Patent Application
20170226511
The present invention relates to nucleic acids. In particular, it relates to aptamers capable of binding to a flavivirus structural protein or a flavivirus non-structural protein, useful as therapeutics for preventing, treating and/or diagnosing a flavivirus infection in a patient.
The Flaviviridae family is composed of seventy enveloped positive single-stranded. RNA viruses. Of the seventy, several are clinically relevant human pathogens, which include Dengue virus (DENV), yellow fever virus (YFV), Japanese encephalitis virus (JEV), West Nile virus (WNV) and tick-borne encephalitis virus (TBEV) (Chavez et al., 2010, Noda et al., 2012). Besides Flavivirus, the Flaviviridae family consists of two other genera, Pestivirus and Hepacivirus (Chavez et al., 2010). Flaviviruses are mostly arboviruses and are transmitted to hosts via infected mosquitoes. The virions of flaviviruses are usually small, in the form of an enveloped particle with a diameter of 40-60 nm. Flaviviruses, specifically Dengue and West Nile have resulted in a wide divergent of diseases with no available vaccines or antiviral specific drugs for human treatment to date (Chavez et al., 2010).
West Nile virus (WNV), a flavivirus (Saxena et al., 2013, Bigham et al., 2011) transmitted by mosquitoes, is a member of the Japanese encephalitis virus (JEV) sero-group within the Flaviviridae family. The other members include Cacipacore virus, Murray Valley encephalitis virus and St. Louis encephalitis virus. Kunjin virus found in Australia and Asia is also a subtype of WNV. WNV was first isolated in 1937 from a woman in the West Nile region of Uganda (Silva et al., 2013, Duan et al., 2009) and was first reported in New York City in 1999 (Silva et al., 2013). WNV is a neurotropic flavivirus and is capable of causing neurological diseases in human, horses and some bird species (Silva et al., 2013). Its genome is a positive single-stranded RNA that is 11,029 nucleotides long and the virions are small, spherical, enveloped, and approximately 50 nm in diameter (Bigham et al., 2011). The most common symptoms of WNV are fever, headache, and/or hepatitis. A recent WNV outbreak in 2012 in the United States reported 5387 cases and 243 deaths (CDC report) (Saxena et al., 2013). No approved vaccine or treatment in human is available to date (CDC report) (Duan et al., 2009). The genomic and proteomic organizations of WNV are very similar to those of Dengue virus. Dengue virus (DENY), a mosquito-borne viral pathogen, is a member of the Flaviviridae family. DENV consists of four serotypes (DENV1, DENV2, DENV3 and DENV4). DENV has a positive-sense, 11-kb RNA genome that contains both structural and non-structural proteins in a single polyprotein (Gromowski et al., 2007, Crill et al., 2001, Lisova et al., 2007, Rajamanonmani et al., 2009). The gene order is C-prM-E-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5. The viral envelope consists of lipid bilayers where envelope (E) and membrane (M) proteins are embedded. The E protein is 495 amino acids in length and is glycosylated in DENV as well as in WNV. In particular, its N-linked glycosylation at Asn-67 is essential for virus propagation and is unique to DENV (Rey, 2003). The functional roles of E protein are its involvement in virus attachment to cells and also in membrane fusion (Clyde et al., 2006, Modis et al., 2004). It has also been demonstrated to be highly immunogenic and is able to elicit production of neutralizing antibodies against wild-type virus. The dengue E protein comprises of 3 regions: Domain-I (DI), Domain-II (DII) and Domain-III (DIII). DI is the central domain; DII is the dimerization and fusion domain, while DIII is an immunoglobulin-like receptor binding domain (Mukhopadhyay et al., 2005, Rey et al., 1995). It has been proven that DIII domain is a receptor recognition and binding domain (Bhardwaj et al., 2001, Chin et al., 2007, Chu et al., 2005, Zhang et al., 2007). Thus DIII is an important target for therapeutic development against DENV. Infected humans can manifest symptoms that vary from being asymptomatic, to a febrile disease, to a potentially fatal internal hemorrhage (Teoh et al., 2012, Noda et al., 2012), Immunity against different dengue serotypes are mediated by serotype-specific antibodies. Hence, patients who have recovered from the infecting serotype are thought to have perennial immunity towards the infecting serotype but short-lived immunity against other serotypes (Teoh et al., 2012). As reported by the Centre for Disease Control and Prevention, there are as many as one hundred million people infected yearly (CDC report). A recent report cautioned that the global distribution of dengue infection might even exceed 390 million per year (Bhatt et al., 2013). To date, no approved vaccine or antiviral therapeutic is available in the clinical market for humans (Teoh et al., 2012).
One way of detecting the WNV and DENV is through the use of antibodies. However, the use of antibody detection has been shown to be non-specific and engineering or inserting a novel detection moiety is difficult.
Therefore, there is a need in the art for alternative methods for detecting, treating and preventing flavivirus infections in patients.
The present invention relates to aptamers capable of binding to a flavivirus structural protein or a flavivirus non-structural protein. Such apatamers are useful as therapeutics for preventing, treating and/or diagnosing a flavivirus infection in a patient. Like antibodies, aptamers are able to bind to the surface of viruses. However, the advantage of aptamers over antibodies is the possibility of the introduction of chemically engineered detection moieties to aptamers. Also, the production cost for aptamers is lower than antibodies, as aptamers are synthesized chemically. Aptamers are also easy to customize, stable, no requirement for cold transport chain and have higher binding affinities to antigens as compared to antibodies.
