Patent Grant
11286502
The present invention relates in general to the field of a ZIKA virus like particle (VLP) based vaccine and microneutralization assay, cell lines, and vectors related to the same.
The present application includes a Sequence Listing, which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Jun. 20, 2018, is named TECH2104WO_SeqList and is 53 kilobytes in size.
Without limiting the scope of the invention, its background is described in connection with viruses belonging to the family Flaviviridae.
Since the identification of Zika virus (ZIKV) in 1947 from a Rhesus Monkey in Uganda until 2010, the virus has predominantly circulated between Aedes mosquitoes and non-human primates. Periodic episodes have been identified in the human population, which were characterized by mild self-limiting febrile disease associated with rash, headache, myalgia and conjunctivitis. However, the recent spread of ZIKV infections in the Western continents has caused much concern due to severe clinical outcome in unborn fetuses including cerebral calcifications, microcephaly and other severe congenital malformations. In adults, neurological manifestations are characterized by an autoimmune condition with symptoms of neuropathy and paralysis, also known as the Guillain-Barre syndrome. While Aedes species of mosquitos are the most common source of transmission, the virus has also been shown to transmit sexually both from women to men and men to women and is capable of persisting in semen and vaginal secretions for up to 6 months after infection.
ZIKV is an enveloped RNA virus belonging to the family Flaviviridae. The 11 Kb positive sense RNA genome is translated in the cytoplasm to generate three structural and seven non-structural proteins. The structural proteins C (Capsid), prM/M (Pre-membrane, membrane), and E (Envelope) aid in virus assembly that predominantly occurs in the lumen of the endoplasmic reticulum (ER). Virus maturation occurs during virus egress via the secretory pathway when the acidic environment in the Golgi cleaves the prM followed by release of pr peptide. The E protein is the major target for neutralizing antibodies and monoclonal antibodies against all 3 E protein domain (DI, DII and DIII) target epitopes have been found. The recent outbreaks of ZIKV infection have sparked efforts in the scientific community towards the development of a safe and effective vaccine. These efforts towards a safe and efficacious vaccine encompass the use of established approaches like purified inactivated virus to more advanced approaches like DNA (Pr-M-E), subunit (E) based vaccines, recombinant adenoviral platforms along with recent development of RNA nanoparticle technology, or modified mRNA (prM-E) as vaccine candidates. The studies have demonstrated a neutralizing antibody response capable of protecting against ZIKV infection both in mice and non-human primates leading several clinical trials currently underway (NCT02963909, NCT02840487, NCT02887482, NCT02809443, NCT02952833). One of the advantages of the development of a ZIKV vaccine is that even though the virus exists as two distinct lineages (the African and Asian/American), the immune response generated against the virus is broadly protective thus obviating the need to incorporate different serotypes in the vaccine.
What is needed is the development of neutralizing antibodies against the infection and availability of a rapid accurate diagnostic assay to quantitate the elicited immune response. A widely used assay for detection of neutralizing antibodies against Flaviviruses is the plaque reduction neutralization test (PRNT) assay, which involves the use of live virus handled under BSL-2 conditions. An ideal assay would be one that could be adapted to a high throughput format with a convenient read out and eliminate the use of live virus making it readily available to laboratories worldwide. With regards to an effective vaccine, the priorities include: safety, efficacy, ease of handling, and economy of production for worldwide dissemination.
Thus, despite many efforts, there is currently no approved vaccine for Zika. A recent report showed a PrME plasmid DNA vaccine to be effective in mouse studies. However, DNA based vaccines includes several risks, e.g., insertional mutagenesis.
In one embodiment, the present invention includes a nucleic acid vector comprising: a recombinant nucleic comprising at least one of: a Zika virus C-prM-E gene, or a recombinant nucleic comprising a portion of a prM-E gene region of Zika virus spanning amino acids 105-795, operably linked to a promoter. In one aspect, the nucleic acid vector is a flavivirus vector. In another aspect, the nucleic acid vector is a plasmid. In another aspect, the C-prM-E, the prM-E gene, or both are codon optimized. In another aspect, the nucleic acid vector is pcDNA3.1™, and the C-prM-E gene, the prM-E gene, or both, are codon optimized for expression in human cells. In another aspect, the nucleic acid vector further comprises an NS2B3 protease gene. In another aspect, the vector is a lentiviral vector pLenti6/5-D-Topo®. In another aspect, the vector further comprises an NS2B3 protease gene. In another aspect, the nucleic acid vector further comprising a selectable marker.
In another embodiment, the present invention includes a method of making a Zika virus Reporter Virus Particles (RVP) comprising: transfecting cells stably expressing a Zika C-prM-E gene; and transfecting the cells stably with a sub-genomic replicon derived from lineage II strain of WNV that also expresses a reporter gene; incubating the cells under conditions in which the proteins are expressed for a period sufficient to form RVPs; and harvesting the RVPs. In one aspect, cells are 293T cells, HeLa cells, MDCK cells, Vero cells, or vaccine-certified cell lines. In another aspect, the reporter gene is selected from at least one of green fluorescent protein; yellow fluorescent protein; blue fluorescent protein; Cerulean fluorescent protein; Cyan fluorescent protein; red fluorescent protein from Zooanthus sp.; red fluorescent protein from Entremacaea quadricolor (RFP), or flavin mononucleotide (FMN)-binding fluorescent proteins (FbFPs). In another aspect, the C-prM-E is codon optimized. In another aspect, the method further comprises transfecting an NS2B3 protease gene into the cell. In another aspect, the vector further a selectable marker and selecting stable expression with a selectable marker.
In another embodiment, the present invention includes a vaccine comprising: a particle that comprises a codon optimized PrM-E protein of Zika virus in a pharmaceutically acceptable carrier or excipient. In one aspect, the particles are manufactured in 293T cells, HeLa cells, MDCK cells, Vero cells, or vaccine-certified cell lines. In another aspect, the particle is a Zika virus like particle. In another aspect, the vector further comprises a selectable marker. In another aspect, the particle is a Zika virus reporter virus particle capable of a single round infection in cells in a manner identical to native Zika virus. In another aspect, the Zika virus reporter virus particle is non-infectious.
In yet another embodiment, the present invention includes a vaccine comprising: a Zika virus like particle comprising a matured C-prM-E expressed from a vector that comprises a codon optimized C-prM-E gene and an NS2B3 protease gene.
