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, ophthalmic, buccal, or rectal administration.
When used in the treatments described herein, a therapeutically effective amount of a Mxra8 inhibiting agent can be employed in pure form or, where such forms exist, in pharmaceutically acceptable salt form and with or without a pharmaceutically acceptable excipient. For example, the compounds of the present disclosure can be administered, at a reasonable benefit/risk ratio applicable to any medical treatment, in a sufficient amount to inhibit a Mxra8-associated alphavirus infection, slow the progress of a Mxra8-associated alphavirus infection, limit the development of a Mxra8-associated alphavirus infection, reduce infection, prevent or block infection, reduce viral load, shorten the duration of the infection, or reduce the symptoms of the infection, including joint or tissue swelling or joint or tissue inflammation.
The amount of a composition described herein that can be combined with a pharmaceutically acceptable carrier to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. It will be appreciated by those skilled in the art that the unit content of agent contained in an individual dose of each dosage form need not in itself constitute a therapeutically effective amount, as the necessary therapeutically effective amount could be reached by administration of a number of individual doses.
Toxicity and therapeutic efficacy of compositions described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals for determining the LD50 (the dose lethal to 50% of the population) and the ED50, (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index that can be expressed as the ratio LD50/ED50, where larger therapeutic indices are generally understood in the art to be optimal.
The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the subject; the time of administration; the route of administration; the rate of excretion of the composition employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the medical arts (see e.g., Koda-Kimble et al. (2004) Applied Therapeutics: The Clinical Use of Drugs, Lippincott Williams & Wilkins, ISBN 0781748453; Winter (2003) Basic Clinical Pharmacokinetics, 4th ed., Lippincott Williams & Wilkins, ISBN 0781741475; Sharqel (2004) Applied Biopharmaceutics & Pharmacokinetics, McGraw-Hill/Appleton & Lange, ISBN 0071375503). For example, it is well within the skill of the art to start doses of the composition at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. If desired, the effective daily dose may be divided into multiple doses for purposes of administration. Consequently, single dose compositions may contain such amounts or submultiples thereof to make up the daily dose. It will be understood, however, that the total daily usage of the compounds and compositions of the present disclosure will be decided by an attending physician within the scope of sound medical judgment.
Again, each of the states, diseases, disorders, and conditions, described herein, as well as others, can benefit from compositions and methods described herein. Generally, treating a state, disease, disorder, or condition includes preventing or delaying the appearance of clinical symptoms in a mammal that may be afflicted with or predisposed to the state, disease, disorder, or condition but does not yet experience or display clinical or subclinical symptoms thereof. Treating can also include inhibiting the state, disease, disorder, or condition, e.g., arresting or reducing the development of the disease or at least one clinical or subclinical symptom thereof. Furthermore, treating can include relieving the disease, e.g., causing regression of the state, disease, disorder, or condition or at least one of its clinical or subclinical symptoms. A benefit to a subject to be treated can be either statistically significant or at least perceptible to the subject or to a physician.
Administration of a Mxra8 inhibiting agent can occur as a single event or over a time course of treatment. For example, a Mxra8 inhibiting agent can be administered daily, weekly, bi-weekly, or monthly. For treatment of acute conditions, the time course of treatment will usually be at least several days. Certain conditions could extend treatment from several days to several weeks. For example, treatment could extend over one week, two weeks, or three weeks. For more chronic conditions, treatment could extend from several weeks to several months or even a year or more.
Treatment in accord with the methods described herein can be performed prior to, concurrent with, or after conventional treatment modalities for viral infections (e.g., a Mxra8-associated alphavirus infection).
A Mxra8 inhibiting agent can be administered simultaneously or sequentially with another agent, such as an antiviral, an antibiotic, an anti-inflammatory, or another agent. For example, a Mxra8 inhibiting agent can be administered simultaneously with another agent, such as an antiviral, an antibiotic or an anti-inflammatory. Simultaneous administration can occur through administration of separate compositions, each containing one or more of a Mxra8 inhibiting agent, an antiviral, an antibiotic, an anti-inflammatory, or another agent. Simultaneous administration can occur through administration of one composition containing two or more of a Mxra8 inhibiting agent, an antiviral, an antibiotic, an anti-inflammatory, or another agent. A Mxra8 inhibiting agent can be administered sequentially with an antiviral, an antibiotic, an anti-inflammatory, or another agent. For example, a Mxra8 inhibiting agent can be administered before or after administration of an antiviral, an antibiotic, an anti-inflammatory, or another agent.
Administration
Agents and compositions described herein can be administered according to methods described herein in a variety of means known to the art. The agents and composition can be used therapeutically either as exogenous materials or as endogenous materials. Exogenous agents are those produced or manufactured outside of the body and administered to the body. Endogenous agents are those produced or manufactured inside the body by some type of device (biologic or other) for delivery within or to other organs in the body.
As discussed above, administration can be parenteral, pulmonary, oral, topical, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, ophthalmic, buccal, or rectal administration.
Agents and compositions described herein can be administered in a variety of methods well known in the arts. Administration can include, for example, methods involving oral ingestion, direct injection (e.g., systemic or stereotactic), implantation of cells engineered to secrete the factor of interest, drug-releasing biomaterials, polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, implantable matrix devices, mini-osmotic pumps, implantable pumps, injectable gels and hydrogels, liposomes, micelles (e.g., up to 30 μm), nanospheres (e.g., less than 1 μm), microspheres (e.g., 1-100 μm), reservoir devices, a combination of any of the above, or other suitable delivery vehicles to provide the desired release profile in varying proportions. Other methods of controlled-release delivery of agents or compositions will be known to the skilled artisan and are within the scope of the present disclosure.
Delivery systems may include, for example, an infusion pump which may be used to administer the agent or composition in a manner similar to that used for delivering insulin or chemotherapy to specific organs or tumors. Typically, using such a system, an agent or composition can be administered in combination with a biodegradable, biocompatible polymeric implant that releases the agent over a controlled period of time at a selected site. Examples of polymeric materials include polyanhydrides, polyorthoesters, polyglycolic acid, polylactic acid, polyethylene vinyl acetate, and copolymers and combinations thereof. In addition, a controlled release system can be placed in proximity of a therapeutic target, thus requiring only a fraction of a systemic dosage.
Agents can be encapsulated and administered in a variety of carrier delivery systems. Examples of carrier delivery systems include microspheres, hydrogels, polymeric implants, smart polymeric carriers, and liposomes (see generally, Uchegbu and Schatzlein, eds. (2006) Polymers in Drug Delivery, CRC, ISBN-10: 0849325331). Carrier-based systems for molecular or biomolecular agent delivery can: provide for intracellular delivery; tailor biomolecule/agent release rates; increase the proportion of biomolecule that reaches its site of action; improve the transport of the drug to its site of action; allow colocalized deposition with other agents or excipients; improve the stability of the agent in vivo; prolong the residence time of the agent at its site of action by reducing clearance; decrease the nonspecific delivery of the agent to nontarget tissues; decrease irritation caused by the agent; decrease toxicity due to high initial doses of the agent; alter the immunogenicity of the agent; decrease dosage frequency, improve taste of the product; or improve shelf life of the product.
Screening
Also provided are methods for screening candidate molecules.
The subject methods find use in the screening of a variety of different candidate molecules (e.g., potentially therapeutic candidate molecules). Candidate substances for screening according to the methods described herein include, but are not limited to, fractions of tissues or cells, nucleic acids, polypeptides, siRNAs, antisense molecules, aptamers, ribozymes, triple helix compounds, antibodies, and small (e.g., less than about 2000 mw, or less than about 1000 mw, or less than about 800 mw) organic molecules or inorganic molecules including but not limited to salts or metals.
Candidate molecules encompass numerous chemical classes, for example, organic molecules, such as small organic compounds having a molecular weight of more than 50 and less than about 2,500 Daltons. Candidate molecules can comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, and usually at least two of the functional chemical groups. The candidate molecules can comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups.
A candidate molecule can be a compound in a library database of compounds. One of skill in the art will be generally familiar with, for example, numerous databases for commercially available compounds for screening (see e.g., ZINC database, UCSF, with 2.7 million compounds over 12 distinct subsets of molecules; Irwin and Shoichet (2005) J Chem Inf Model 45, 177-182). One of skill in the art will also be familiar with a variety of search engines to identify commercial sources or desirable compounds and classes of compounds for further testing (see e.g., ZINC database; eMolecules.com; and electronic libraries of commercial compounds provided by vendors, for example: Chem Bridge, Princeton BioMolecular, Ambinter SARL, Enamine, ASDI, Life Chemicals etc.).
Candidate molecules for screening according to the methods described herein include both lead-like compounds and drug-like compounds. A lead-like compound is generally understood to have a relatively smaller scaffold-like structure (e.g., molecular weight of about 150 to about 350 kD) with relatively fewer features (e.g., less than about 3 hydrogen donors and/or less than about 6 hydrogen acceptors; hydrophobicity character xlogP of about −2 to about 4) (see e.g., Angewante (1999) Chemie Int. ed. Engl. 24, 3943-3948). In contrast, a drug-like compound is generally understood to have a relatively larger scaffold (e.g., molecular weight of about 150 to about 500 kD) with relatively more numerous features (e.g., less than about 10 hydrogen acceptors and/or less than about 8 rotatable bonds; hydrophobicity character xlogP of less than about 5) (see e.g., Lipinski (2000) J. Pharm. Tox. Methods 44, 235-249). Initial screening can be performed with lead-like compounds.
When designing a lead from spatial orientation data, it can be useful to understand that certain molecular structures are characterized as being “drug-like”. Such characterization can be based on a set of empirically recognized qualities derived by comparing similarities across the breadth of known drugs within the pharmacopoeia. While it is not required for drugs to meet all, or even any, of these characterizations, it is far more likely for a drug candidate to meet with clinical successful if it is drug-like.
Several of these “drug-like” characteristics have been summarized into the four rules of Lipinski (generally known as the “rules of fives” because of the prevalence of the number 5 among them). While these rules generally relate to oral absorption and are used to predict bioavailability of compound during lead optimization, they can serve as effective guidelines for constructing a lead molecule during rational drug design efforts such as may be accomplished by using the methods of the present disclosure.