In a first aspect of the invention, there is provided a nucleic acid aptamer comprising a DNA molecule that binds specifically to a flavivirus structural protein or a flavivirus non-structural protein.
Preferably, the flavivirus is selected from the group consisting of West Nile virus, Dengue virus, yellow fever virus, Japanese encephalitis, and tick-borne encephalitis virus.
In a preferred embodiment, the aptamer binds specifically to a West Nile virus envelope protein, and preferably the aptamer binds specifically to the Domain III region of the West Nile virus envelope protein. In this embodiment, the DNA molecule is preferably a modified DNA molecule based on one of three native aptamer sequences: (a) the sequence of the West Nile Virus envelope protein DIII 5′-ACGCTGCCACAAGTCCTGGTTCCCTG-3′ (SEQ ID NO: 1); (b) the sequence of the West Nile Virus envelope protein DIII 5′-CCTCCCAAACATGTAGAGTCTCACAT-3′ (SEQ ID No: 2); or (c) the sequence of the West Nile Virus envelope protein DIII 5′-CCAAATTGCCGCAGACTCGTTGTGAA-3′ (SEQ ID NO: 3) and comprising amino acid side chains. Preferably, the modified DNA molecule comprises a sequence selected from the group consisting of:
In an alternative preferred embodiment, the aptamer binds specifically to a Dengue virus envelope protein, and preferably the aptamer binds specifically to the Domain III region of the Dengue virus envelope protein. In this embodiment, the DNA molecule is preferably a modified DNA molecule based on one of three native aptamer sequences: (a) the sequence of DENV 2 envelope protein DIII TCACATTCAGATATGTTGGTTCCCAC-3′(SEQ ID NO: 4); (b) the sequence of DENV 2 envelope protein DIII 5′-AAATGTGACGTTCACAGACAAGTCC-3″ (SEQ ID No: 5); or (c) the sequence of DENV 2 envelope protein DIII 5′-GATACACTGAAGTGTTCTGATTG-3′ (SEQ ID NO: 6) and comprising amino acid side chains. Preferably the modified DNA molecule comprises a sequence selected from the group consisting of:
In both embodiments, the DNA molecule may further comprise a detectable moiety. The detectable moiety may be selected from the group consisting of biotin, enzymes, chromophores, fluorescent molecules, chemiluminescent molecules, phosphorescent molecules, coloured particles and luminescent molecules. Preferably, the detectable moiety is biotin.
Preferably, the aptamer further comprises a drug of interest, wherein the binding of the DNA molecule to a flavivirus structural or non-structural protein targets the drug of interest to its intended site of action and/or releases the drug of interest from the aptamer. Preferably, the drug is selected from the group consisting of a pharmaceutical compound, a nucleotide, an antigen, a steroid, a vitamin, a hapten, a metabolite, a peptide, a protein, a peptidomimetic compound, an imaging agent, an anti-inflammatory agent, a cytokine, and an immunoglobulin molecule or fragment thereof.
Methods of attaching various agents or drugs to antibodies or aptamers and other target site-delivery agents are well known in the art, and so methods of preparing aptamers of the invention comprising a drug of interest will be readily apparent to the person skilled in the art.
The drug or agent may be chemically or biologically conjugated to the aptamer of the invention. In particular, any method for conjugating a drug or agent to a DNA molecule also can be used. However, it is recognized that, regardless of which method of producing a conjugate of the invention is selected, a determination must be made that the DNA molecule maintains its targeting ability and that the drug maintains its relevant function.
The drug or agent may be released from the aptamer after the binding of the aptamer to its specific target. The release of the drug or agent may be by any method known to the skilled person. For example, the drug or agent may be cleaved by the host by way of a trigger molecule or mechanism. Alternatively, the drug or agent may be released by photo-activation. Radiation for the release of the drug in its active form can be provided by one of a variety of means, depending upon the photo sensitivities of the chosen photolabile bond, the DNA molecule and the drug. This may comprise the use of electromagnetic radiation, for example infrared, visible or ultraviolet radiation, supplied from incandescent sources, natural sources, lasers including solid state lasers or even sunlight.
In a second aspect of the invention, there is provided an aptamer according to the first aspect of the invention for use in diagnosis. Preferably, the aptamer of the invention is used in diagnosis of a flavivirus infection in a patient. The patient is preferably human but may be any animal, mammal, primate or the like.
In a third aspect of the invention, there is provided an aptamer according to the first aspect of the invention for use in therapy. Preferably the aptamer of the invention is used in the treatment or prevention of flavivirus infection in a patient.
In a fourth aspect of the invention, there is provided an immunogenic composition or vaccine comprising an aptamer according to the first aspect of the invention. Generally, a vaccine refers to a therapeutic material, treated to lose its virulence and containing antigens derived from one or more pathogenic organisms, which on administration to a patient, will stimulate active immunity and protect against infection with these or related organisms, whilst an immunogenic composition refers to any pharmaceutical composition containing an antigen, for example, a microorganism, or a component thereof, which composition can be used to elicit an immune response in a patient.
In a fifth aspect of the invention, there is provided a composition comprising an aptamer according to the first aspect of the invention and an excipient or carrier. Pharmaceutically-acceptable excipient may be, for example, antiadherents, binders, coatings, disintegrants, flavours, colours, lubricants, gildants, sorbents, preservatives and sweeteners. An example of a pharmaceutically-acceptable carrier is a carrier protein which facilitates the diffusion of different molecules across a biological membrane.