In another embodiment, the present invention includes a cell line comprising: a codon optimized C-prM-E gene of Zika virus that is stably expressed. In one aspect, the cell line comprises 293T, Raji, or Vero cells. In another aspect, the cell line is transduced with Lentiviral particles made in cells expressing lentiviral prME and php-dl-NA, and VSVG Env. In another aspect, the cell line is stably transduced selected using a selectable marker. In another aspect, the stable expression is by blasticidin selection. In another aspect, the cell line is further transfected with an NS2B3 protease gene.
In another embodiment, the present invention includes a cell line comprising: a codon optimized prM-E gene region of Zika virus spanning amino acids 105-795 that is stably expressed. In one aspect, the cell line is 293T cells, HeLa cells, MDCK cells, Vero cells, or vaccine-certified cell lines.
In another embodiment, the present invention includes a method of detecting Zika virus microneutralization using reporter virus particles (RVP) or Virus Like Particles (VLP) comprising: (a) incubating serial dilutions of sera or antibodies with a pre-determined amount of ZIKV RVPs or VLPs comprising a detectable marker at room temperature to make an antibody-RVP mix; (b) adding the antibody-RVP or VLP mix to cells under conditions in which the detectable marker is expressed; (c) measuring infection after a pre-determined amount of time by counting the number of detectable marker positive cells; and (d) comparing the level of the detectable marker in a first set of cells where no sera or neutralizing antibody was used to a second set of cells where the sera or neutralizing antibodies were used, wherein a decrease in the number of marker positive cells in the second set of cells when compared to the first set of cells is indicative of microneutralization that correlates to a level of neutralizing antibodies in the biological sample of the patient. In one aspect, the cells are 293T, Raji, or Vero cells. In another aspect, the method further comprises obtaining another biological sample from the patient after a pre-determined time and comparing a titer of neutralizing antibodies between an earlier and a later biological sample to determine antibody titers. In another aspect, the method is adapted for use in 4, 6, 8, 12, 24, 48, 96, 384, 1538, 6114, or 9,600 well plates. In another aspect, the ZIKV VLPs comprise a PrM-E protein expressed by a codon-optimized construct. In another aspect, the detectable marker in the ZIKV RVPs comprises a fluorescent protein expressed in conjunction with a WNV sub-genomic replicon. In another aspect, the ZIKV VLP is adapted to be an antigen in a diagnostic assay.
In another embodiment, the present invention includes a method of making stably transduced cell lines comprising: transfecting cells with lentiviral vector expressing ZIKV prME, CprME, or both prME and CprME, and php-dl-NA and VSVG Env to produce Lentiviral particles; transducing cells with the Lentiviral particles; and selecting stable transduced cells with a selection agent to create the stably transduced cell lines. In one aspect, the cell line is prME-F4. In another aspect, the cell line is CprME (F6). In another aspect, the selection agent is Blasticidin. In another aspect, the transfected cells are selected from 293T cells, HeLa cells, MDCK cells, Vero cells, or vaccine-certified cell lines. In another aspect, the ZIKV prME, CprME, or both prME and CprME are codon optimized.
In another embodiment, the present invention includes a reporter Zika virus Reporter Virus Particles (RVP) made by expressing a matured, codon optimized ZIKV CprME made in a cell line that expressed an NS2B3 protease.
For a more complete understanding of the features and advantages of the present invention, reference is now made to the detailed description of the invention along with the accompanying figures and in which:
While the making and using of various embodiments of the present invention are discussed in detail below, it should be appreciated that the present invention provides many applicable inventive concepts that can be embodied in a wide variety of specific contexts. The specific embodiments discussed herein are merely illustrative of specific ways to make and use the invention and do not delimit the scope of the invention.
To facilitate the understanding of this invention, a number of terms are defined below. Terms defined herein have meanings as commonly understood by a person of ordinary skill in the areas relevant to the present invention. Terms such as “a”, “an” and “the” are not intended to refer to only a singular entity, but include the general class of which a specific example may be used for illustration. The terminology herein is used to describe specific embodiments of the invention, but their usage does not limit the invention, except as outlined in the claims.
The present invention meets the criteria for an effective vaccine, namely, safety, efficacy, ease of handling, and economy of production for worldwide dissemination. The VLPs-based vaccines of the present invention are safe for manufacturing and handling and generate an effective immune response and can be readily scaled up for cost-effective production. The ZIKV VLPs taught herein can be readily produced in cells expressing the prM-E proteins and the particles although non-infectious resemble the live virus in morphology. Further, addition of the Capsid has been shown to promote virion stability and is effective in inducing a cell-mediated immune response against Flaviviruses. VLP-based vaccines have been successful against viral diseases like Hepatitis B (GlaxoSmithKline's ENGERIX® and Merck and Co., Inc.'s RECOMBIVAX HB®) and human papilloma virus (Merck and Co., Inc.'s GARDISIL®) with others in the pipeline for diseases like influenza, Parvovirus, Norwalk virus etc.
As used herein, the terms “antigen,” “antigenic,” and “antigenically active,” refer to any substance that can be recognized by a specific humoral and/or cell-mediated immune response. As used herein, the terms “immunogen,” “immunogenic” and “immunologically active” refer to any substance that is capable of inducing a specific humoral and/or cell-mediated immune response. An antigen or immunogen generally contains at least one epitope. Antigens and immunogens include but are not limited to molecules, including small molecules, peptides, polysaccharides, nucleic acids, and/or lipid, that trigger an immune response. Complexes of peptides with lipids, polysaccharides, or with nucleic acid sequences are also contemplated, including (without limitation) glycopeptide, lipopeptide, glycolipid, etc. These complexes are particularly useful immunogens where smaller molecules with few epitopes do not stimulate a satisfactory immune response by themselves. In certain aspects, the virus like particles of the present invention can be used as an antigen in a diagnostic assay.
As used herein, the term “adjuvant” refers to the addition of an agent that enhances an immune response in an animal. For example, the antigen may be mixed or emulsified in saline, for example, Freund's complete adjuvant (“FCA”), Freund's incomplete adjuvant, alum, CpG, and the mixture is injected parenterally, intraperitoneally, subcutaneously, intramuscularly, orally, etc. The animal is generally boosted 2-6 weeks later with one or more injections of the antigen with or without an adjuvant. Antibodies may also be generated by in vitro immunization, using methods known in the art. When isolated from an animal, polyclonal antisera is then obtained from the immunized animal.