The four “rules of five” state that a candidate drug-like compound should have at least three of the following characteristics: (i) a weight less than 500 Daltons; (ii) a log of P less than 5; (iii) no more than 5 hydrogen bond donors (expressed as the sum of OH and NH groups); and (iv) no more than 10 hydrogen bond acceptors (the sum of N and O atoms). Also, drug-like molecules typically have a span (breadth) of between about 8 Å to about 15 Å.
In Silico Screening
As described herein, the atomic coordinates for Mxra8 alone has been discovered by x-ray crystallography and its complexes with the Mxra8 entry receptor have been discovered by single particle cryo-EM. These coordinates can be used in any in silico screening method known in the art (e.g., Miteva (2011) In silico Lead Discovery; Kortagere (2013) In Silico Models for Drug Discovery).
As used herein, three-dimensional (3D) structure refers to the Cartesian coordinates corresponding to an atom's spatial relationship to other atoms in a macromolecule, for example a protein macromolecule. In the methods provided herein, an estimated three-dimensional (3D) structure can be determined by computer modeling, nuclear magnetic resonance (MR), X-ray crystallography, electron microscopy, or homology modeling.
Many in-silico methods for structure prediction and modeling can be used in connection with the methods described herein, including, without limitation, comparative protein modeling methods (e.g., homology modeling methods such as those described in Marti-Renom et al. 2000. Annu Rev Biophys Biomol Struct 29: 291-325), protein threading modeling methods (such as those described in Bowie et al. 1991. Science 253: 164-170; Jones et al. 1992. Nature 358: 86-89), ab initio or de novo protein modeling methods (Simons et al. 1999. Genetics 37: 171-176; Baker 2000, Nature 405: 39-42; Wu et al. 2007. BMC Biol 5: 17), physics-based prediction (Duan and Kollman 1998, Science 282: 740-744; Oldziej et al. 2005, Proc Natl Acad Sci USA 102: 7547-7552); or any combination thereof. Comparative modeling methods can be performed using a number of modeling programs, including, but not limited to, Modeller (Fiser and Sali 2003, Methods Enzymol 374: 461-91) or Swiss-Model (Arnold et al. 2006, Bioinformatics 22: 195-201). Protein threading modeling methods can be performed using a number of modeling programs, including, but not limited to, HHsearch (Soding 2005, Bioinformatics 21: 951-960), Phyre (Kelley and Sternberg. 2009, Nature Protocols 4: 363-371), or Raptor (Xu et al. 2003, J Bioinform Comput Biol 1: 95-117). Ab initio or de novo protein modeling methods can be performed using a number of modeling programs including, but not limited to, Rosetta (Simons et al. 1999. Genetics 37: 171-176; Baker 2000, Nature 405: 39-42; Bradley et al. 2003, Proteins 53: 457-468; Rohl 2004, Methods in Enzymology 383: 66-93) and I-TASSER (Wu et al. 2007. BMC Biol 5: 17).
Compositions and methods described herein utilizing molecular biology protocols can be according to a variety of standard techniques known to the art (see, e.g., Sambrook and Russel (2006) Condensed Protocols from Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ISBN-10: 0879697717; Ausubel et al. (2002) Short Protocols in Molecular Biology, 5th ed., Current Protocols, ISBN-10: 0471250929; Sambrook and Russel (2001) Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, ISBN-10: 0879695773; Elhai, J. and Wolk, C. P. 1988. Methods in Enzymology 167, 747-754; Studier (2005) Protein Expr Purif. 41(1), 207-234; Gellissen, ed. (2005) Production of Recombinant Proteins: Novel Microbial and Eukaryotic Expression Systems, Wiley-VCH, ISBN-10: 3527310363; Baneyx (2004) Protein Expression Technologies, Taylor & Francis, ISBN-10: 0954523253).
Definitions and methods described herein are provided to better define the present disclosure and to guide those of ordinary skill in the art in the practice of the present disclosure. Unless otherwise noted, terms are to be understood according to conventional usage by those of ordinary skill in the relevant art.
In some embodiments, numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth, used to describe and claim certain embodiments of the present disclosure are to be understood as being modified in some instances by the term “about.” In some embodiments, the term “about” is used to indicate that a value includes the standard deviation of the mean for the device or method being employed to determine the value. In some embodiments, the numerical parameters set forth in the written description and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the present disclosure are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable. The numerical values presented in some embodiments of the present disclosure may contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements. The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein.
In some embodiments, the terms “a” and “an” and “the” and similar references used in the context of describing a particular embodiment (especially in the context of certain of the following claims) can be construed to cover both the singular and the plural, unless specifically noted otherwise. In some embodiments, the term “or” as used herein, including the claims, is used to mean “and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive.
The terms “comprise,” “have” and “include” are open-ended linking verbs. Any forms or tenses of one or more of these verbs, such as “comprises,” “comprising,” “has,” “having,” “includes” and “including,” are also open-ended. For example, any method that “comprises,” “has” or “includes” one or more steps is not limited to possessing only those one or more steps and can also cover other unlisted steps. Similarly, any composition or device that “comprises,” “has” or “includes” one or more features is not limited to possessing only those one or more features and can cover other unlisted features.
All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g. “such as”) provided with respect to certain embodiments herein is intended merely to better illuminate the present disclosure and does not pose a limitation on the scope of the present disclosure otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the present disclosure.
Groupings of alternative elements or embodiments of the present disclosure disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.
All publications, patents, patent applications, and other references cited in this application are incorporated herein by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application or other reference was specifically and individually indicated to be incorporated by reference in its entirety for all purposes. Citation of a reference herein shall not be construed as an admission that such is prior art to the present disclosure.
Having described the present disclosure in detail, it will be apparent that modifications, variations, and equivalent embodiments are possible without departing the scope of the present disclosure defined in the appended claims. Furthermore, it should be appreciated that all examples in the present disclosure are provided as non-limiting examples.
The following non-limiting examples are provided to further illustrate the present disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent approaches the inventors have found function well in the practice of the present disclosure, and thus can be considered to constitute examples of modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the present disclosure.
The following example describes the discovery that a receptor for Mxra8 on the surface of arthritogenic alphaviruses is an entry receptor. Furthermore, this example describes inhibition of Mxra8 on the alpha virus or inhibition of the Mxra8 receptor on a cell mitigates infection by alphaviruses.
Arthritogenic alphaviruses comprise a group of enveloped RNA viruses that are transmitted to humans by mosquitoes and cause debilitating acute and chronic musculoskeletal disease1. The host factors required for alphavirus entry remain poorly characterized2. This example describes the use of a genome-wide CRISPR-Cas9-based screen to identify the cell adhesion molecule Mxra8 as an entry mediator for multiple emerging arthritogenic alphaviruses, including chikungunya, Ross River, Mayaro and O'nyong nyong viruses. Gene editing of mouse Mxra8 or human MXRA8 resulted in reduced levels of viral infection of cells and, reciprocally, ectopic expression of these genes resulted in increased infection. Mxra8 bound directly to chikungunya virus particles and enhanced virus attachment and internalization into cells. Consistent with these findings, Mxra8-Fc fusion protein or anti-Mxra8 monoclonal antibodies blocked chikungunya virus infection in multiple cell types, including primary human synovial fibroblasts, osteoblasts, chondrocytes and skeletal muscle cells. Mutagenesis experiments suggest that Mxra8 binds to a surface-exposed region across the A and B domains of chikungunya virus E2 protein, which are a speculated site of attachment. Finally, administration of the Mxra8-Fc protein or anti-Mxra8 blocking antibodies to mice reduced chikungunya and O'nyong nyong virus infection as well as associated foot swelling. Pharmacological targeting of Mxra8 could form a strategy for mitigating infection and disease by multiple arthritogenic alphaviruses.
A genome-wide screen was performed for host factors required for chikungunya virus (CHIKV) infection, using the CRISPR-Cas9 system3,4 and lentiviruses delivering single-guide RNAs (sgRNA) targeting 20,611 mouse genes (see e.g.,
The requirement of Mxra8 for infection by other alphaviruses was tested. Whereas Mayaro, Ross River, O'nyong nyong and Barmah Forest arthritogenic alphaviruses showed reduced infection in ΔMxra8 3T3 cells, Semliki Forest and Getah viruses had partial phenotypes, and other related alphaviruses (Sindbis, Bebaru, Una and Middleburg viruses) showed little dependence on Mxra8 (see e.g.,
To determine whether Mxra8 is required for replication, CHIKV genomic RNA was transfected into control and ΔMxra8 MEF cells in the presence of NH4Cl to inhibit virus maturation and further rounds of infection. As no difference in CHIKV gene expression was detected (see e.g.,
To corroborate an effect of Mxra8 on binding and entry, Fc fusion proteins with the extracellular domains of mouse Mxra8 (Mxra8-Fc) or human MXRA8-2 (MXRA8-2-Fc) were generated, along with a control osteoprotegerin protein (OPG-Fc) (see e.g.,
To determine whether Mxra8 directly binds to CHIKV, virions or virus-like particles18 were captured with a human anti-CHIKV mAb19, and added Mxra8-Fc or MXRA8-2-Fc in an enzyme-linked immunosorbent assay. Both Mxra8-Fc and MXRA8-2-Fc, but not OPG-Fc, bound to CHIKV virions and virus-like particles (see e.g.,
It was evaluated whether human anti-CHIKV mAbs that bound epitopes within the E2 protein19 altered Mxra8-Fc binding to CHIKV. Several mAbs recognizing epitopes in the A domain (2H1, 8G18, 3E23 and 106) and mAbs recognizing shared epitopes in the A and B domains (1H12 and 4J14) inhibited binding, whereas other mAbs that localize to distinct sites had less effect (see e.g.,
To begin to assess the physiological importance of MXRA8 interaction with CHIKV, surface expression of MXRA8 on primary human keratinocytes, dermal fibroblasts, synovial fibroblasts, osteoblasts, chondrocytes and skeletal muscle cells were tested (see e.g.,
These studies establish that mouse Mxra8 contributes to CHIKV entry and is required for infection and disease. Human MXRA8 also bound CHIKV and supported infection, and MXRA8 expression in primary human cells overlapped with the tropism of CHIKV in vivo. Infection of several arthritogenic alphaviruses—including CHIKV, O'nyong nyong virus, Mayaro virus and Ross River virus—was reduced in ΔMxra8 cells, which suggests that Mxra8 may serve as a shared receptor. The disclosed data contrasts with those relating to natural resistance-associated macrophage protein (NRAMP2), which is an entry receptor for Sindbis virus but not for CHIKV or Ross River virus25. Nonetheless, residual CHIKV infection in the absence of Mxra8 in cells and in mice, and the absence of an apparent mosquito orthologue, suggests that additional unidentified host factors contribute to cell binding and entry.