In a sixth aspect of the invention, there is provided a kit comprising an aptamer according to the first aspect of the invention and a carrier. Preferably, the carrier may be biodegradable nano-particles containing chemotherapeutic agents, photo-agents or quantum dots. The carrier may be conjugated with the aptamer for use in diagnostic/therapeutic applications or therapeutics development. Also, preferably, the kit is used for detecting a flavivirus infection in a patient.
In a seventh aspect of the invention, there is provided an ex vivo method for diagnosing or detecting a flavivirus infection in a patient, the method comprising: (a) obtaining a biological sample from a patient; (b) contacting the biological sample with an aptamer according to the first aspect of the invention; (c) detecting the formation of the binding complex between the aptamer and a flavivirus structural protein and/or a flavivirus non-structural protein, wherein the presence of the binding complex indicates that the patient has a flavivirus infection. The step of detecting the formation of the binding complex may be carried out by conjugating an agent or drug chemically or biologically to the aptamer of the invention. The SELEX (Systematic Evolution of Ligands by Exponential Enrichment) procedure may be used to obtain high affinity and highly specific aptamers against the target protein. The major advantage of the aptamer is that the value of the dissociation constant (KD) towards the target protein lies in the nanomolar ranges. The sequence with the high affinity is taken and conjugated with the biotin molecule which may be detected by streptavidin HRP (horseradish peroxidase).
Preferably, the flavivirus is selected from the group consisting of West Nile virus, Dengue virus, yellow fever virus, Japanese encephalitis and tick-borne encephalitis virus.
Preferably, the biological sample is a blood sample, saliva or urine. As used herein, the term “blood sample” includes blood cells, serum and plasma. More preferably, the biological sample is a blood sample.
In an eighth aspect of the invention, there is provided a method for treating or inducing an immune response to a flavivirus infection in a patient, the method comprising administering to the patient a therapeutically effective dose of the composition or vaccine according to the fifth and sixth aspects of the invention. The mode of administration may be by way of intravenous, oral, pulmonary, ocular, parental, depot or topical. Preferably, the mode of administration is intravenous.
The present invention aims to develop a new platform using modified aptamers for diagnostic and therapeutic applications to flaviviruses, in particular West Nile and Dengue viruses.
Advantageously, the present invention utilizes a modified aptamer rather than the conventional DNA or RNA aptamer, whereby the DNA strands contain modified amino acid side-chains. These amino acid side chains form additional intermolecular interactions between the aptamer and target protein, thus resulting in higher affinity interactions. The modified aptamer technology may be used to develop new therapeutics, as well as a new platform for the diagnosis of flavivirus infections.
As a proof of concept, the West Nile virus and Dengue virus serotype 2 envelope Domain III (DIII) proteins were used as antigens/target proteins for the designing of modified aptamers. For each protein, binding of the protein was screened against a random library of 1013 aptamers, followed by identifying the specific and strong binding aptamers to each of the proteins. By evaluating the binding characteristics of the selected aptamers with each of the purified DIII protein and the full length E protein in the virus, aptamers that can be utilized for diagnostic and therapeutic applications were identified. Ten potential aptamer candidates for each protein were evaluated and the results are discussed below.
The invention will now be further described with reference to the following non-limiting examples.
Material and Methods
Construction of pET28a WNE-BNDIII Plasmid
WNE-DIII gene (Wengler strain) was sub-cloned from the lab original plasmid which harbors the WN-DIII gene. The DIII gene was previously amplified from cDNA synthesized from West Nile virus Wengler strain. Primers Biotin_F, BiotinWNDIII_F, Biotin_WNDIII_R, and WNDIII_R (Table 1) were used to join the biotinylation signal peptide gene containing an enterokinase cleavage site with the WNEDIII gene via overlap extension PCR (OE-PCR) as shown in
GACGACGACAAGAGCC
TTGTCGTCGTC
Protein Expression and Extraction.
pET28aBNWNDIII plasmid was transformed into BL-21-DE3 expression competent cells (Agilent Technologies, USA) and grown on Luria-Bertani (LB) agar containing 30 μg/ml kanamycin. Selected clones were cultured in 1 L LB broth (30 μg/ml kanamycin) at 30° C. until an absorbance OD600 of 0.6. Expression of BN-WNDIII protein was induced with 1 mM isopropyl β-D-thiogalactoside (IPTG) overnight at 16° C. Bacterial cells were pelleted down with centrifugation at 8,000 rpm for 15 mM at 4° C. The protein expressed was targeted to inclusion bodies. In order to isolate the inclusion bodies, the pellet was resuspended in lysis buffer (20 mM Tris pH 8.0, 500 mM NaCl, 10 mM imidazole), followed by sonication in ice bath (15 mM, 10 Amp). The lysate was centrifuged at 12,000 rpm for 15 min at 4° C. A small white translucent pellet of inclusion body was obtained. The inclusion body pellet was then washed with the same lysis buffer followed by incubation with extraction buffer (8 M urea, 20 mM Tris, 300 mM NaCl, 10 mM imidazole, pH 8.0) at room temperature for 30 mM. The lysate was subsequently clarified by centrifugation at 13,500 rpm for 20 min.
Purification.