As used herein, the term “cell culture” refers to any in vitro culture of cells, including, e.g., continuous cell lines (immortal), primary cell cultures, transformed cell lines, finite cell lines (e.g., non-transformed cells), and any other cell population capable of being maintained in vitro. Cells may include bacterial, fungal, mammalian, insect, etc.
As used herein, the term “codon optimized” refers to a nucleic acid sequence or polynucleotides that is expressed into a polypeptide wherein the codon usage is optimized for a specific host. Codon optimized sequences are engineered to increase the expression of the polypeptide in a given species. To provide optimized polynucleotides coding for the viral and other proteins described herein, the DNA sequence of the gene is modified to 1) include codons preferred by highly expressed genes in a particular species; 2) include an A+T or G+C content in nucleotide base composition to that substantially found in the target species; 3) form an initiation sequence of the target species; or 4) eliminate sequences that cause destabilization, inappropriate polyadenylation, degradation and termination of RNA, or that form secondary structure hairpins or RNA splice sites. Increased expression of the proteins described herein is achieved by using a distribution frequency of codon usage in eukaryotes and prokaryotes, or in a particular species. The term “frequency of codon usage” refers to the usage of nucleotide codons found in a specific host cell to express a given amino acid. There are 20 natural amino acids, most of which are specified by more than one codon. Therefore, all degenerate nucleotide sequences can be codon optimized so as long as the amino acid sequence of the polypeptide encoded by the nucleotide sequence is functionally unchanged.
As used herein, the term “gene” refers to a nucleic acid (e.g., DNA or RNA) sequence that comprises coding sequences necessary for the production of a polypeptide, polypeptide precursors, or RNA (e.g., rRNA, tRNA, RNAi). The polypeptide can be encoded by a full length coding sequence or by any portion of the coding sequence so long as the desired activity or functional properties (e.g., immunogenicity) of the full-length or fragment are retained. The term can also encompasses the coding region of a structural gene and the sequences located adjacent to the coding region on both the 5′ and 3′ ends on either end such that the gene corresponds to the length of the full-length mRNA, e.g., when engineered into a nucleic acid vector. Sequences located 5′ of the coding region and present on the mRNA are referred to as 5′ non-translated sequences. Sequences located 3′ or downstream of the coding region and present on the mRNA are referred to as 3′ non-translated sequences. The term “gene” encompasses both cDNA and genomic forms of a gene, e.g., a viral genome. A genomic form or clone of a gene contains the coding region interrupted with non-coding sequences termed “introns” or “intervening regions” or “intervening sequences.” Introns are segments of a gene that are transcribed into heterogenous nuclear RNA (hnRNA); introns may contain regulatory elements such as enhancers. Introns are removed or “spliced out” from the nuclear or primary transcript; introns therefore are absent in the messenger RNA (mRNA) transcript. The mRNA functions during translation to specify the sequence or order of amino acids in a nascent polypeptide.
As used herein, the term “heterologous gene” refers to a gene that is not in its natural environment, e.g., a gene from one species introduced into another species. A heterologous gene also includes a gene native to an organism that has been altered in some way (e.g., mutated, added in multiple copies, linked to non-native regulatory sequences, etc). Heterologous genes are distinguished from endogenous genes in that the heterologous gene sequences are typically joined to DNA sequences that are not found naturally associated with the gene sequences in the chromosome or are associated with portions of the chromosome not found in nature (e.g., genes expressed in loci where the gene is not normally expressed).
As used herein, the term “host cell” refers to any cell capable of replicating and/or transcribing and/or translating a heterologous gene. Thus, a “host cell” refers to any eukaryotic or prokaryotic cell (e.g., such as 293T, Raji, or Vero cells, or bacterial cells, E. coli, yeast cells, mammalian cells, avian cells, amphibian cells, plant cells, fish cells, and insect cells), whether located in vitro or in vivo.
As used herein, the term “in vitro” refers to events that occur in an artificial environment, e.g., in a plate, well, test tube or reaction vessel, in cell culture, etc., rather than within a multi-cellular organism or tissue.
As used herein, the term “in vivo” refers to events that occur within a multi-cellular organism. For example only and not meant to be limiting, such as a mammal more particularly a human and/or non-human animal.
As used herein, the term “nucleic acid sequence” refers to an oligonucleotide, a nucleotide or a polynucleotide, and fragments or portions thereof, including, DNA or RNA of genomic or synthetic origin, which may be single or double-stranded, and represent the sense or antisense strand. As used herein, the term “amino acid sequence” as used herein refers to peptide or protein sequence.
As used herein, the terms “operably linked”, “in operable combination,” or “in operable order,” refer to the linkage of a nucleic acid sequence in such a manner that a nucleic acid molecule capable of directing the transcription of a given gene and/or the synthesis of a desired protein molecule is produced. The terms also refer to the linkage of amino acid sequences in such a manner so that a functional protein is produced.
As used herein, the phrase “pharmaceutically acceptable” refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
As used herein, the term “purified” refers to molecules, either polynucleotides or polypeptides that are removed from their natural environment, isolated or separated. An “isolated nucleic acid sequence” is therefore a purified nucleic acid sequence. “Substantially purified” molecules are at least 60% free, preferably at least 75% free, and more preferably at least 90% free from other components with which they are naturally associated.
As used herein, the term “purified” refers to the removal of contaminants from a sample. The removal of contaminating proteins results in an increase in the percent of polypeptide of interest in the sample. In another example, recombinant polypeptides are expressed in plant, bacterial, yeast, or mammalian host cells and the polypeptides are purified by the removal of host cell proteins; the percent of recombinant polypeptides is thereby increased in the sample.
As used herein, the term “reporter gene” refers to a gene that, when expressed as a protein, produces a polypeptide that is capable of being identified in an assay. One example of a reporter gene includes that are fluorescent, e.g., luciferase, green fluorescent protein, red fluorescent protein, that can be visually identified (e.g. with marked probes or antibodies) as known to those skilled in the art. Further, while specific examples are given any other means of fluorescent, bioluminescent, luminescent, and related reporter proteins useful for tracking are contemplated by the present invention. Other reporter genes include enzymes that can be used to metabolize or perform a reaction that creates a change in color (β-galactosidase) or the presence of a new metabolite (e.g., acetylation of chloramphenicol). Non-limiting examples of fluorescent proteins include: green fluorescent protein (GFP) PDB designation 1GFL; yellow fluorescent protein (YFP) PDB designation 3DPW; blue fluorescent protein (BFP) PDB designation 1BFP; Cerulean fluorescent protein (CFP) PDB designation 2WSO; Cyan fluorescent protein (CFP) PDB designation 2WSN; red fluorescent protein from Zooanthus sp. (RFP) PDB designation 2ICR; red fluorescent protein from Entremacaea quadricolor (RFP) PDB designation 2PJB, flavin mononucleotide (FMN)-binding fluorescent proteins (FbFPs).