The mutagenesis mapping studies suggest that amino acids in the E2 A and B domains contribute to the interaction of CHIKV with Mxra8. Higher-resolution structural experiments are needed to define the complete footprint of binding between Mxra8 and CHIKV E2 protein. Such studies could facilitate the development of small molecules or biological agents that disrupt Mxra8 interaction with E2 protein, which could form the basis of therapeutic strategies for the amelioration of diseases caused by multiple emerging alphaviruses.
Methods
Cells and Viruses.
Vero, NIH-3T3, MEF, HEK 293T, A549, HeLa (ATCC #CCL-2), MRC-5 (provided by D. Wang, Washington University), HFF-1 (ATCC #SCRC-1041), Hs 633T (Sigma-Aldrich #89050201), Huh7, RPE (provided by M. Mahjoub, Washington University), JEG3 (provided by I. Mysorekar, Washington University), U2OS (provided by S. Cherry, University of Pennsylvania), HT1080 (provided by J. Cooper, Washington University), Raji and K562 cells all were cultured at 37° C. in Dulbecco's Modified Eagle medium supplemented with 10% fetal bovine serum (FBS), 10 mM HEPES, 1 mM sodium pyruvate, 1× non-essential amino acids, and 100 U/ml of penicillin—streptomycin. HTR8/SV.neo cells (provided by I. Mysorekar, Washington University) were cultured in RPMI 1640 supplemented with 5% FBS and 1% penicillin and streptomycin. Jurkat cells were cultured at 37° C. in RPMI 1640 supplemented with 10% FBS and 10 mM HEPES. hCMEC/D3 cells (provided by R. Klein, Washington University) were cultured in EBM-2 medium (Lonza, 00190860) supplemented with 5% FBS, 5 μg/ml ascorbic acid, 10 mM HEPES, 1% lipid concentrate (Gibco, 11905-031) in plates pre-coated with collagen. All cell lines were tested and found to be free of mycoplasma contamination using a commercial kit. Cell lines were not authenticated.
Primary human keratinocytes (#102-05n), synovial fibroblasts (#408-05a), osteoblasts (#406-05f), chondrocytes (#402-05f) and skeletal muscle cells (#S150-05f) were purchased from Cell Applications. Primary human dermal fibroblasts (#CC-2509) were obtained from Lonza. Cells were thawed and cultured in specified medium according to the instructions of the manufacturers, and used within one week.
The following alphaviruses were used: CHIKV (strains 2006 La Reunion OPY1, 37997, AF15561, 181/25, and 181/25-mKate2 (rescued from pJM6 CHIKV-181/25 mKate2 cDNA clone, provided by T. Morrison and M. Heise), RRV (T48), MAYV (BeH407), ONNV (MP30), SFV (Kumba), SINV (Toto1101, Girdwood), Bebaru virus (BEBV, MM 2354), Middleburg virus (MIDV, 30037), Getah virus (GETV, AMM-2021), Una virus (UNAV, CoAr2380) and Barmah Forest virus (BRV, K10521). Additional viruses tested included chimaeric encephalitic alphaviruses (SINV-EEEV and SINV-WEEV), VEEV-GFP (TC-83)), a flavivirus (WNV, New York 2000), a bunyavirus (RVFV-GFP, MP-12), a rhabdovirus (VSV-GFP, Indiana) and a picornavirus (encephalomyocarditis virus, EMCV). Replication-competent SINV chimaeric viruses were constructed by replacing the SINV TR339 structural protein genes with EEEV FL93-939 or WEEV Fleming structural protein genes under control of the SINV subgenomic promoter in the TR339 cDNA clone27. All viruses were propagated in Vero cells and titrated by standard plaque or focus-forming assays28.
Pooled sgRNA Screen and Data Analysis.
A GeCKOv2 CRISPR knockout pooled library encompassing 130,209 different sgRNAs against 20,611 mouse genes29 was made available by F. Zhang (Addgene #1000000053), and amplified in Endura cells (Lucigen #60242) as previously described29,30. The sgRNA library was divided in half (A+B), packaged into lentiviruses and used for independent screening. The sgRNA plasmid library was packaged in 293FT cells after co-transfection with psPAX2 (Addgene #12260) and pMD2.G (Addgene #12259) at a ratio of 2:2:1 using FugeneHD (Promega). Approximately 48 h after transfection, supernatants were collected, clarified by centrifugation (3,500 r.p.m.×20 min) and aliquotted for storage at −80° C.
For the CRISPR screen, a clonal 3T3-Cas9 cell line was generated by transduction with a packaged lentivirus (lentiCas9-Blast, Addgene #52962), blasticidin selection and limiting dilution. 3T3-Cas9 cells were expanded and transduced with CRISPR sgRNA lentivirus library at a multiplicity of infection (MOI) of 0.3 by spinoculation (1,000 g) at 32° C. for 30 min in 12-well plates. After selection with puromycin for 7 days, ˜1×108 cells were inoculated with CHIKV-181/25-mKate2 (MOI of 1) and then incubated for 24 h to allow nearly all cells to become infected. Cells were sorted for an absence of mKate2 expression using a Sony Biotechnology Synergy SY3200 Cell sorter (Siteman Flow Cytometry Core, Washington University). To enrich for the cell population that was resistant to CHIKV infection and increase the signal-to-noise ratio, the mKate2-negative cells were expanded in culture. Given the rapid replication rate and cytopathic effect of CHIKV, two humanized neutralizing mAbs5, CHK-152 and CHK-166 (2 μg/ml of each), were added to block infection by any residual virus. The expanded cells were re-infected with CHIKV-181/25-mKate2 in the absence of mAbs, sorted for mKate2 negative cells and the procedure was repeated for one additional round.
Genomic DNA was extracted from the uninfected cells (5×107) or the mKate2-negative sorted cells (1×107), and sgRNA sequences were amplified31 and subjected to next generation sequencing using an Illumine HiSeq 2500 platform (Genome Technology Access Center, Washington University). The sgRNA sequences against specific genes were determined after removal of the tag sequences using the FASTX-Toolkit (http://hannonlab.cshl.edu/fastx_toolkit/) and cutadapt 1.8.1. sgRNA sequences were analyzed using a published computational tool (MAGeCK)6 (see e.g., TABLES 1A, 1B, 2A, and 2B in provisional application Nos. 62/671,680 and 62/672,080, incorporated herein by reference).
Gene Validation.
Mxra8 was validated by using three independent sgRNAs as follows: Mxra8 sgRNA1, 5′-CTTGTGGATATGTATTCGGC-3′ (SEQ ID NO: 34); Mxra8 sgRNA2, 5′-TGTGCGCCTCGAGGTTACAG-3′ (SEQ ID NO: 35); Mxra8 sgRNA3, 5′-GCTGCATGATCGCCAGCGCG-3′ (SEQ ID NO: 36). The sgRNAs were cloned into the plasmid lentiCRISPR v.2 (Addgene #52961) and packaged with a lentivirus express system. 3T3 or MEF cells were transduced with lentiviruses expressing individual sgRNA and selected with puromycin for seven days before infection with different viruses. For some validation experiments, clonal cells edited by sgRNA1 were isolated by limiting dilution. To validate the human orthologue MXRA8, two different sgRNAs targeting all four isoforms were used: MXRA8 sgRNA1, 5′-GGCGCGGATGCCTTTGAGCG-3′ (SEQ ID NO: 37); MXRA8 sgRNA2, 5′-GTCCGCCTGGAGGTCACCGA-3′ (SEQ ID NO: 38). The sgRNAs were cloned similarly as above, and the gene-edited bulk cells were used for validation studies.
For flow cytometric analyses, gene-edited 3T3 cells were inoculated with different viruses as follows: CHIKV-181/25 (MOI of 3, 9.5 h), CHIKV-AF15561 (MOI of 10, 24 h), CHIKV-37997 (MOI of 3, 10 h), CHIKV-LR 2006 (MOI of 1, 9.5 h), ONNV (MOI of 3, 12 h), RRV (MOI of 3, 32 h), MAYV (MOI of 3, 24 h), SFV (MOI of 1, 9 h), SINV (MOI of 10, 6 h), SIN-WEEV (MOI of 10, 10 h), SIN-EEEV (MOI of 10, 10 h), VEEV-GFP (MOI of 3, 6.5 h), RVFV-GFP (MOI of 10, 8 h), VSV-GFP (MOI of 3, 6 h), WNV (MOI of 10, 25 h) and EMCV (MOI of 3, 6 h). Gene-edited MEF cells were inoculated with CHIKV-181/25 (MOI of 3, 8 h), CHIKV-AF15561 (MOI of 10, 10 h), CHIKV-37997 (MOI of 1, 10 h) and CHIKV-LR 2006 (MOI of 1, 8 h). Gene-edited MRC-5, RPE and HFF-1 cells were inoculated with CHIKV-181/25 (MOI of 10, 10 h), CHIKV-AF15561 (MOI of 10, 10 h) and CHIKV-LR 2006 (MOI of 1, 10 h). Gene-edited Hs 633 T cells were inoculated with CHIKV-181/25 (MOI of 15), CHIKV-AF15561 (MOI of 15) and CHIKV-LR 2006 (MOI of 3) for 11.5 h. At the indicated times, cells were collected with trypsin, and fixed with 1% paraformaldehyde (PFA) diluted in PBS for 15 min at room temperature and permeabilized with Perm buffer (Hank's Balanced Salt Solution (HBSS) supplemented with 10 mM HEPES, 0.1% (w/v) saponin, and 2% FBS) for 10 min at room temperature. Cells were then incubated for 30 min at room temperature with 1 μg/ml of the following virus-specific antibodies: CHIKV (mouse CHK-115), ONNV and MAYV (mouse CHK-48), RRV (human 119); SINV (ascites fluid, ATCC VR-1248AF), SFV (mouse CHK-124), VEEV (mouse 1A4A), WEEV (mouse 11A1), EEEV (mouse EEEV-10), WNV (human E1632) and EMCV (mouse serum). After washing, cells were incubated with 2 μg/ml of Alexa Fluor 647-conjugated goat anti-mouse or anti-human IgG (Invitrogen) for 30 min at room temperature. Cells were processed on a MACSQuant Analyzer 10 (Miltenyi Biotec), and analyzed using FlowJo software (Tree Star).