The extracted inclusion body containing the BN-WNDIII protein was incubated with nickel-nitrilotriacetic acid (Ni-NTA) resin (Bio-Rad, USA) for binding in a chromatography column overnight at 4° C. Ten column volumes of wash buffer (8 M urea, 20 mM Tris, 300 mM NaCl, 20 mM imidazole, pH 8.0) was used to wash away non-specific binding proteins. BN-WNDIII protein was eventually eluted out with elution buffer (8 M urea, 20 mM Tris, 300 mM NaCl, 500 mM Imidazole, pH 8.0) in six fractions. Next, all eluates were combined for refolding. Briefly, eluates were pooled into a SnakeSkin dialysis membrane tubing (Thermo Scientific, USA) and 0.5% of Tween-20 was added into the samples. The dialysis tubing was incubated in 1 L of 6 M urea for 6-12 hrs at 4° C., and 250 ml of 25 mM Tris (pH 8.0) was added into the solution every 6-12 hrs. When the final volume reached 3 L, the dialysis tubing was transferred into 2 L of 25 mM Tris and 150 mM NaCl (pH 8.0) for 6 hr. Refolded WNDIII protein was collected from the dialysis tubing. Fractions containing the protein-of-interest were injected into a FPLC machine and further purified via size-exclusion chromatography in PBS.
Protein Identity Analysis.
Samples collected from the flow through, wash, and eluates were analyzed by SDS-PAGE and Western blot. 12% Tris-tricine polyacrylamide denaturing gel was used to separate proteins in the samples and it was subsequently stained with Coomassie blue for protein detection. The presence of biotinylated WNDIII protein was confirmed by Western blot via two different approaches. First, the identity of WNDIII protein was determined with anti-His antibody. Briefly, separated proteins were transferred from the polyacrylamide gel onto a PVDF membrane using iBlot® Dry Blotting System (Life Technologies, USA). Blocking was done with 5% BSA for 1 hr at room temperature. Next, the membrane was incubated with 0.1 μg/ml mouse anti-His antibody (Qiagen, Germany) overnight at 4° C. The membrane was then washed with 1×TBST and incubated with 0.1 μg/ml goat anti-mouse secondary antibody conjugated with HRP (Thermo Scientific, USA) for 1 hr at room temperature. After washing with 1×TBST, the membrane was developed using SuperSignal® West Pico chemiluminescent substrate (Thermo Scientific, USA). For the second approach, WNDIII protein was detected directly using streptavidin conjugated with HRP. After transferring the samples onto a PVDF membrane, it was blocked with 4% BSA for 1 hr at room temperature. Then, the membrane was incubated with HRP-conjugated streptavidin (Millipore, USA) for another hour at room temperature. Subsequently, the membrane was washed thoroughly with 1×PBST for 1 hr at room temperature and developed with chemiluminescent substrate. A similar purification procedure was used for the production of non-biotinylated WNDIII.
Sample Preparation for Mass Spectrometry.
Purified protein (BN-WNDIII and WNDIII) was electrophoresed through SDS-PAGE using 12% Tris-tricine polyacrylamide denaturing gel and stained with Coomassie blue. The background of Coomassie-stained gel was removed with destaining solution (40% methanol, 10% glacial acetic acid, 50% distilled H2O). The BN-WNDIII protein-corresponding band was excised from the gel and kept in eppendorf tube containing distilled water. Samples were submitted to Protein and Proteomics Centre, Department of Biological Sciences, NUS for mass spectrometry analysis.
Enzyme Linked Immunosorbent Assay (ELISA) for Biotinylation.
Samples and standards were added into the wells of a MaxiSorp plate (eBioscience, USA) in triplicate. The plate was covered with aluminum foil and incubated for 2 hrs. All incubating and washing steps were carried out at room temperature. After washing with 1×PBST, blocking buffer was added into each well and incubated for another hour. Next, streptavidin-HRP enzyme conjugates was added and incubated for 1 hr. The plate was washed with 1×PBST to remove unbound conjugates and then substrate solution, tetramethyl benzidine (TMB), was added for development. The reaction was stopped by adding 0.5 M H2SO4 solution. The absorbance was measured immediately at 450 nm. Every batch of FPLC purified BN-WNDIII protein was tested by ELISA to ensure that the protein is biotinylated.
Biotinylated Protein Binding Assay.
The binding affinity of purified biotinylated WNDIII protein was tested using streptavidin magnetic beads (GE Healthcare, UK) according to manufacturer's protocol. Briefly, samples were mixed with streptavidin magnetic beads and incubated for 30 min with gentle mixing. Unbound proteins were removed with wash buffer while biotinylated proteins were eluted out with elution buffer provided in the kit. Eluted proteins were analyzed by Western blot and ELISA.
Selex procedure for aptamer designing: Apta Biosciences Pte Ltd, (Adaptamer Biosolutions) www.aptabiosciences.com, 31 Biopolis Way, #02-25 Nanos, Singapore 138669, Phone: +65-3109-0178, Fax: +65-6779-6584, Mobile+65-9184-7323) formerly known as Fujitsu Biolaboratories. Bio-laboratories, R&D Division, (Fujitsu Asia Pte Ltd, Fujitsu Laboratories Ltd., Nanotechnology Business Creation Initiative, 31 Biopolis Way, #02-25 Nanos, Singapore).
Aptamer designing and synthesis: Fujitsu, Biolaboratories, Singapore.
Surface Plasma Resonance (SPR) Analysis using BN-WNDIII protein: Fujitsu (
SPR analysis using WNDIII for affinity calculation: Fujitsu (Table 2)
Ten aptamers received from Fujitsu for evaluation are as follows:
Non-Biotinylated aptamers: N03, N66, N67, N71, N73, N74, N76, N79, N97, N99
Biotinylated aptamers: B03, B66, B67, B71, B73, B74, B76, B79, B97, B99
Enzyme Linked Modified Aptamer Sorbent Assay (ELMASA) for Surface Screening.