As used herein, the term “subject” or “patient” refers to any organism immunized with the nucleic acid constructs, polypeptides, cells, or Reporter Virus Particles (RVP) invention are administered, e.g., for experimental, diagnostic, prophylactic, and/or therapeutic purposes. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans; insects; worms; etc.). In vitro systems may also be used (e.g. to express ZIKV proteins or portions thereof for study within the target cell and/or for isolation).
As used herein, the term “vaccine” refers to an immunogenic composition that is administered to a host to provide some degree of protection from an infection and/or disease from a target virus or pathogen, e.g., ZIKV. Immunogenic agents are those that trigger an immune response in an animal. Moreover, some degree of protection includes but is not limited to decreasing, reducing, modifying, and/or ameliorating one or more symptoms of an infection and/or disease.
Vaccines can be administered alone or in combination with various adjuvants/carriers. Pharmaceutical carriers for immunization are known to those skilled in the art. These most typically would be standard carriers for administration of vaccines to humans, including solutions such as sterile water, saline, and buffered solutions at physiological pH. Other ingredients can include, e.g., adjuvants, excipients, carriers, thickeners, diluents, buffers, preservatives, and surface-active agents.
As used herein, the term “vaccine-certified cells”, refers to those cell lines that are routinely used for vaccine manufacturing purposes. A certification process is used, e.g., that taught by Donis, “Performance characteristics of qualified cell lines for isolation and propagation of influenza viruses for vaccine manufacturing”, Vaccine, Volume 32, Issue 48, 12 Nov. 2014, Pages 6583-6590, relevant portions incorporated herein by reference.
As used herein, the term “virus-like particle” (VLP) refers to a membrane-surrounded viral core structure having viral envelope proteins. In addition, additional molecules can be expressed in or on the VLP, e.g., adjuvants, antigens, or reporter proteins or enzymes. Typically, viral core proteins are located within the membrane of the VLP and do not contain intact viral nucleic acids, and are non-infectious.
The present invention includes a vaccine against Zika virus as well as a high throughput assay to test anti Zika neutralizing antibodies and anti-viral compounds. The present invention provides for the first time a VLP-based vaccine, due to their protein composition only. Moreover, no VLP-based methodologies have been shown or published for Zika virus. VLP based approaches are both safer and more effective than DNA vaccines. The VLP system of the present invention overcomes the deficiencies in nucleic acid based approaches.
Development of Virus like Particle Vaccine and Reporter Assay for Zika Virus. Recent worldwide outbreaks of Zika virus (ZIKV) infection and the lack of an approved vaccine raise serious concerns regarding preparedness to combat this emerging virus. The present inventors used a Virus Like Particle (VLP) based approach to develop a vaccine and a microneutralization assay against ZIKV. Synthetic C-prM-E gene construct of ZIKV was used to generate Reporter Virus Particles (RVP) that package GFP reporter expressing WNV replicon. The assay was adapted to 96-well format, similar to the Plaque Reduction Neutralization assay (PRNT) and showed high reproducibility with specific detection of ZIKV neutralizing antibodies. Furthermore, C-prM-E and prM-E VLPs were tested as vaccine candidates in mice and compared to DNA vaccination. While flaviviral prM-E constructs alone are sufficient for generating VLPs; it was found that efficient VLP production with the C-prM-E constructs could be achieved in the presence of WNV NS2B-3 protease that cleaves C from prM allowing for VLP release. Immunization studies in mice showed that VLPs generated higher neutralizing antibody titers than DNA vaccine with C-prM-E VLPs slightly better than prM-E VLPs. The superiority of C-prM-E VLPs demonstrates that inclusion of Capsid may have benefits for ZIKV and other flaviviral VLP vaccines. To facilitate the VLP platform, the present inventors generated stable cell line expressing high levels of ZIKV prM-E proteins that constitutively produce VLPs as well as a cell line expressing ZIKV C-prM-E proteins for RVP production. Thus, the present invention provides a scalable platform to tackle the emerging threat of ZIKV by developing a safe, effective and economical ZIKV vaccine.
The present invention addresses two areas highly relevant to Zika virus research-development of an economical, efficacious and safe VLP-based vaccine and a rapid versatile diagnostic assay. The cell lines stably expressing the Zika prM-E were generated to produce high amounts of VLPs in the supernatants and a Zika C-prM-E expressing cell line was developed that produces reporter virus particle upon transfection with GFP replicon plasmid. It was found that the prM-E VLPs induced a strong neutralizing antibody response in mice that was better when the capsid protein was included in the VLPs. The VLP-based vaccines of the present invention showed significantly better neutralizing antibody response when compared to their DNA counterparts. Finally, a RVP-based microneutralization assay was developed that works similar to the plaque reduction neutralization test (PRNT) assay with a rapid GFP readout in a 96 well format. Thus, the VLP based platform provides a source for Zika vaccine, detection, and diagnosis that can be rapidly adapted to current outbreaks.
The present inventors determined the immune response in mice vaccinated with ZIKV VLP generated using a prM-E or the C-prM-E construct and compared it to the DNA based counterparts. To generate PrM-E VLPs, the inventors developed a cell line stably expressing the prM-E proteins while for C-prM-E, VLPs were generated by cotransfection of cells with C-prM-E and NS2B-3 protease of WNV to allow cleavage of Capsid (C) from prM-E. VLPs were purified via ultracentrifugation and used to immunize mice. Concomitantly, the inventors also developed a rapid and quantitative GFP based micro-neutralization assay using Reporter Virus Particles (RVPs) for measuring the neutralizing immune response generated in mice. These data demonstrates that both prM-E and C-prM-E based VLP vaccines were highly effective in generating neutralizing antibodies with the latter being more potent. Interestingly, while the prM-E DNA based vaccine was less effective than both the VLPs, the C-prM-E DNA construct was unable to generate significant neutralizing antibodies, most likely due to the lack of VLP formation in the absence of NS2B-3 protease. The RVP based neutralization assay of the present invention is safe for regular clinical laboratories because it does not require use of live virus and yielded results in <72 hrs compared to the PRNT assay that may require a week for completion. The assay was highly reproducible and effective in measuring the neutralizing antibody response against ZIKV that could be conducted in 96-well format using simple fluorescent microscopy. Thus, this study demonstrates for the first time the use of a VLP platform to tackle the emerging threat of ZIKV by providing a scalable source for a VLP based vaccine and RVP based diagnostic assay.