Validation also was performed by an infectious virus yield assay. Gene-edited 3T3 cells were plated 12 h before infection. Cells were inoculated with CHIKV-181/25, CHIKV AF15561, CHIKV-LR 2006 at an MOI of 0.01 or other alphaviruses (BEBV, MOI 0.001; BFV, GETV, UNAV, MIDV, and SFV, all at MOI of 0.01) for 1 h, then washed once and maintained in reduced 2% FBS culture medium. Supernatants were collected at specific times after infection for titration on Vero cells by focus-forming assay (CHIKV) or standard plaque assay (all other viruses).
Genomic RNA Transfection and Analysis.
To assess for effects of Mxra8 on CHIKV replication, capped viral genomic RNA was transfected into MEF cells. Capped genomic RNA was generated using an mMESSAGE mMACHINE SP6 Transcription Kit according to the manufacturer's instructions (Thermo Fisher #AM1340) from the NotI-linearized CHIKV-181/25 cDNA clone. One microgram of purified RNA was transfected into control or ΔMxra8 cells using the Neon transfection system according to the manufacturer's instructions (Thermo Fisher Scientific). Cells were then incubated in 15 mM NH4Cl to prevent subsequent rounds of infection. At specified times, cells were collected with trypsin and processed for E2 expression levels by flow cytometry.
Pseudotyped Virus Experiments.
MLV-GFP pseudoviruses were made as described33,34 except plasmids encoding structural proteins of CHIKV (strain 37997), VEEV (strain TrD) and EEEV (strain FL91-4697) were used. Pseudovirus entry in 3T3 cells expressing or lacking Mxra8 was assessed 36 h later by measuring GFP expression by flow cytometry.
Plasmid Construction.
The C-terminal Flag-tagged mouse Mxra8 corresponding to the transcript (NM_024263) was synthesized (Integrated DNA Technologies) and cloned into the lentivirus vector pCSII-EF1-IRES-Venus with restriction sties NotI/BamHI. The Mxra8 sgRNA target sequences were mutated (cttgtggatatgtattcggcg (SEQ ID NO: 39) to ctGgtCgaCatgtaCAGCgcg (SEQ ID NO: 40)) to avoid re-cutting by Cas9 protein for the trans-complementation. Based on this plasmid, a truncation lacking the cytoplasmic domain was constructed by PCR-mediated mutagenesis, the ΔC-tail (378-442). To express the GPI-anchored Mxra8, the N-terminal 336 amino acids missing the transmembrane and cytoplasmic domains were fused with PLAP
To assess the function of different human MXRA8 isoforms, the cDNA of isoform 2 (NM_032348.3; SEQ ID NO: 43) containing C-terminal Myc and Flag tags was purchased from OriGene (Cat. No. RC200955), and cloned into the lentivirus vector pCSII-EF1-IRES-Venus. Isoform 1 (NM_001282585.1, SEQ ID NO: 44), isoform 3 (NM_001282584.1, SEQ ID NO: 45) and isoform 4 (NM_001282583.1, SEQ ID NO: 46) were created by either mutagenesis of isoform 2 or gene synthesis (Integrated DNA Technologies), and cloned into the lentivirus vector pCSII-EF1-IRES-Venus containing C terminus Myc and Flag tags. Trans-complementation and ectopic expression experiments.
The plasmids constructed above were packaged using the lentivirus expression system. Cells transduced with these lentiviruses were sorted for Venus-positive cells by flow cytometry. 3T3 cells were inoculated with CHIKV-181/25 (MOI 3) for 9.5 h or CHIKV-AF15561 (MOI 10) for 20 h. HeLa and A549 cells were inoculated with CHIKV-181/25 (MOI 3), CHIKV-AF15561 (MOI 10) or CHIKV-LR 2006 (MOI 1) for 14 h. 293T cells were inoculated with CHIKV-181/25 (MOI 1), CHIKV-AF15561 (MOI 3) or CHIKV-LR 2006 (MOI 0.5) for 11.5 h. CHO cells (K1 and 745) were inoculated with CHIKV-181/25 (MOI 0.3), CHIKV-AF15561 (MOI 10) or CHIKV-LR 2006 (MOI 0.3) for 12 h. Cells were then collected and processed for E2 expression by flow cytometry.
Generation and Production of Mxra8-Fc and MXRA8-2-Fc.
A cDNA fragment encoding the mouse Mxra8 extracellular domain (residues 22-336, GenBank accession number NM_024263 (SEQ ID NO: 32)) or the human MXRA8-2 extracellular domain (residues 20-337, GenBank accession number NM_032348.3 (SEQ ID NO: 33)) and the mouse IgG2b-Fc were synthesized (Integrated DNA Technologies) and inserted into the pCDNA3.4 vector (Thermo Fisher) downstream of an IL-2 signal peptide sequence. After confirmation by Sanger sequencing, Mxra8-Fc and MXRA8-2-Fc were expressed into Expi293 cells (Thermo Fisher). Cells were seeded at 5×106 cells per ml one day before transfection. Two hundred micrograms of plasmid was diluted in Opti-MEM (Thermo Fisher) and complexed with HYPE-5 transfection reagent before addition to cells. Transfected cells were supplemented daily with Expi293 medium and 2% (w/v) Hyclone Cell Boost. Four days after transfection, supernatant was collected, centrifuged at 3,000 g for 15 min, and purified by protein A sepharose 4B (Thermo Fisher) chromatography. After elution and buffer neutralization, the purified protein was dialysed into 20 mM HEPES, 150 mM NaCl (pH 7.5), filtered through a 0.20-μm filter, and stored at −80° C. Mxra8 that was cleaved from the IgG backbone was generated by inserting an HRV cleavage sequence (LEVLFQGP) (SEQ ID NO: 52) into Mxra8-Fc downstream of the Mxra8 coding sequence and before the mouse IgG sequence. Mxra8-HRV-Fc was expressed in Expi293 cells as described above. After purification, Mxra8-HRV-Fc was cleaved using HRV 3C protease (Thermo Fisher) at a 1:10 ratio overnight at 4° C. Cleaved Fc fragments were depleted using protein A sepharose chromatography, and the purity of Mxra8 was confirmed using SDS-PAGE analysis.
Expression of CHIKV Virus-Like Particles.
The CHIKV virus-like particles plasmid (strain 37997) was provided by G. Nabel (via the Vaccine Research Center, NIH) and expressed in Expi293 cells as previously described18. The supernatant was collected four days after transfection, 0.2-μm filtered and stored at 4° C.
ELISA-Based Mxra8-Fc Binding Assays.
Anti-CHIKV human mAb 4N1219 or anti-EEEV human mAb 53 (J.E.C., unpublished data) were immobilized (50 μl, 2 μg/ml) onto Maxisorp ELISA plates (Thermo Fisher) overnight in sodium bicarbonate buffer, pH 9.3. Plates were washed four times with PBS and blocked with PBS supplemented with 4% BSA for 1 h at room temperature. CHIKV-181/25 or SINV-EEEV was diluted to 1.5×107 FFU per ml in PBS and 50 μl per well was added for 1 h at room temperature. Mxra8-Fc and respective positive (CHK-115 and EEEV-10 (A.S.K. and M.S.D., unpublished results) and negative OPC-Fc (D.H.F., unpublished results) controls were diluted in PBS supplemented with 2% BSA and incubated for 1 h at room temperature. After serial washes with PBS, plates were incubated with horseradish peroxide conjugated goat anti-mouse IgG (H+L) (1:2000 dilution, Jackson ImmunoResearch) for 1 h at room temperature. After washing with PBS, plates were developed with 3,3′-5,5′ tetramethylbenzidine substrate (Dako) and 2N H2SO4. Absorbance was read at 450 nm with a TriStar Microplate Reader (Berthold). The mAb competition binding assay was performed by incubating 10 μg/ml of indicated anti-CHIKV human mAbs19 for 30 min before the addition of mMxra8-Fc, as described above; the anti-CHIKV human mAbs were mapped previously to different epitopes by alanine scanning mutagenesis and evaluated for neutralizing activity19. Humanized anti-WNV mAb E1632 was included as a negative control in competition binding assays.
Surface Plasmon Resonance Based Mxra8 Binding Assay.
Surface plasmon resonance binding experiments were performed on a Biacore T200 system (GE Healthcare) to measure the kinetics and affinity of Mxra8 binding to CHIKV virus-like particles. Experiments were performed at 30 μl/min and 25° C. using 0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% v/v surfactant P20 as running buffer. CHK-265 mAb5 was immobilized onto a CM5 sensor chip (GE Healthcare) using standard amine coupling chemistry, and CHIKV virus-like particles were captured. Recombinant Mxra8 was injected over a range of concentrations (1 μM to 20 nM) for 5 min followed by a 10 min dissociation period. As a negative control, murine norovirus was captured with mAb A6.2, as previously described35. Real-time data were analysed using BIAevaluation 3.1 (GE Healthcare). Kinetic profiles and steady-state equilibrium concentration curves were fitted using a global 1:1 binding algorithm with drifting baseline.
Cell-Based Mxra8-Fc Binding Assay.