The modified aptamers consist of amino acid side-chains incorporated into the DNA backbone in order to enhance the binding of the aptamer molecule to the target protein. In order to select the suitable surface for the analysis of the modified aptamer, four different ELISA surfaces were tested. (Nunc-Multisorp, Polysorp, Medisorp and Maxisorp). Briefly, 50 ng of biotinylated aptamer and different concentrations of BN-WNDIII proteins were added to each well and incubated at 4° C. overnight. Blocking with 4% BSA was carried out after overnight incubation, followed by washing with PBS. Then, 1:2000 dilution of streptavidin-HRP enzyme conjugates was added and incubated for 1 hr. The plate was washed 6 times with 1×PBST to remove unbound conjugates. Then, tetramethyl benzidine (TMB) substrate solution was added for development and incubated for 15 min at room temperature. 0.5 M H2SO4 solution was added to stop the reaction. The absorbance was measured immediately at 450 nm.
Protein Coated Enzyme Linked Modified Aptamer Sorbent Assay for Affinity Screening.
100 ng of purified non-biotinylated WNDIII protein was coated on maxisorp plate overnight at 4° C. Following the coating, the ELISA plate was washed three times with PBS and incubated for 1 hour with different concentrations (1.65 nM to 26 nM/well) of biotinylated aptamers solubilized in RNase free TE buffer (Invitrogen). Then, 1:2000 dilution of streptavidin-HRP enzyme conjugates was added and incubated for 1 hr following the standard procedure as mentioned above.
Virus Coated Enzyme Linked Modified Aptamer Sorbent Assay.
Instead of using DIII protein, West Nile virus Wengler strain was coated onto the ELISA plate. Briefly, 1000 PFU of virus was coated in each well followed by overnight incubation at 4° C. The wells were washed with 1× PBST followed by blocking with 4% BSA. Following this step, the wells were incubated with different concentrations (0.3 nM to 26 nM/well) of biotinylated aptamers (1-10) for 1 hr. Then, 1:2000 dilution of streptavidin-HRP enzyme conjugates was added and incubated for 1 hr following the standard procedure as mentioned earlier. Coating, Washing, aptamer addition and developing were carried out in the BSC class 2.
Plaque Reduction Neutralization Test (PRNT).
Baby hamster kidney (BHK) cells were seeded in a 24-well plate overnight before use. Frozen virus stocks were carefully thawed and diluted to 1000 PFU/ml. To 50 PFU/50 μl West Nile virus Wengler strain, various concentrations (1.25 nM, 2.5 nM, 5 nM, 10 nM, 20 nM, 40 nM, 80 nM, 165 nM, 330 nM, 660 nM, 13.33 μM, 5 μM and 10 μM/well) of non biotinylated aptamers were added in duplicates and allowed to incubate for 1.5 hrs for binding. Cell growth medium was removed from the 24-well plate, the cell monolayers briefly washed with 2% RPMI and then infected with 100 μl of the aptamer+virus incubated mixture. The plate was incubated at 37° C. and 5% CO2 for 1 hr with constant rocking of the plate at every 15 min interval. The inoculum was aspirated, briefly washed with 2% RPMI and each well overlaid with 1 ml overlay medium. The plate was incubated at 37° C. and 5% CO2 for 4.5 days until plaques were formed. The cell monolayer was stained with a solution of 0.1% crystal violet in PBS for 24 hrs. The crystal violet solution was removed, the plates washed in distilled water and plaques were counted.
Aptamer Stability Assay:
Stability of the aptamers was tested by incubating a fixed concentration (400 ng/ml) of aptamer at three different temperatures (−20° C., room temperature and 37° C.) for 1 to 5 days. After each time point, the integrity of the aptamers was analysed by running a 1.5% agarose gel which was premixed with GEL-RED. The sample was ran 40 V for 4 hr and viewed on a Gel-doc under ultraviolet (UV) light.
ApoTox-Glo Triple Assay.
The assay was performed using ApoTox-Glo Triple Assay kit (Promega) and readings were taken using Glomax Instrument. Briefly, BHK cells were seeded in a 96-well assay plate with cell density of 5000 cells/well (5000 cells/0.1 ml) and cultured overnight. After 24 hrs, cells were treated with aptamers (3.3 to 26 nM concentration/well) and positive controls for cytotoxicity (digitonin detergent, MPER, membrane protein extraction reagent). At day 1 and day 4, the cells were incubated with 20 μl of Viability/Cytotoxicity Reagent. The plate was briefly mixed by orbital shaking at 300 rpm for 30 seconds and incubated at 37° C. for 30 min. Fluorescence was measured at two wavelength sets, 400Ex/505Em (Viability) and 485Ex/520Em (Cytotoxicity). For luminescence reading, the plate was inoculated with 100 μl of Capase-Glo 3/7 Reagent in each well. The plate was briefly mixed by orbital shaking at 300 rpm for 30 sec and incubated at room temperature for 30 min.
Alamar Blue Viability Assay.
The assay was performed using alamarBlue Cell Viability Assay (Invitrogen) and readings were taken using Glomax Instrument. BHK cells were seeded in a 96-well assay plate with cell density of 5000 cells/well (5000 cells/0.1 ml) and cultured overnight. After 24 hrs, cells were treated with aptamers (3.3 to 26 nM concentration), and positive controls for cytotoxicity (digitonin detergent, MPER, membrane protein extraction reagent). At day 1, 2, 3 and 4, the cells were incubated with 10 μl of alamar Blue reagent. The plate was briefly mixed by orbital shaking at 300 rpm for 30 sec and incubated at 37° C. for 1-4 hrs, protected from direct light. Fluorescence of the plate was measured at 570Ex/585Em.