Cell culture and reagents. 293T and Vero cells were obtained from ATCC and cultured in DMEM supplemented with 10% FBS. All transfections were performed using Turbofect reagent (Thermo Fisher) as per the manufacturer's instructions. The WNV C-prM-E and Rep/GFP plasmids have been described previously (39) and were kindly provided by Dr. Ted Pierson (NIAID). Plasmid containing the WNV NS2B-3 accessory fusion protein expressing the active protease has been described previously (52) and was a kind gift from Dr. Frank Scholle (NC State Univ). The ZIKV-117 antibody was kindly provided by Dr. James Crowe (Vanderbilt University Medical Center, Nashville Tenn.) and ZIKV mouse polyclonal sera against the isolate MR766 was kindly provided by the Centers for Disease Control and Prevention (Fort Collins, Colo.). The ZIKV isolate PRVABC59 derived from a human serum specimen from Puerto Rico in December 2015 was obtained from ATCC and propagated in Vero cells strictly following the ATCC recommendations.
Generation of vectors expressing ZIKV C-prM-E and prM-E: ZIKV C-prM-E construct was synthesized using the complete ZIKV sequence available from the current outbreak in Americas (accession number KU312312.1). This most current sequence was used to synthesize a codon optimized version of the C-prM-E gene using the Gene Synthesis Technology by GenScript, Piscataway, N.J., USA. The synthesized gene was subcloned into pcDNA3.1™ vector (Invitrogen) using BamH1 and Xhol restriction sites. The C-prM-E cassette was also cloned into the lentiviral vector pLenti6/V5® vector (Invitrogen) using the above restriction sites to generate plasmid pLenti-C-prM-E. The prM-E construct was generated by PCR amplification of prM-E region spanning amino acids 105-795 using the Phusion® high fidelity PCR kit (New England BioLabs) and cloned into the pcDNA3.1+™ vector. The prM-E cassette was also subcloned into pLenti6/V5 vector using the BamH1-Xhol sites to generate plasmid pLenti-prM-E. Plasmids were sequenced to confirm sequence identity.
Detection of ZIKV E protein expression. Detection of ZIKV virus E protein was conducted either via immunofluoresscnce or Western blotting. For this, cells were stained using ZIKV E specific antibody MAB10216 (clone 4G2, EMD Millipore) that reacts with Flavivirus group specific antigens followed by secondary antibody Alexa 488 (Invitrogen) and analyzed by fluorescence microscopy. The monoclonal antibody MAB8150 (clone 3.67G, EMD Millipore) was used as control and reacts with E protein of West Nile/Kunjin virus. For Western blotting, lysates were resolved on an SDS-PAGE gel, transferred onto PVDF membranes and probed with ZIKV virus E antibody (GTX133314, GeneTex, 1:3000) followed by HRP conjugated anti mouse secondary antibody and bands visualized via enhanced chemiluminescence using the Super signal West Femto substrate (Pierce).
Metabolic labeling and immunoprecipitation. The protocol for radiolabeling and immunoprecipitation of cell and virus lysates has been described in detail previously (Garg, 2013. Briefly, transfected cells were washed with RPMI medium lacking Met and Cys. Thereafter, cells were incubated in RPMI medium supplemented with FBS and [35S]Met/Cys. Culture supernatants were filtered and subjected to ultracentrifugation at 100,000×g for 45 min. Cell and virion samples were lysed with Triton X containing lysis buffer (0.5% Triton X-100, 300 mM NaCl, 50 mM Tris [pH 7.5] containing protease inhibitors [Complete; Roche]). Thereafter, lysates were immunoprecipitated with anti-WNV serum (kindly provided by Dr. Robert B. Tesh, University of Texas Medical Branch, Galveston) or MAB10216 coated Protein A beads. Immunoprecipitated cell lysates were washed three times with TritonX-100 wash buffer and once with SDS-DOC wash buffer (0.1% sodium dodecyl sulfate, 300 mM NaCl, 50 mM Tris [pH 7.5], 2.5 mM deoxycholic acid), resolved by SDS-PAGE followed by PhosphorImager analysis.
Production of RVPs. ZIKV RVPs were generated using protocol described previously (38) with some modifications and originally described by Pierson et al (39). 293T cells were co-transfected with the ZIKV C-prM-E construct along with plasmid containing the sub-genomic GFP expressing replicon derived from lineage II strain of WNV (39). The RVPs were harvested 48 h post transfection, aliquoted and stored for future use. RVPs were titrated in Vero cells plated in 96 well clear bottom black plates at 5,000 cells per well. Thereafter, cells were infected with serial two-fold dilutions of the RVPs and incubated for 72 h. The plates were fixed using 4% formalin/PBS, images acquired using a Nikon EclipseTi microscope and number of GFP positive cells counted using the NIS elements software (Nikon).
RVP based and clinical ZIKV based microneutralization assay. For neutralization assays, mouse sera or antibodies were serially diluted in DMEM in technical duplicates in a volume of 50 μl. Thereafter, a predetermined concentration of virus producing 200 to 500 GFP positive cells was added to each well in a volume of 50 μl. The sera and RVPs were incubated for 1 h at room temperature. Subsequently, the virus:sera mix (100 μl) was added to Vero cells after removing all the media and incubated for ˜72 h after which the number of GFP positive cells was quantitated. Statistical analysis was performed using the unpaired t test. The limit of detection for the RVP assay was defined as the highest concentration of sera (1:20 dilution) used in the neutralization experiments.
For neutralization assays using the ZIKV isolate PRVABC59, mouse sera was serially diluted in serum free media in technical duplicates in a volume of 50 μl. Thereafter, diluted ZIKV was added to each sera sample at an MOI of ˜5 and incubated for 2 h at 37° C. The virus antibody mixtures were then added to Vero cells plated in 96-well plates and incubated for another 2 h at 37° C. The virus antibody mix was then removed and cells incubated in DMEM containing 10% FBS for 48 h. The cells were then fixed and stained using MAB10216. Images were acquired using fluorescence microscopy and number of antibody stained cells quantitated as described above.