3T3 cells were inoculated with different viruses at the following MOI: CHIKV-181/25 (MOI of 3, 9.5 h), ONNV (MOI of 5, 12 h), MAYV (MOI of 3, 24 h), RRV (MOI of 3, 32 h), SINV (MOI of 10, 9 h) and VEEV (MOI of 3, 6.5 h). Cells were detached using TrypLE (Thermo Fisher #12605010), washed twice with cold HBSS supplemented with 15 mM HEPES and 2% FBS. Cells were incubated with 1 μg/ml of Mxra8-Fc, OPG-Fc or viral E2-specific antibodies at 4° C. for 25 min. After washing, cells were stained with goat anti-mouse IgG (H+L) conjugated with Alexa Fluor 647 for 25 min at 4° C. After washing twice, cells were fixed with 2% PFA for 10 min at room temperature. After two additional washes, cells were subjected to flow cytometry analysis.
Virus Binding and Internalization Assays.
The assays were conducted in suspension. MEF cells were collected using TrypLE and washed twice with ice-cold medium supplemented with 2% FBS. CHIKV-AF15561 virions were purified through a 25% glycerol cushion at 25,000 r.p.m. for 2 h. For the binding assay, cells (5×105) and virions (MOI of 20) were mixed in a 1.5-ml microcentrifuge tube and incubated on ice for 45 min. After five cycles of centrifugation and washing, cells were lysed in RLT buffer for RNA extraction using an RNeasy Mini Kit (QIAGEN #74104). For the internalization assay, after 5 cycles of centrifugation and washing, cells were resuspended into medium supplemented with 2% FBS and 15 mM NH4Cl and then incubated at 37° C. for 1 h. Cells were chilled on ice and treated with 500 ng/ml proteinase K on ice for 1 h. After three additional washes, cells were lysed in RLT buffer for RNA extraction. qRT-PCR was conducted with Gapdh as an internal control using a TaqMan RNA-to-CT 1-Step Kit (Thermo Fisher #4392938). Primers and probes used are as follows:
For the flow cytometry-based binding assay, experiments were conducted as above but with 5×104 cells and an MOI of 200. After binding and washing, cells were fixed and stained with a mixture of mAbs (CHK-11, CHK-84, CHK-124 and CHK-1665) (1 μg/ml) at room temperature for 25 min. Cells were washed once and stained with 2 μg/ml of goat anti-mouse IgG (H+L) conjugated with Alexa Fluor 647 (Thermo Fisher #A21235) for 25 min. After two additional washes, cells were subjected to flow cytometry analysis.
Surface Staining of Mouse Mxra8 and Human MXRA8.
Mouse or human cells were collected with TrypLE and washed twice with cold HBSS supplemented with 15 mM HEPES and 2% FBS. Cells were incubated with anti-Mxra8 (mouse) Armenian hamster serum (1:300), anti-Mxra8 (mouse) hamster mAbs (1 μg/ml) or anti-MXRA8 (human) mAb (1 μg/ml) (MBL International #W040-3) at 4° C. for 25 min. After washing, cells were stained with 2 μg/ml goat anti-Armenian hamster IgG (H+L) conjugated with Alexa Fluor 647 (Abcam #ab173004) or goat anti-mouse IgG (H+L) conjugated with Alexa Fluor 647 (Thermo Fisher #A21235) for 25 min at 4° C. After two additional washes, cells were fixed with 2% PFA for 10 min at room temperature. Cells were then washed twice and subjected to flow cytometry analysis.
Cell Viability Assay.
A CellTiter-Glo Luminescent Cell Viability Assay (Promega) was performed according to the manufacturer's instructions. In brief, 2×104 3 T3 or MEF cells in 100 μl culture medium were seeded into opaque-walled 96-well plates. 24 h later, 100 μl of CellTiter-Glo reagent was added to each well and allowed to shake for 2 min. After a 10-min incubation at room temperature, luminescence was recorded by using a Synergy H1 Hybrid Plate Reader (Biotek) with an integration time of 0.5 s per well.
Western Blotting.
Cells seeded in 6-well plates were washed once with PBS, chilled on ice in PBS and detached with a cell scraper. After spinning at 300 g for 5 min, cell pellets were lysed in 45 μl RIPA buffer (Cell Signaling #9806S) with a cocktail of protease inhibitors (Sigma-Aldrich #S8830). Samples were prepared in LDS buffer (Life Technologies) under reducing (+dithiothreitol) conditions. After heating (70° C., 10 min), samples were electrophoresed using 10% Bis-Tris gels (Life Technologies) and proteins were transferred to PVDF membranes using an iBlot2 Dry Blotting System (Life Technologies). Membranes were blocked with 5% non-fat dry powdered milk and probed with hamster mAb 3G2.F5 (0.5 μg/ml) against mouse Mxra8. Western blots were developed using SuperSignal West Pico Chemiluminescent Substrate or SuperSignal West Femto Maximum Sensitivity Substrate (Life Technologies).
Alanine Scanning Mutagenesis for Mapping.
A CHIKV E2, 6K and E1 envelope protein expression construct (strain S27, Uniprot Reference #Q8JUX5) with a C-terminal V5 tag was subjected to alanine scanning mutagenesis to generate a comprehensive mutation library36. Each residue of the envelope proteins was mutated to alanine, with alanine codons mutated to serine. One hundred and forty-one mutations within the E2 A and B domains were screened for binding by Mxra8-Fc. Binding of Mxra8-Fc to each mutant expressed in HEK-293T cells was determined by immunofluorescence detected with a high-throughput flow cytometer (Intellicyt), as previously described36. Residues of domains A or B were identified as contributing to the binding site if their mutation eliminated Mxra8-Fc binding, but supported binding of CHIKV mAbs that bind to the appropriate domain (control mAbs were CHKV-84, CHKV-88, IM-CKV063, IM-CKV065 and C95,36,37).
Mapping of Mutations onto the CHIKV p62-E1 Crystal Structure.
Figures were prepared using the atomic coordinates of CHIKV p62-E1 (RCSB accession number 3N41) using the program PyMOL (The PyMOL Molecular Graphics System, Version 1.7.4 Schrödinger, LLC).
mAB Generation.
Four-week-old male Armenian hamsters were immunized intravenously with 100 μg of purified Mxra8-Fc. After two boosts (˜7 months of age), spleens were collected for hybridoma fusion and mAb production. Hybridoma supernatants were initially screened by ELISA using Mxra8-human Fc (mouse Fc was replaced by human Fc). As a second assay, the binding of hybridoma supernatants to Mxra8 on the surface of 3T3 cells was examined by flow cytometry. Finally, a tertiary screen evaluating blockade of CHIKV-181/25 infection by hybridoma supernatants was performed in 3T3 cells. After limiting dilution subcloning, the seven clones with the strongest blocking activities were selected and expanded. Antibodies were purified using protein A sepharose 4B (Invitrogen #101042), dialysed in PBS, concentrated and filtered for in vitro and in vivo experiments.
Blocking assays with Mxra8-Fc, MXRA8-2-Fc or anti-Mxra8 mAbs.
Twenty-five thousand 3T3 or MRC-5 cells were seeded into 96-well plates 12 h before treatment. CHIKV-181/25 virions were purified through a 25% glycerol cushion at 25,000 r.p.m. for 2 h. Serially diluted Mxra8-Fc or MXRA8-2-Fc protein was incubated with purified virions (MOI of 3) for 1 h at 37° C. in a volume of 100 μl. The mixture was added to 3T3 or MRC-5 cells for 9.5 h or 11.5 h, respectively. Cells were then collected for intracellular E2 expression as measured by flow cytometry. For hamster mAb blocking experiments, 3T3 or MRC-5 cells were pre-incubated with serially diluted mAbs for 1 h at 37° C. in a volume of 50 μl, and then purified virions (MOI of 3) in 50 μl were added and incubated for 9.5 or 11.5 h, respectively. Cells were collected and intracellular E2 expression was analysed by flow cytometry. For hamster mAb blocking experiments on primary human cells (keratinocytes, dermal fibroblasts, synovial fibroblasts, osteoblasts, chondrocytes and skeletal muscle cells), 2×104 cells were seeded into 96-well plates 12 h before treatment. Cells were pre-incubated with Armenian hamster isotype control (Bio X Cell #6E0260), 1H1.F5 or 9G2.D6 mAb for 1 h at 37° C. in a volume of 50 μl (50 μg/ml) and, subsequently, purified CHIKV-AF15561 virions (MOI of 15) in 50 μl were added and incubated for 10.5 h. Cells were collected and intracellular E2 expression was analysed by flow cytometry.
Phosphatidylinositol-Specific Phospholipase C Treatment.
3T3 cells (105) expressing GPI-anchored Mxra8 were collected using TrypLE and washed twice with PBS. Cells were then treated with 1 U/ml of phosphatidylinositol-specific phospholipase C (PI-PLC) (Sigma-Aldrich #P8804) in 50 μl PBS at 37° C. for 1 h. After two more cycles of washing, cells were stained for Mxra8 expression and processed by flow cytometry analysis as described above.
Mouse Experiments.
Experiments were carried out in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health after approval by the Institutional Animal Care and Use Committee at the Washington University School of Medicine (Assurance number A3381-01). Mxra8-Fc or an IgG control (JEV-13 mAb) (250 μg per mouse in PBS) was administered via an intraperitoneal route to four week-old wild-type male C57BL/6J mice 6 h before subcutaneous inoculation in the footpad with 103 FFU of CHIKV-AF15561. Alternatively, in co-injection experiments, CHIKV or ONNV was mixed directly with Mxra8-Fc or JEV-13 (25 μg per mouse in PBS), and incubated at 37° C. for 30 min before inoculation. At 12 h, 24 h and 72 h post-infection, animals were euthanized, and after perfusion with PBS, indicated tissues were collected. For antibody pre- or post-treatment experiments, 300 μg of purified hamster mAbs 1G11.E6+7F1.D8 or 4E7.D10+8F7.E1, and isotype control PIP (Bio X cell #BE0260) in PBS were administered via an intraperitoneal route to four week-old wild-type male C57BL/6 mice 12 h prior to, or 8 or 24 h after, subcutaneous inoculation in the footpad with 103 FFU of CHIKV-AF15561. Virus was titrated by focus forming assay as described5 using mouse CHK-115 for CHIKV or mouse CHK-48 for ONNV as the detection antibody. Joint swelling was monitored at 72 h post-infection via left foot measurements (width×height) using digital calipers as previously described38. Samples sizes were estimated to determine a difference of three- to fivefold, depending on data distribution. Blinding and randomization were not performed.