Determination of Stability of the Modified Aptamers in Serum by ELISA Method:
Known amount (40 ng/well) of biotinylated aptamers were coated on the Maxisorp plate and incubated at RT for 2 hours. Then the plates were incubated with and without 100% and 20% serum for varying time points (1, 20, 48 and 120 hours). Positive controls (Just aptamer) were incubated with 4% Bovine serum albumin (BSA). At the end of each time point the serum and BSA were removed. Streptavidin-HRP enzyme conjugates (1:5000 dilution) was added and incubated for 1 hr. The plate was washed 6 times with 1×PBST to remove unbound conjugates. Then, tetramethyl benzidine (TMB) substrate solution was added for development and incubated for 15 min at room temperature. 0.5 M H2SO4 solution was added to stop the reaction. The absorbance was measured immediately at 450 nm. As a negative control the aptamer (303) was boiled at 95° C. for 48 hours. If the aptamer is degraded by the serum or heating, then the aptamer will not be detected by the streptavidin-HRP.
To obtain the biotinylated protein of West Nile virus envelope protein domain III (WNE-BNrDIII) for aptamer screening, a new plasmid construct was designed by engineering in the biotinylation acceptor peptide (BAP) on the N-terminus, and an enterokinase cleavage site between the BAP and the WNDIII gene. The DNA sequence corresponding to the BAP was chemically synthesized (Cull et al., 2000), whereas the WNDIII sequence was obtained from the previous construct, which was derived from the cDNA of WNV Wengler strain. Later, the BAP sequence with the enterokinase cleavage site was linked to WNDIII at the 5′ end through overlapping extension PCR (OE-PCR) as illustrated in
To express the recombinant protein, the engineered plasmid was transformed into a commercial E. coli strain AVB-100 obtained from Genecopoeia. The AVB 100 E. coli strain has been incorporated with an overexpressing BR A (Biotin ligase) gene within the genomic DNA. This enzyme specifically adds a biotin molecule to the lysine residue of the BAP. Initially, the protein (BAP-WNDIII) of interest was not expressed in E. coli K12 AVB-100 (
As the attempt to express the construct in K12 Strain AVB100 was unsuccessful, in vitro biotinylation using Bir-A enzyme was carried out. In vitro biotinylated WNDIII was tested using ELISA, and the result shows absorbance at 450 nm, indicating that the recombinant protein was biotinylated and binds to streptavidin-HRP conjugate in both experimental conditions (1 hr and overnight reaction set up). Interestingly, the control experiment, i.e. the sample without Bir A enzyme, also showed high absorbance at 450 nm, indicating that it also binds to the streptavidin-HRP conjugate (
After it has been proved that the BAP containing WNDIII protein was endogenously in vivo biotinylated during the expression of the protein itself, there was an interest to understand how the biotinylation could have taken place endogenously, and where is the source for the biotin in the cell for the biotinylation. A bioinformatics search for the Bir A enzyme in the genomic DNA sequence of E. coli BL 21(DE3) was carried out and it was discovered that the gene encoding Bir A was found in the E. coli strain, which have been used for expression. In addition, biotin has been found to be present in the medium, which has been used to cultivate the bacterial cells (Tolaymat et al., 1989). Thus, the protein is endogenously biotinylated by the Biotin ligase enzyme already present in the cell, utilizing the biotin in the culture medium. Therefore, attaching a BAP to a gene-of-interest and expressing it in E. coli BL 21 (DE3) will result in the production of biotinylated protein endogenously, hence eliminating the need for a commercial expression strain or in vitro biotinylation. Thus, a platform to obtain endogenously biotinylated, purified protein for biological applications, like aptamer screening, has been established. Every batch of purified protein for biotinylation was checked and was found to be consistent. It was also tested to determine whether endogenous biotinylation is universal for other proteins by cloning the BAP for dengue virus capsid protein and it was confirmed that the capsid protein was found to be endogenously biotinylated. This showed that this platform can be potentially extended to other biotinylated proteins, which have commercial applications in diagnostics and drug development. This has been filed as a provisional patent by Exploit Technologies (Singapore Patent Application No. 201208602-1, Entitled: Biotinylated Protein, Filing Date: 22 Nov. 2012, contents of which are incorporated herein by reference).
In order to test the binding efficiency of aptamers, suitability of the four different surfaces were tested by coating with 50 ng biotinylated modified aptamers (1 to 10) followed by detection with streptavidin-HRP conjugate. Similarly, varying concentrations of biotinylated WNDIII (10, 25, 50 and 100 ng/well) protein was also coated. The results are shown in the
In order to evaluate specific binding of aptamers to WNDIII protein, protein-coated ELISA was carried out for the ten aptamers. WNDIII protein (100 ng/well) was coated overnight and incubated with biotinylated aptamers of various concentrations (0 to 26 nM), followed by probing with streptavidin-HRP conjugate. If an aptamer were to bind to the WNDIII protein, it would be detected through the enzyme substrate reaction. In this case, it was observed that aptamers B03, B79 and B99 bound to the WNDIII protein as their absorbance were significantly higher when compared to the control and the other aptamers (
Once it had been confirmed that a modified aptamer was able to bind to purified WNDIII protein, it was evaluated whether the aptamer could bind to the West Nile envelope protein if the whole virus was coated. West Nile virus Wengler strain (1000 PFU/well) was coated in the ELISA plate overnight, followed by incubating with different concentrations of aptamers. It was still observed that the aptamers B03, B79 and B99 bind specifically to domain III in the native envelope protein present on the virus (
As it has been established that the modified aptamers were able to bind to purified WNDIII and native DIII in the envelope protein of wildtype West Nile virus, the ability of the aptamers to neutralize WNV was then tested. The virus was incubated with different concentrations of aptamers followed by infecting BHK cells with the aptamer-treated or untreated virus. Both the treated and untreated virus were removed after an hour. The plate was stained on day 4 after the infection and formation of plaques were observed. In the lower concentrations of aptamer treatment, there was no neutralizing activity. There was visible reduction in the number of plaques in the 5 μM and 10 μM aptamer treatment.