Generation of stable cell lines expressing ZIKV C-prM-E or prM-E: Lentiviral vectors expressing ZIKV C-prM-E and prM-E were packaged in 293T cells by transfecting with pLenti-C-prM-E or pLenti-prM-E along with the helper construct php-dl-NA (NIH AIDS Reagent program) and VSVG Env. The viral supernatants were collected at 48 h post transfection, aliquoted and stored. To generate stable cell lines, 293T cells were transduced with the lentiviral particles and the cells were selected using blasticidin at a concentration of 10 μg/ml. Bulk selected cells were passaged 8-10 times and stained for ZIKV E protein expression using monoclonal Ab MAB10216 at regular intervals to confirm selection. Subsequently, single cell clones were generated from the bulk selected cells using limiting dilution cloning in 96 well plates. Up to 6 single cell clones were selected for both C-prM-E and prM-E constructs from wells that showed single colony formation. Each single cell clone was further characterized for ZIKV E protein expression using immunostaining followed by flow cytometry.
Production of DNA and VLPs for immunization: For DNA immunizations in mice, the pcDNA-C-prM-E and pcDNA-prM-E plasmids were purified using the endotoxin free plasmid maxi kit (Qiagen) following the manufacturer's protocol. VLPs for immunization were generated and purified. For C-prM-E VLPs, 293T cells were transfected with pcDNA3.1-CprM-E construct along with WNV NS2B-3 plasmid. The viral supernatants were harvested at 24 h and 48 h post transfection. For production of prM-E virus particles, the 293T-Lenti-prM-E bulk cell line was cultured in the absence of Blasticidin and supernatants harvested at 72 and 96 hrs. VLPs were concentrated as follows. Harvested supernatants (25-30 ml) were transferred into ultracentrifuge tubes and carefully underlayed with 5 ml of 25% glycerol in TNE buffer. VLPs were pelleted by centrifugation at 110,500×g for 3 hrs at 4° C. Thereafter, the supernatant was carefully removed and the VLP pellet resuspended in PBS or TNE buffer. The total protein content in the VLP prep was measured using the micro BCA kit (Pierce) and specific E protein in the prep was detected by western blotting using the GTX133314 antibody.
Electron microscopy. VLPs were concentrated as above and imaged by electron microscopy after negative staining. Purified VLPs (3 μl) were applied to glow-discharged carbon-coated 300 mesh grid. After ˜1 min, the grid was blotted with filter paper and 3 μl of 2% uranyl acetate aqueous solution was added for 30 sec. After blotting off excess liquid and drying, images were acquired using the Transmission Electron Microscope JEOL1010 with a Hamamatsu digital camera and AMT Advantage image capture software at 100× magnification.
Mice studies. For immunization studies, 6 to 8 week old Balb/c mice were purchased from Jackson laboratory and housed in pathogen free animal facility at Texas Tech University Health Sciences Center, El Paso. Mice were divided into groups of six mice each and immunized with different preparations. For DNA immunization, a total of 50 μg of DNA in a volume of 100 μl PBS was injected intramuscularly in each thigh. Mice received two additional boosts at week 2 and 4 of primary immunization as described above. For VLPs, the first immunization consisted of VLP prep (approximate total protein content 1.7-2.3 mg/ml) mixed with TiterMax Gold adjuvant (Sigma) at a 1:1 ratio in total volume of 100 μl injected intramuscularly in each thigh. Mice received two additional boosts of VLPs at week 2 and 4 without adjuvant. Control mice were sham injected with PBS. Blood was collected from mice at week 9 post first immunization under terminal isoflurane anesthesia followed by intracardiac puncture. Blood samples were collected in serum separator tubes as per the manufacturer's recommendations. After coagulation, the tubes were centrifuged, sera harvested, aliquoted and stored at −80° C. until further use.
Expression of ZIKV C-prM-E using codon optimized synthetic construct: To develop a VLP based platform for ZIKV vaccine and RVP assay, the inventors used the complete ZIKV sequence available from the current outbreak in Americas (KU312312.1) (6) to synthesize a codon optimized C-prM-E gene. The isolate was derived from a 52 year male hospitalized with symptoms of conjunctivitis and exanthema in Paramaribo (Suriname) in 2015. The synthesized gene was cloned into pcDNA3.1™ vector that contains a CMV promoter (
RVP based microneutralization assay for ZIKV using a 96 well format and GFP readout. To make and test ZIKV RVPs, the inventors used the method described previously by Garg et al (38) and adapted from Pierson et al (39). 293T cells were co-transfected with the ZIKV C-prM-E construct along with the WNV replicon reporter plasmid Rep/GFP (29, 39, 40) that provides the WNV accessory proteins and the GFP reporter gene (
Although ZIKV RVPs have recently been used by other labs, the current method of choice is infection of Raji or Vero cells, followed by detection of GFP+ cells via flow cytometry (29, 39, 40). On the other hand, the infectious virus based PRNT utilizes Vero cells in 96 well format and can be tedious and time consuming. The inventors adapted The RVP based assay to a 96 well plate using Vero cells similar to the PRNT method. Cells plated in 96 well clear bottom black tissue culture plates were infected with serial dilutions of the RVPs. The plates were fixed 72 h later, analyzed by fluorescent microscopy and the number of GFP positive cells counted using an automated software (NIS Elements, Nikon). Serial dilutions (1-6) of the input virus showed a dose dependent decrease in the number of GFP positive cells (
The inventors next tested whether the assay could detect neutralization of ZIKV RVPs via antibodies or polyclonal mouse sera. Experiments were carried in a manner similar to the standard PRNT, where sera/antibody dilutions were incubated with RVPs for 1 h prior to addition to Vero cells. As shown in
Establishment of a stable cell line expressing ZIKV C-prM-E. The sudden outbreaks of ZIKV infection have caught the research community off guard and the limited number of reagents and resources available for ZIKV studies has hampered research efforts. The inventors hence developed a stable cell line expressing the ZIKV C-prM-E to facilitate generation of RVPs. For this, the codon optimized ZIKV C-prM-E cassette was subcloned into a lentiviral vector (pLenti6/V5®) carrying a Blasticidin resistance gene (
Generation of prM-E cell line for VLP production. Extensive studies with different flaviviruses including ZIKV have shown that expression of prM-E in the absence of capsid can produce sub viral particles that can be used for immunization and elicit protective antibodies (44-48). These prM-E based constructs are also the basis of DNA and mRNA nanoparticle vaccines currently in development for ZIKV (20-25). The inventors hence developed a stable cell line that would constitutively produce prM-E VLPs in the supernatant and can be used for large-scale production of VLPs for immunization studies. For this, the inventors used the original C-prM-E construct and PCR amplified and cloned the prM-E region spanning amino acids 105-795 (