Statistical Analysis.
Statistical significance was assigned when P values were <0.05 using Prism Version 7 (GraphPad). Cell culture experiments were analysed by multiple t-tests with a Holm-Sidak correction or ANOVA with a multiple comparison correction. Analysis of levels of joint swelling or viral burden in vivo was determined by a Mann-Whitney, ANOVA, Kruskal-Wallis or unpaired t-test depending on data distribution and the number of comparison groups.
The following example shows that Mxra8 can be used to assess an alphavirus vaccine or a diagnostic antigen integrity.
Because Mxra8 is a receptor for multiple arthritogenic alphaviruses, it can be used to physically assess the integrity of alphavirus vaccine or diagnostic antigens, which is important to define lot-to-lot integrity or variation in the context of commercial production. Alphavirus vaccine or diagnostic antigens can include, but are not limited to, live-attenuated viruses, virus-like particles, viral structural proteins, or nucleic acids or vectors producing these viral proteins or particles.
The format of the integrity assay can include capturing Mxra8 or Mxra8 fusion proteins (e.g., Mxra8-Fc) on a substrate (e.g., microtiter well plate, chip, or pin) and then incubation of the alphavirus vaccine or diagnostic antigen and detection with a monoclonal or polyclonal anti-vaccine/diagnostic antigen antibody or even Mxra8-Fc itself. Alternatively, the alphavirus vaccine or diagnostic antigen can be captured in the solid phase (directly or via a bridging antibody) and then incubated with Mxra8 or Mxra8-Fc fusion proteins for binding. The assay could be performed by ELISA, biolayerinterferometry, surface plasmon resonance, or any other detection based system for binding.
The following example describes a small insertion in Mxra8 renders it resistant to infection by alphaviruses. Thus, it is possible to make Mxra8 resistant to infection.
CRISPR-Cas9 based gene editing of Mxra8 resulted in markedly diminished alphavirus infection in vitro and in vivo, and viral infectivity was restored after trans-complementation with either mouse Mxra8 or human MXRA8. Because some alphaviruses can infect a range of vertebrate hosts in epizootic cycles, we explored whether Mxra8 orthologs from different mammalian species could support infection by arthritogenic alphaviruses. 3T3 cells lacking Mxra8 expression (ΔMxra8) were trans-complemented via lentivirus transduction with Mxra8 genes from mouse (Mus musculus, positive control), rat (Rattus norvegicusdog (Canis lupus familiaris), cow (Bos taurus), goat (Capra hircus), sheep (Ovis aries), horse (Equus caballus), chimpanzee (Pan troglodytes). Surface expression of the different Mxra8 orthologs was confirmed with an oligoclonal pool of species cross-reactive monoclonal antibodies (mAbs) against Mxra8 (see e.g.,
To begin to investigate why cow Mxra8 in particular failed to restore alphavirus infectivity, we aligned its protein sequence with other Mxra8 gene sequences (see e.g.,
The structural analysis suggested that the 15-amino acid insertion in Mxra8 of cows might disrupt interactions with the CHIKV virion. To begin to evaluate this hypothesis, we generated mouse Mxra8-Fc fusion proteins that lacked or contained the additional 15 residues from cow (mouse Mxra8 and mouse Mxra8+moo) and reciprocally cow Mxra8-Fc fusion protein that lacked or contained the 15 residues (cow Mxra8 and cow Mxra8 Amoo) (see e.g.,
To further evaluate the significance of the 15-amino acid insertion to alphavirus infectivity, we transduced ΔMxra8 3T3 cells with lentiviruses encoding mouse Mxra8, mouse Mxra8+moo, cow Mxra8, and cow Mxra8 Δmoo. The addition of the 15 residues to mouse Mxra8 abolished infectivity by CHIKV, MAYV, and RRV, whereas deletion of the 15 residues from cow Mxra8 facilitated infection by these viruses (see e.g.,
The following example describes the generation of Mxra8 knockout mice and reduction of alphavirus infection in the KO mice.
Generation of Mxra8mut/mut mice by CRISPR-Cas9 gene targeting has been demonstrated (see e.g.,
Mxra8 is a receptor for multiple arthritogenic alphaviruses that cause debilitating acute and chronic musculoskeletal disease in humans. Here, we present a 2 Å X-ray crystallographic structure of Mxra8 and 4 to 5 Å resolution cryo-electron microscopy reconstructions of Mxra8 bound to chikungunya (CHIKV) virus-like particles (VLPs) and infectious virus. Mxra8 wedges into a cleft created by adjacent CHIKV E2 proteins in one trimeric spike and engages E1 protein on an adjacent spike. Two binding modes are observed with the fully mature VLP, with one of the Mxra8 binding sites having unique E1 contacts. Only this high-affinity binding mode was observed in the complex with infectious CHIKV, as viral maturation and E3 occupancy appear to influence receptor binding site usage. Our studies provide structural insight into how Mxra8 binds CHIKV and creates a path for developing alphavirus entry inhibitors.
Introduction
Alphaviruses are positive-sense, single-stranded RNA, enveloped viruses and are among the most important arthropod-borne viruses causing disease in humans (Powers et al., 2001). This genus includes chikungunya (CHIKV), Mayaro (MAYV), O'nyong'nyong (ONNV), and Ross River (RRV) viruses, which are emerging beyond their historical boundaries and now cause debilitating acute and chronic polyarthritis affecting millions of people in Africa, Asia, Europe, and the Americas. Despite their epidemic potential, there are no specific therapies or licensed vaccines for any alphavirus infection.
Alphavirus genomes encode four non-structural and five structural proteins. The non-structural proteins are required for virus replication, protein modification, and immune evasion. The structural proteins (capsid (C)-envelope (E)3-E2-6K-E1) are synthesized from a subgenomic promoter and cleaved co- and post-translationally. The E1 envelope glycoprotein participates in cell fusion (Lescar et al., 2001), whereas the E2 envelope glycoprotein binds to entry factors (Smith et al., 1995; Zhang et al., 2005) and initiates clathrin-dependent endocytosis (DeTulleo and Kirchhausen, 1998; Lee et al., 2013; Ooi et al., 2013). The E3 protein is essential for the proper folding of p62 (precursor to E2) and the formation of the p62-E1 heterodimer (Carleton et al., 1997; Mulvey and Brown, 1995) but is cleaved by furin-like proteases during the maturation process in the trans-Golgi network (Heidner et al., 1996). Mature alphaviruses are ˜700 Å icosahedral particles that assemble at the plasma membrane and contain a lipid bilayer with 240 embedded E2-E1 heterodimers assembled into 80 trimeric spikes with T=4 icosahedral symmetry (Cheng et al., 1995; Kostyuchenko et al., 2011; Paredes et al., 1993), and a nucleocapsid containing a single copy of genomic RNA.
Crystallographic studies of the precursor p62-E1, the mature E2-E1 glycoprotein complex, and the E1 protein (Lescar et al., 2001; Li et al., 2010; Roussel et al., 2006; Voss et al., 2010) have elucidated the glycoprotein structures. Several alphavirus virions structures also have been elucidated by cryo-electron microscopy (cryo-EM) (Cheng et al., 1995; Kostyuchenko et al., 2011; Lee et al., 1998; Li et al., 2010; Pletnev et al., 2001; Zhang et al., 2011; Zhang et al., 2002). The E1 ectodomain consists of three n-barrel domains termed Domain I (DI), DII, and DIII. The fusion peptide is located at the distal end of DII. E1 monomers lie at the base of the surface spikes and form trimers surrounding the icosahedral axes. E2 localizes to a long, thin, leaf-like structure on the top of the spike. The mature E2 protein contains three domains with immunoglobulin (Ig)-like folds: the N-terminal domain A, located at the center; domain B at the lateral tip; and the C-terminal domain C, located close to E1 and the viral membrane.
We recently used a genome-wide CRISPR/Cas9-based screen to identify the two immunoglobulin (Ig)-like domain containing cell adhesion molecule, Mxra8 as a receptor for multiple arthritogenic alphaviruses including CHIKV, RRV, MAYV, and ONNV (Zhang et al., 2018). Importantly, the human gene ortholog, MXRA8, also bound to CHIKV and other alphaviruses, and its cell surface expression facilitated infection of primary human target cells including fibroblasts, skeletal muscle cells, and chondrocytes. Mxra8 bound directly to CHIKV particles and enhanced attachment and internalization into cells, and Mxra8-Fc fusion protein or anti-Mxra8 monoclonal antibodies (mAbs) blocked CHIKV infection of several cell types. Administration of Mxr8a-Fc protein or anti-Mxra8 blocking mAbs to mice reduced CHIKV or ONNV infection and associated joint swelling. Despite defining several biological characteristics of CHIKV interaction with this receptor, structural insight as to how Mxra8 engages the spike proteins on the virion is lacking.
Here, we describe the X-ray crystallographic structure of Mxra8 and cryo-EM reconstructions of CHIKV virus-like particles (VLPs) (produced as capsid-E3-E2-6K-E1 but lacking viral RNA) and fully infectious CHIKV in complex with Mxra8. These are the first structures of alphavirus virions with their cognate host cell binding partner. Mxra8 has an unusual architecture, as its two Ig-like domains are oriented in a disulfide-linked head-to-head arrangement, with domain 2 emanating from the loops of domain 1 and the N- and C-terminal residues proximal to each other. Mxra8 binds into a cleft formed between two E2-E1 heterodimers within a trimeric spike, making dominant contacts with E2 domain A residues. Mxra8 also engages a determinant in E1 domain II on an adjacent glycoprotein spike. Two binding modes for Mxra8 were observed with mature CHIKV VLPs, which was consistent with a high and low affinity binding site model supported by surface plasmon resonance measurements. The low affinity binding sites, however, were sterically obscured by the retention of E3 on partially mature infectious CHIKV. Overall, this structural analysis defines how CHIKV engages its receptor Mxra8 to facilitate attachment and infection of cells. This information may inform the basis of therapies and improved vaccine designs that mitigate disease of multiple emerging alphaviruses.