As the possibility for aptamers to be developed for therapeutics is very high, it was tested whether treating mammalian cells with the modified aptamers causes cytotoxicity to the cells. In order to check the outcome of cell viability during aptamer treatment, two different sets of viability experiments were performed. The first involved the use of the apotox-glo triple assay while the second involved the use of the alamar blue viability assay. The cells were treated with different concentrations of aptamers followed by testing the viability at various time points (24, 36, 48 and 60 hours post-treatment). The results obtained by the two methods are shown in
The stability of the aptamers were tested by incubating them at three different temperatures (−20° C., room temperature and 37° C.) for different periods of time (1 to 5 days), followed by checking the integrity of the modified aptamers in a gel-red stained agarose gel.
Testing the stability of the modified aptamers was extended in the presence of serum as a initial step towards the exploring the possibility of these aptamers for therapeutic application. The biotinylated aptamer was coated followed by incubating the human serum for different time points (1, 20, 48 and 120 hours).
The following Example evaluates the stability and functionality of the modified aptamers for WNV DIII in the human and fetal bovine serum. Comparison studies with other modified and unmodified aptamers, and commercially available aptamer and antibody have also been carried out.
In order to test the stability of the modified aptamers by ELISA, biotinylated WNDIII aptamers (B03, B66, B71, B73, B74, B76 and B79 obtained from, Apta Biosciences Pte Ltd www.aptabiosciences.com, 31 Biopolis Way, #02-25 Nanos, Singapore 138669, Phone: +65-3109-0178, Fax: +65-6779-6584, Mobile: +65-9184-7323) formerly known as Fujitsu Biolaboratories) were coated on a maxisorp plate (40 ng/well) followed by incubation with human serum for different durations. If the aptamer was unstable, it would degrade and be removed during washing. Otherwise, the stable modified aptamer would remain bound to the maxisorp plate. The presence of the biotinylated aptamer would then be detected by a streptavidin-HRP conjugate, thereby resulting in TMB substrate conversion. The serum stability of the modified aptamers was monitored for up to 14 days, and was found to vary between 50% and 90% when compared to their respective serum-free controls as shown in
Based on results from the stability studies of aptamers in human serum, the modified aptamers could be classified into Type 1: Moderately stable (B74), Type 2: Highly stable (B03, B66, B71, B73, B76 and Type 3: Very highly stable—(B79). This implied that the backbone of modified aptamer B79 can be used as the starting template to generate highly stable aptamers in the future. Although modified aptamer B79 was shown to have the highest stability, as can be seen from
Comparison on the stability of modified aptamer B03 in fetal bovine serum (FBS) for 5 days was also made.
Using ELISA as the platform, maxisorp plates were coated with either WNV DIII protein or WNV. Different concentrations of biotinylated WNV DIII modified aptamer B03 was then added and incubated for 2 hours to allow the modified aptamer to bind to the target. Neat human serum or FBS was subsequently added and incubated for different durations. After incubation, the presence of modified aptamers was probed with streptavidin-HRP conjugate, followed by TMB substrate development.
Polynucleotides corresponding to the DNA backbone of the WNV DIII modified aptamers B03 and B99 (i.e. unmodified aptamers) were synthesized (Sigma Aldrich, USA) for comparison with the modified aptamers (which have peptide side chains) in terms of stability and functionality. The nucleotide sequences corresponding to the DNA backbone of the WNV DIII modified aptamers B03 and B99 are listed below.
For the stability comparison study, known amounts of unmodified DNA aptamers were incubated at room temperature (RT) for varying durations in human serum or FBS. Their stability was then determined through detection using streptavidin-HRP conjugate in ELISA.
Based on the stability study as shown in
Comparison of Aptamer Binding with WNV DIII Commercial Antibody
Using the ELISA platform, the same concentration (33 nM) of aptamers (B03, B79, B99, B66, B67, B71) and WNV-specific antibody (Millipore MAB8151) were coated onto a maxisorp plate to capture biotinylated WNV DIII protein.
The following Example evaluates the binding characteristics of a separate set of selected modified aptamers (generated by Adaptamer Solutions, www.aptabiosciences.com, Apta Biosciences Pte Ltd, 31 Biopolis Way, #02-25 Nanos, Singapore 138669, Phone: +65-3109-0178, Fax: +65-6779-6584, Mobile: +65-9184-7323) against purified DENV2 DIII protein and the native envelope protein on wildtype DENV. The best aptamer which can be utilized for diagnostic and therapeutic applications was then identified. Ten potential aptamer candidates against DENV2 DIII protein were evaluated and the results are also discussed.
Overlapping Extension-Polymerase Chain Reaction (OE-PCR).
Two fragments were used in the cloning of DENV1-4 BN-rEDIII protein. The biotin acceptor peptide (BAP) (Fragment 1) was synthesized chemically. Domain III of the envelope glycoprotein (Fragment 2) of each DENV serotypes was derived from the cDNA of DENV1-4, respectively.