Immunization studies in mice: Lack of an approved vaccine for ZIKV amidst the recent outbreaks and the association of ZIKV infections with severe congenital birth defects, warrants development of a safe and efficacious vaccine against the virus. For a vaccine to be available worldwide, especially in underdeveloped countries, it should be both easy to prepare and cost effective. In this regard, stable cell lines constitutively producing the ZIKV VLPs would be optimal as they can be readily scaled up with minimal scientific infrastructure and can provide an economical alternative to other forms of vaccination. The inventors hence tested the immunogenicity of the ZIKV VLPs in mice. Although prM-E VLPs are most commonly used in flavivirus vaccine research, the inventors also generated VLPs incorporating the capsid protein using the C-prM-E construct. While prM-E particles can be readily be generated using The stable cell lines (293T-prM-E), for generation of C-prM-E VLPs the WNV NS2B-3 protease was needed for cleavage of C from prM-E in the ER (35, 50). As shown in
The purified VLPs were subsequently used to immunize mice. The immunization schedule is shown in
Anti-ZIKV virus immune response in mice immunized with prM-E/C-prM-E DNA and VLPs. The inventors next determined the immune response generated in mice upon immunization with the DNA or VLP based vaccines. Two-fold serial dilutions of the individual mice sera were tested in the RVP based microneutralization assay described in
The inventors have previously seen that immune sera from WNV infected mice is capable of binding to ZIKV E protein and cross neutralize ZIKV RVPs (
The ZIKV genome consists of a single stranded positive sense RNA and an open reading frame encoding a polyprotein 5′-C-prM-E-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5-3′ (53, 54). This polyprotein is subsequently cleaved into Capsid (C), precursor of membrane (prM), Envelope (E) and seven non-structural proteins (53, 55). The E protein (˜53 Kd) is the major virion surface protein involved in virus binding to the cell surface and membrane fusion (53, 55). Co-expression of the flaviviral proteins prM and E leads to secretion of VLPs that do not contain viral RNA and resemble empty particles produced during viral infection (48, 50, 56-58). These VLPs induce neutralizing antibody responses that are more potent than purified proteins (59, 60). One of the major obstacles in flavivirus research is the requirement of high level biosafety containment to undertake infectious virus studies with BSL-2 or higher needed for ZIKV studies. The persistence of ZIKV in body for prolonged periods (12) and the reports of sexual transmission (10, 11) highlight the risks associated with working with infectious virus. The use of VLPs has recently emerged as a powerful technology not only to study basic virus biology but also for vaccine and diagnostic assay development (59, 60). VLPs resemble viruses and are largely composed of viral structural proteins (53, 55). VLPs contain minimal or no genetic material, are non-replicating, and may also contain a reporter gene, thus allowing for easy detection (39, 61). Moreover, the requirement of high level biosafety containment to undertake infectious flavivirus studies (61, 62) can be overcome by using recombinant VLP based assays. This eliminates the use of infectious virus while still allowing testing of various aspects of the pathogen including: (1) mechanism of viral entry; (2) neutralizing antibody sensitivity; (3) vaccine efficacy; and (4) screening compounds that inhibit viral entry (63).
In this study, a construct expressing the C-prM-E polyprotein of ZIKV was used to package GFP reporter containing WNV replicon to generate RVPs similar to those generated with WNV (38, 39). WNV based RVPs have been used by the present inventors and others previously to study WNV E biology (29, 38, 39). ZIKV RVPs were generated using a codon optimized synthetic ZIKV C-prM-E construct that was co-transfected with WNV replicon construct (Rep/GFP). These RVPs were infectious in multiple cell lines and could be neutralized using anti-ZIKV antibodies or cross-neutralizing WNV immune sera. Although similar ZIKV RVPs have recently been used for anti-ZIKV response, the current approach involves detection of infected cells by flow cytometry (29, 40). While flow cytometry has its benefits, especially in minimizing human error and bias and can be readily used for large sample sizes, the plate-based assay may be more helpful in resource limiting conditions where flow cytometry is not available.
The inventors adapted the RVP assay to a 96-well plate format with a GFP readout that could be quantitated using a simple fluorescent microscope. The objective was to develop an assay that can replace PRNT, requires less time to complete, does not involve use of live ZIKV, is highly reproducible and can be used in resource limiting situations. The inventors tested the assay extensively in 96 well format and found that both the number of GFP+ cells and the neutralization sensitivity was highly reproducible. Both automated counting using imaging software or manual counting yielded similar results suggesting that the assay needs minimum infrastructure. For further ease and to facilitate the use of the assay, the inventors generated a stable cell line expressing ZIKV structural proteins (293T-C-prM-E-F6) that can be used for RVP production by transfecting with a GFP replicon construct, in the case WNV replicon. However, the same cell line could also be utilized to package ZIKV reporter replicons when those become available. Besides the elimination of the use of infectious virus, the assay also provides several key advantages that make it attractive for resource limiting areas. The assay requires simple to grow and maintain cell lines (293T-C-prM-E-F6 and Vero), standard transfection protocols, 96 well plates and a basic fluorescent microscope with a 4× objective. As in the case of PRNT, the plates can be fixed using formalin and saved for extended periods for quantitation of GFP positive cells at a later time. In The hands, the inventors have saved the fixed plates for up to 6 months without loss of GFP signal and quantified at different time points with comparable accuracy. With rapid spread of ZIKV in countries with limited scientific infrastructure, this assay would be a valuable tool to assess ZIKV neutralizing antibodies in response to either vaccination drives and/or natural infection with ZIKV or related flaviviruses. Recently, a number of studies have investigated vaccine candidates for ZIKV. These include conventional approaches like use of purified inactivated virus (PIV) (20, 21), DNA (20-22), adenoviral-based subunit vaccines incorporating prM-E or M-E regions of ZIKV (20, 23) as well as lipid nanoparticle (LNP) encapsulated RNA or modified mRNA as vaccine candidates (24-26). These studies have demonstrated effective neutralizing antibody responses capable of protecting against ZIKV infection in various animal models. Recruitment of human subjects for testing some of these vaccine candidates is ongoing (NCT02963909, NCT02840487, NCT02887482, NCT02809443, NCT02952833). The geographical distribution of ZIKV is largely in developing and underdeveloped areas of the world (64). Hence, besides safety and efficacy being the top priorities for a successful vaccine, an important practical aspect for ZIKV vaccine is cost effectiveness and ease of production. In this regard, VLP based vaccines especially from cells lines stably expressing and releasing ZIKV proteins are highly attractive. The 293T-prM-E cell lines provide a much needed resource to take ZIKV VLP vaccines from the bench to the bedside. Not only do these cell lines release copious amount of E protein in the supernatant, but VLPs produced from these cells generate a robust neutralizing antibody response in mice making it ideal for further vaccine development.