Results
CHIKV VLPs and Mxra8 protein. Previous studies have obtained high-resolution structural information that elucidated how neutralizing antibodies engage CHIKV using VLPs, which contain the structural proteins but lack infectious viral RNA and can be imaged under lower biosafety conditions (Long et al., 2015; Sun et al., 2013). To begin to define the structural basis for interaction between Mxra8 and arthritogenic alphaviruses (Zhang et al., 2018), we produced CHIKV VLPs (strain 37997) after transient transfection of HEK-293 cells with a plasmid encoding C-E3-E2-6K-E1 (Akahata et al., 2010); equivalent preparation of VLPs were used in a phase 1 or are being used in phase 2 human clinical trials for vaccine protection against CHIKV disease ((Chang et al., 2014), NCT02562482 and NCT03483961). Soluble VLPs were collected, buffer exchanged, and monitored for purity by SDS-PAGE and antigenicity by Western blotting with anti-capsid and anti-E2/E1 antibodies (see e.g.,
X-ray crystallographic structure of Mxra8. The ectodomain residues of Mxra8, corresponding to positions 23 through 295 of NCBI reference sequence: NP_077225, were crystallized and the structure solved by molecular replacement. The resulting protein consists of two Ig-like domains &ranged in a head to head β-strand-swapped dimer that is connected by an interchain disulfide bond (see e.g.,
Cryo-EM reconstruction of Mxra8 bound to CHIKV VLPs. Electron micrographs of CHIKV VLPs with or without bound Mxra8 were recorded using a 300 kV Titan Krios cryo-electron microscope with Gatan K2 detector at the Washington University Center for Cellular Imaging (WUCCI) (see e.g.,
The three-dimensional reconstructions showed no large conformational changes in the structure of the CHIKV VLP when Mxra8 is bound (see e.g.,
Refinement of the model. We iteratively built an atomic model of the CHIKV VLP structural proteins and Mxra8 using a combination of published CHIKV crystal structures and de novo modelling. As a starting point, we used the structures of the CHIKV capsid (PDB: 5H23), CHIKV p62-E1 (PDB: 3N42), Venezuelan equine encephalitis virus (VEEV) transmembrane domains of E1 and E2 (PDB: 3J0C), and our structure of Mxra8 (see e.g.,
Our model details the residues of the CHIKV E2-E1 heterodimers at the Mxra8 binding interface. At all four sites of the icosahedral asymmetric unit, Mxra8 engages the A and B domains of the E2 protein (residues 26-29, 116-121, 191-194, 221-224), as well as the A domain and pinker of the counter-clockwise, adjacent E2 neighbor within the same trimeric spike (residues 5, 62-66, 158-161). Mxra8 is also positioned near the fusion loop of E1 (residues 85 and 87) as well as E1 domain II of an adjacent spike (residues 72, 211, 212) (see e.g.,
Many of the structurally identified contact residues are coincident with previous alanine scanning mutagenesis mapping data, which identified W64, D71, T116, and 1121 in the A domain and Y199 and 1217 in the B domain of E2 as important for optimal Mxra8 binding (Zhang et al., 2018) (see e.g.,
This finding prompted us to evaluate the effect on CHIKV-Mxra8 interactions of CHK-265, a murine mAb that cross-neutralizes infection of CHIKV, MAYV, RRV, and ONNV (Fox et al., 2015), all of which can use Mxra8 as an entry receptor (Zhang et al., 2018). CHK-265 cross-links trimeric spikes via bridging residues in the A and B domains of adjacent E2 proteins (Fox et al., 2015). Although the epitope of CHK-265 does not overlap directly the Mxra8 binding site, we hypothesized that it might still hinder interaction since the Mxra8 binding groove lies beneath the antibody epitope. Order-of-addition binding experiments showed that CHK-265 can bind CHIKV VLPs when Mxra8 is pre-bound, but that Mxra8 cannot bind when CHK-265 is bound (see e.g.,
Modes of Mxra8 binding to CHIKV VLPs and infectious virus. We also generated a 4.99 Å cryo-EM reconstruction of CHIKV infectious particles with Mxra8 and built an atomic model (see e.g.,
Discussion
Cell culture infection experiments with mouse and human cells and in vivo pathogenesis studies in mice defined Mxra8 as a cell surface receptor required for optimal infectivity and induction of musculoskeletal disease by multiple arthritogenic alphaviruses (Zhang et al., 2018). Here, our single particle cryo-EM analysis of CHIKV VLPs and infectious virus provides structural insight into how CHIKV engages Mxra8 to facilitate interactions with target cells. Our study adds to the limited structural knowledge of how receptors bind to enveloped icosahedral virions. Only one other structure of an enveloped, icosahedral virus complexed with its receptor exists. In a 25 Å resolution cryo-EM structure, Dengue virus (DENV) was complexed with the carbohydrate recognition domain of DC-SIGN; the only contacts were with protruding N-linked glycans in domain II of the E protein (Pokidysheva et al., 2006). In contrast, we observed a complex network of quaternary protein-protein interactions involving Mxra8 engaging two E2-E1 heterodimers within one trimeric spike and E1 on an adjacent spike. These sites are coincident with alanine scanning mutagenesis analysis of E2 protein interactions with Mxra8 and epitope maps of neutralizing human mAbs against CHIKV that directly block engagement of Mxra8. Our structures indicate that Mxra8 can bind at four distinct sites in the icosahedral asymmetric unit of the CHIKV VLP but only one site in the infectious virus, which retains E3.
The quaternary interactions formed between Mxra8 and multiple envelope proteins would effectively cross-link CHIKV spikes in a manner analogous to a previously defined broadly neutralizing mAb (CHK-265) that binds domain B on one trimer and domain A on an adjacent spike (Fox et al., 2015). The cross-linking of the viral structural proteins by Mxra8, while facilitating attachment and entry, might create a conundrum for viral fusion in the endosome, which requires domain B on E2 to undergo a substantive conformational shift to expose the underlying hydrophobic fusion loop in domain II of E1 (Li et al., 2010; Voss et al., 2010). After clathrin-mediated endocytosis, fusion of CHIKV occurs within the acidic environment of early endosomes (Hoornweg et al., 2016). Although further studies are required, we speculate that some of the Mxra8 binding interactions with CHIKV E1 and E2 may be sensitive to acidic pH, such that upon transiting to the early endosome the cross-linked trimers can separate and allow the structural transitions required for fusion to occur. In preliminary experiments, we have observed deceased affinity of binding of Mxra8 to recombinant pE2-E1 under conditions of mildly acidic pH (K. Basore, unpublished results). It is plausible that the strength of Mxra8 interactions with viral proteins may regulate the stage of endocytosis and pH of fusion of some arthritogenic alphaviruses.
The alphavirus structural polyprotein is processed In the endoplasmic reticulum to yield E3-E2 (p62) and E1, which form heterodimers and oligomerize as trimers to generate the immature spike (Uchime et al., 2013). In the Golgi network, furin-like proteases cleave E3 from E2 (yielding E2-E1) to render the spikes optimally fusogenic. However, this cleavage event can be variable in a cell-type and virus type-specific manner, as E3 remains associated with the mature virus in some alphaviruses, including Sindbis virus, Semliki Forest virus, and VEEV (Heidner et al., 1996; Zhang et al., 2011; Ziemiecki and Garoff, 1978). The comparison of our cryo-EM structures of Mxra8 bound to CHIKV VLPs and infectious virus revealed a difference in stoichiometry of binding, with 240 Mxra8 proteins bound to CHIKV VLP and only 60 bound to our partially mature, infectious CHIKV particles. One reason for the decreased occupancy on the infectious CHIKV was the retention of the E3 protein, which appears to occlude Mxra8 binding at three of four chemical environments in the asymmetric unit. Biochemical and structural analysis confirmed that E3 was absent from our CHIKV VLP preparation, possibly because of the mildly alkaline buffers used in the chromatography purification steps. Our binding studies with soluble mouse or human Mxra8 and CHIKV VLPs defined two classes of sites, one of high affinity (˜60 nM) and a second of lower affinity (˜300 nM). In comparison, infectious CHIKV had only the high-affinity binding site. These data suggest that while Mxra8 can bind mature CHIKV virions in two binding modes with full occupancy, regions of partial maturity which retain E3 will bind in only a single mode. At present, the contribution of the low affinity site for Mxra8 to infectivity remains unclear although these same infectious virus preparations still showed a strong Mxra8-dependence (Zhang et al., 2018). It is plausible that E3 retention on some arthritogenic (e.g., SINV) alphaviruses could result in a lack of contribution of Mxra8 to entry and infection. Future studies with fully mature, infectious alphaviruses propagated in Vero cells over-expressing furin (Mukherjee et al., 2016) and lack E3 might address this hypothesis directly.
Our cryo-EM map of Mxra8 bound to CHIKV was corroborated by mutational analysis and coincidence mapping of neutralizing antibodies that blocked Mxra8 binding. We previously identified residues W64, D71, T116, and 1121 in the A domain and Y199 and 1217 in the B domain of E2 as essential for optimal Mxra8 engagement using an E2 alanine scanning mutagenesis library (Fox et al., 2015; Smith et al., 2015; Zhang et al., 2018). Our cryo-EM analysis identified all of these A domain amino acids as contact residues and several B domain residues in close proximity. In the original study, mutations in E1 were not evaluated for binding to Mxra8 because it was expected that E2 and not E1 contributed to receptor engagement (Strauss et al., 1994; Tucker and Griffin, 1991). In further support of the structurally determined Mxra8 binding site on CHIKV, neutralizing human mAbs recognizing epitopes in the A domain or shared epitopes in the A and B domains inhibited Mxra8 binding, whereas others localizing to distinct sites had less effect (Zhang et al., 2018). The epitopes of the mAbs that blocked Mxra8 binding, as determined by alanine scanning mutagenesis (Smith et al., 2015), directly overlap the structural binding sites of Mxra8 and CHIKV E2.
Mxra8 is a receptor for multiple arthritogenic but not encephalitic alphaviruses (Zhang et al., 2018). An alignment of amino acid sequences corresponding to residues comprising the Mxra8 binding site revealed that contact residues generally are conserved among arthritogenic alphaviruses but are located in regions containing or proximal to insertions in encephalitic alphaviruses, which likely explains the negligible interaction or binding requirement for infectivity of Mxra8 with VEEV or related encephalitic alphaviruses (Zhang et al., 2018). Cryo-EM structures with other alphaviruses including ONNV, MAYV, and RRV should provide further insight into the critical contacts on E2, E1, and Mxra8 that facilitate attachment, entry, and infection. Such analysis could identity targets on either the viral structural proteins or Mxra8 to facilitate the development of agents capable of disrupting these interactions. This approach could form the basis of a therapy that mitigates infection caused by multiple arthritogenic alphaviruses.
Experimental Models
Viruses. CHIKV strain 181/25 was obtained from the World Reference Center for Emerging Viruses and Arboviruses (S. Weaver and K. Plante, Galveston, Tex.). The virus was propagated in Vero cells (ATCC) in DMEM supplemented with 10% FBS, concentrated by sucrose gradient ultracentrifugation, and titrated by standard focus-forming assays (Brien et al., 2013; Pal et al., 2013).
VLP production and purification. CHIKV VLPs were produced via transient transfection of C-E3-E2-6K-E1 (CHIKV strain 37997) plasmid DNA (Akahata et al., 2010) into HEK293 cells (obtained from Vaccine Research Center, NIH), and purified via Q Sepharose XL (GE Healthcare, GE17-5072-01) anion chromatography. The peak of interest was diafiltered into a buffer containing 218 mM sucrose, 10 mM potassium phosphate, and 25 mM citrate, pH 7.2. The material was sterile-filtered using a 0.2 μM filter, 500 μL aliquots were made and stored at −80° C. The final material was analyzed by BCA assay for protein concentration, Coomassie-stained SDS-PAGE gel with densitometry, and Western blotting with rabbit polyclonal anti-CHIKV 181/25 (04-0008, IBT Bioservices) and a secondary horseradish peroxidase conjugated goat anti-rabbit antibody (65-6120, ThermoFisher Scientific).
SPR-based Mxra8 binding assay. SPR binding experiments were performed on a Biacore T200 system (GE Healthcare) to measure the kinetics and affinity of Mxra8 binding to CHIKV VLPs. Experiments were performed at 30 μl/min and 25° C. using HBS-EP (0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% v/v Surfactant P20) as running buffer. CHK-265 mAb (Pal et al., 2013) was immobilized onto a CM5 sensor chip (GE Healthcare) using standard amine coupling chemistry, and CHIKV VLPs were captured. Recombinant Mxra8 was injected over a range of concentrations (2 μM to 20 nM) for 200 sec, followed by a 600 sec dissociation period. As a negative control, murine norovirus was captured with mAb A6.2, as described previously (Taube et al., 2010). Real-time data was analyzed using BIAevaluation 3.1 (GE Healthcare). For the equal affinity binding sites model, kinetic profiles and steady-state equilibrium concentration curves were fitted using a global 1:1 binding algorithm with drifting baseline.
For the model of Mxra8 binding to 1 of 4 sites with higher affinity, the following fits were used:
Equilibrium Analysis:
Kinetic Analysis:
Differential Equations:
BLI-based binding Assay. Binding of CHK-265 and Mxra8 to mAb-captured CHIKV VLP was monitored in real-time at 25° C. using an Octet-Red96 device (Pall ForteBio). CHK-265 (100 μg) was mixed with biotin (EZ-Link-NHS-PEG4-Biotin, Thermo Fisher) at a molar ratio of 20:1 biotin:mAb, incubated at room temperature for 30 min, then unreacted biotin was removed by passage through a desalting column (5 mL Zeba Spin 7K MWCO, Thermo Fisher). Biotinylated-CHK-265 was loaded onto streptavidin biosensors (ForteBio) until saturation, typically 10 μg/ml for 2 min, in 10 mM HEPES (pH 7.4), 150 mM NaCl, 3 mM EDTA, and 0.005% P20 surfactant with 1% BSA. CHIKV VLP was then added for 5 min. The biosensors were dipped sequentially into saturating concentrations of Mxra8 and CHK-265 (2 μM and 100 nM, respectively) for 5 min or vice-versa, with 30 sec baseline measurements in between associations, followed by a final 10 min dissociation.
Cryo-EM sample preparation, data collection, and single particle reconstruction. CHIKV VLP with and without cleaved Mxra8 and CHIKV infectious virus (181/25 strain) with cleaved Mxra8 in molar excess were flash frozen on holey carbon EM grids in liquid ethane under BSL-2 containment conditions using an FEI Vitrobot (ThermoFisher). Movies of the samples were recorded with the software EPU (Thermo Fisher) using a K2 Summit electron detector (Gatan) mounted on a Bioquantum 968 GIF Energy Filter (Gatan) attached to a Titan Krios microscope operating at 300 keV with an electron dose of 50 e−/Å2 and a magnification of 18,000×. The movies (30 frames, 300 msec exposure per frame) were corrected for beam-induced motion using MotionCor2 (Zheng et al., 2017). Contrast transfer function parameters of the electron micrographs were estimated using CTFFIND4 (Rohou and Grigorieff, 2015) in cisTEM (Grant et al., 2018). Particles were auto-picked, and underwent reference-free 2D classification, ab initio 3D reconstruction, and 3D refinement in cisTEM. Local resolution was estimated using RELION-3 (Zivanov et al., 2018). Additional information regarding the number of images and particles is listed in TABLE 2. All structural analyses of the 3D reconstructions were performed using the programs Chimera (http://www.cgl.ucsf.edu/chimera) and PyMOL (Bramucci et al., 2012).
Model building. The initial models of the CHIKV structural proteins (E1, E2, TM regions, and Capsid) with or without Mxra8 were built into the density of a subunit by docking the components from the crystal structures of CHIKV p62-E1 (PDB: 3N42), the CHIKV capsid (PDB: 5J23), and the modeled TM regions of VEEV (PDB: 3J0C) using the fitmap command in Chimera. Amino acid substitutions and connections were added manually in COOT to reflect the strain of CHIKV used for VLP production (CHIKV-37997) and infectious virus (CHIKV-181/25) for cryo-EM studies. The subunit models then underwent real-space refinement using PHENIX with default parameters plus rigid body refinement. Rigid bodies for E1 were divided into domains I and II (residues 1-292), domain III (293-381), stem (382-412), and transmembrane region (413-442). E2 was divided into the N-linker region (residues 5-15), domain A (residues 16-134), domain B (residues 173-231), domain C (residues 269-341), pinker (residues 135-172, 232-268), and the stem, transmembrane region, and cytoplasmic tail (residues 342-423). The capsid protein was divided into two rigid bodies (residues 111-176 and residues 177-261), and Mxra8 was divided into D1 (residues 44-174) and D2 (10-43; 175-269). The refined subunits were used to build the asymmetric unit with adjacent subunits to prevent clashes and optimize interactions. These models underwent further real-space refinement. COOT was used to fix regions manually with poor geometry. After optimization, coordinates of the asymmetric units were checked by MolProbity (TABLE 4). Contact residues were identified, and buried surface areas were calculated using PDBePISA (www.ebi.ac.uk/pdbe/pisa/).
Mapping of antibody or Mxra8 contact residues onto the CHIKV p62-E1 crystal structure. Figures were prepared using the atomic coordinates of CHIKV pE2-E1 (RCSB accession number 3N42) using the program PyMOL (The PyMOL Molecular Graphics System, Version 1.7.4 Schrödinger, LLC). Amino acid contact residues for neutralizing anti-human or anti-mouse mAbs that block Mxra8 binding were derived from published studies (Fox et al., 2015; Smith et al., 2015).
Data Availability. All structures described below are deposited in the PDB and EMDB databases and are incorporated by reference in their entireties: Crystal structure of murine Mxra8 ectodomain (PDB code for this deposition is 6NK3); electron Cryo-Microscopy Of Chikungunya VLP alone (PDB code for this deposition is 6NK5; EMDB code for this deposition is EMD-9393); Electron Cryo-Microscopy of Chikungunya VLP in complex with mouse Mxra8 receptor (PDB code for this deposition is 6NK6; EMDB code for this deposition is EMD-9394); Electron Cryo-Microscopy of Chikungunya in complex with mouse Mxra8 receptor (PDB code for this deposition is 6NK7; EMDB code for this deposition is EMD-9395).
Statistics for the highest-resolution shell are shown in parentheses.
The microscope settings for image collection were: Dose: 50 e−/Å2; Magnification: 18,000×; Pixel size: 1.4; and Voltage: 300 keV. Micrographs were corrected for the contrast transfer function using CTFFIND4 (Rohou and Grigorieff, 2015) in the program cisTEM (Grant et al., 2018).
Contact residues were identified using PDBePISA (www.ebi.ac.uk/pdbe/pisa/). Mxra8 is numbered according to NCBI reference sequence NP_077225.4. In bold are contacts unique to Mxra8 at binding site 2, and in italics are contacts at each Mxra8 position except binding site 2.
Li, L., Jose, J., Xiang, Y., Kuhn, R. J., and Rossmann, M. G. (2010). Structural changes of envelope proteins during alphavirus fusion. Nature 468, 705-708.
This application claims priority from U.S. Provisional Application Ser. Nos. 62/613,658 filed on 4 Jan. 2018; 62/671,680 filed on 15 May 2018; and 62/672,080 filed on 16 May 2018, which are incorporated herein by reference in their entireties.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US19/12435 | 1/4/2019 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62613658 | Jan 2018 | US | |
| 62671680 | May 2018 | US | |
| 62672080 | May 2018 | US |