The inventors also found that VLP vaccines were more efficacious than their DNA counterparts in inducing a neutralizing antibody response. In contrast, other studies have reported higher antibody titers upon immunization with a single dose of prM-E DNA (22) or prM-E modified RNA (25). This could be due to use of heterologous signal sequence from JEV to improve expression, use of stem/transmembrane region of the E protein from JEV to improve particle secretion (22) or use of signal peptide from MHC class II (25). Moreover, while C-prM-E VLPs worked better than prM-E VLPs, the C-prM-E DNA based vaccine was relatively non-efficacious. This is largely because the flavivirus NS3 enzyme along with the cofactor NS2B forms an active protease that cleaves the flavivirus C protein that spans the ER membrane producing its mature form (35, 50). Processing of the C protein by the viral protease in the ER is important for subsequent cleavage of E protein from prM by ER resident signal peptidases (50, 65). Interestingly, this study finds that inclusion of capsid in VLPs generates a better neutralizing immune response. However, it has been proposed that in the absence of an RNA genome, the nucleocapsid does not form and hence no capsid would be released into the supernatant (66) debating the benefit of a C-prM-E based vaccine.
Studies have suggested that T cells play an important role in generating a functional immune response in the presence of the viral capsid for Hepatitis B and C viruses (67, 68). Similarly, for DENV-4, epitopes in the capsid were shown to be recognized by CTLs that were cross reactive with other dengue serotypes (33). In fact, immunization by capsid alone was shown to generate a protective immune response independent of neutralizing antibodies and largely dependent on cell-mediated immunity (34). Moreover, CD4 T cells may also be involved in protection as specialized subsets have been implicated in lysing flavivirus infected cells (33, 69). Interestingly, The study shows that inclusion of capsid in VLPs requires a functional flavivivral protease, in the case WNV NS2B-3 fusion protein. The NS2B-3 fusion protein sequence itself is about 2Kb and can be easily included in VLP platforms, DNA vaccines as well as modified mRNA vaccines.
In summary, the inventors describe the development, testing and efficacy of a VLP based vaccine against ZIKV and the generation of stable cell lines to facilitate this platform. The inventors also describe the optimization of a RVP based microneutralization assay using ZIKV C-PrM-E cell line and WNV replicon/GFP. This assay recapitulates the standard PRNT routinely used by virologists with several advantages including ease of use, reproducibility and eliminating infectious virus use. Thus, the study addresses two most relevant aspects of ZIKV infection, a safe, effective and economical vaccine and a neutralization assay that could be employed in the fight against the current ZIKV outbreaks.
It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method, kit, reagent, or composition of the invention, and vice versa. Furthermore, compositions of the invention can be used to achieve methods of the invention.
It will be understood that particular embodiments described herein are shown by way of illustration and not as limitations of the invention. The principal features of this invention can be employed in various embodiments without departing from the scope of the invention. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures described herein. Such equivalents are considered to be within the scope of this invention and are covered by the claims.
All publications and patent applications mentioned in the specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.
The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and/or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.” The use of the term “or” in the claims is used to mean “and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and/or.” Throughout this application, the term “about” is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects.
As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. In embodiments of any of the compositions and methods provided herein, “comprising” may be replaced with “consisting essentially of” or “consisting of”. As used herein, the phrase “consisting essentially of” requires the specified integer(s) or steps as well as those that do not materially affect the character or function of the claimed invention. As used herein, the term “consisting” is used to indicate the presence of the recited integer (e.g., a feature, an element, a characteristic, a property, a method/process step or a limitation) or group of integers (e.g., feature(s), element(s), characteristic(s), property(ies), method/process steps or limitation(s)) only.
The term “or combinations thereof” as used herein refers to all permutations and combinations of the listed items preceding the term. For example, “A, B, C, or combinations thereof” is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, AB, BBC, AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan will understand that typically there is no limit on the number of items or terms in any combination, unless otherwise apparent from the context.
As used herein, words of approximation such as, without limitation, “about”, “substantial” or “substantially” refers to a condition that when so modified is understood to not necessarily be absolute or perfect but would be considered close enough to those of ordinary skill in the art to warrant designating the condition as being present. The extent to which the description may vary will depend on how great a change can be instituted and still have one of ordinary skilled in the art recognize the modified feature as still having the required characteristics and capabilities of the unmodified feature. In general, but subject to the preceding discussion, a numerical value herein that is modified by a word of approximation such as “about” may vary from the stated value by at least ±1, 2, 3, 4, 5, 6, 7, 10, 12 or 15%.
All of the compositions and/or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.
This application is the National Stage of International Application No. PCT/US2018/038551, filed on Jun. 20, 2018, which claims priority to U.S. Patent Appl. Ser. No. 62/522,655, filed Jun. 20, 2017, the content of each of which is incorporated by reference herein.
This invention was made with government support under 1R21A1131696-01 awarded by the National Institutes of Health. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2018/038551 | 6/20/2018 | WO | 00 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2018/237039 | 12/27/2018 | WO | A |
| Number | Date | Country |
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| 2016210127 | Dec 2016 | WO |
| 2017015463 | Jan 2017 | WO |
| 2018152526 | Aug 2018 | WO |
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| Number | Date | Country | |
|---|---|---|---|
| 20200140891 A1 | May 2020 | US |
| Number | Date | Country | |
|---|---|---|---|
| 62522655 | Jun 2017 | US |