Protein Expression and Extraction.
pET28a-DENV2 BN-rEDIII plasmid was transformed into BL-21-DE3 expression competent cells (Agilent Technologies, USA) and grown in Luria-Bertani (LB) agar containing 30 μg/ml kanamycin. Selected clones were cultured in 1 L LB broth (30 μg/ml kanamycin) at 30° C. until an OD600 of 0.6. Expression of DENV2 BN-rEDIII protein was induced with 1 mM isopropyl β-D-thiogalactoside (IPTG) for 6 hours. Bacterial cells were pelleted down with centrifugation at 8,000 rpm for 15 min at 4° C. The protein expressed was targeted to inclusion bodies (IB). IBs were isolated in the subsequent steps. The bacterial cell pellet was first resuspended in lysis buffer (20 mM Tris pH 8.0, 500 mM NaCl, 10 mM imidazole), followed by sonication in ice bath (10 min, 10 Amp). The lysate was then centrifuged at 12,000 rpm for 15 min at 4° C. to obtain a small white translucent pellet of inclusion body. The inclusion body pellet was then washed with the same lysis buffer, incubated in extraction buffer (8 M urea, 20 mM Tris, 300 mM NaCl, 10 mM imidazole, pH 8.0) at room temperature for 30 min, and its extract clarified by centrifugation at 13,500 rpm for 20 min.
Immobilised Metal Ion Affinity Chromatography (IMAC) Purification of BN-rEDIII Protein.
The inclusion body extract containing DENV2 BN-rEDIII protein was incubated with nickel-nitrilotriacetic acid (Ni-NTA) resin (Bio-Rad, USA) for binding in a chromatography column overnight at 4° C. Five column volume of wash buffer (8 M urea, 20 mM Tris, 300 mM NaCl, 20 mM imidazole, pH 8.0) was used to remove non-specific binding proteins. BN-D2DIII protein was then eluted out with elution buffer (8 M urea, 20 mM Tris, 300 mM NaCl, 500 mM Imidazole, pH 8.0) in eight 1.5-ml fractions. All the eluates were pooled into a SnakeSkin dialysis membrane tubing (Thermo Scientific, USA) and 0.05% of Tween-20 was added to the samples. The dialysis tubing was incubated in 4 M urea for 6-12 hrs at 4° C., and the urea diluted stepwise to 0.5M. The refolded DENV2 BN-rEDIII protein was finally collected from the dialysis tubing and injected into a FPLC machine to be further purified via size-exclusion chromatography into PBS. DENV1, 3 and 4 BN-rEDIII proteins were also purified in a similar manner.
Protein Identity Analysis.
The flow through, wash, and eluates from the IMAC purification were analyzed by SDS-PAGE and Western blot. 12% Tris-tricine polyacrylamide denaturing gel was used to separate proteins and was subsequently stained with Coomassie blue for protein detection. For Western blotting, proteins were transferred from the polyacrylamide gel onto a PVDF membrane using iBlot® Dry Blotting System (Life Technologies, USA). Blocking was done with 5% BSA overnight in 4° C. The membrane was then incubated with streptavidin conjugated-HRP to detect for DENV BN-rEDIII for 2 hours at room temperature. The membrane was washed thoroughly with 1×PBST for 1 hour at room temperature and developed with SuperSignal® West Pico chemiluminescent substrate (Thermo Scientific, USA). A schematic flowchart representing the expression, purification and evaluation of recombinant purified DENV1-4 BN-rEDIII proteins is shown in
Protein-Coated Enzyme-Linked Modified Aptamer Sorbent Assay (ELMASA) for Affinity screening.
100 ng of purified non-biotinylated DENV2 rEDIII protein was coated onto each well of a maxisorp plate overnight at 4° C. On the following day, the ELISA plate was washed three times with Phosphate-buffered saline (PBS) and incubated for 1 hour with different concentrations (1 to 32 nM/well) of biotinylated (DENV) aptamers solubilized in RNase free TE buffer (Invitrogen) in triplicates. Blocking with 4% BSA in PBS was then carried out overnight, followed by washing with PBS. 1:2000 (v/v) dilution of streptavidin-HRP enzyme conjugate (Millipore) was subsequently added and the plate was incubated for 1 hour. The plate was washed 6 times with 1×PBST, before 50 μl of tetramethyl benzidine (TMB) substrate solution was added and incubated for 15 min at room temperature. Finally, 50 μl of 0.5 M H2SO4 solution was added to stop the reaction and absorbance was measured immediately at 450 nm.
Virus-Coated ELMASA.
Instead of using DENV2 rEDIII protein, 1,000 PFU of DENV2 wildtype virus was coated onto the ELISA plate and incubated overnight at 4° C. The wells were washed with 1×PBST followed by blocking with 4% BSA. Following this step, the wells were incubated with different concentrations (1 to 32 nM) of biotinylated aptamers (1-10) for 1 hour. 1:2000 (v/v) dilution of streptavidin-HRP enzyme conjugate was then added and the rest of the experiment was performed as described in the protein-coated ELMASA above. All procedures were carried out in a class 2 Biological Safety Cabinet (BSC).
Virus Blocking Assay.
BHK cells were seeded in a 24-well plate overnight at 50000 cells/well. 50 μl of 2 μM aptamers solubilized in RNase-free TE buffer (Invitrogen) were added to 50 PFU/50 μl DENV2 in triplicates. The mixture was incubated for 1.5 hrs for binding (final aptamer working concentration is 1 μM/well). A negative control was set up similarly without any virus. Following which, growth medium was removed from the 24-well plate, and the cell monolayer in each well was washed with RPMI containing 2% FCS and infected with the 100 μl of aptamer-virus mixture. The plate was incubated at 37° C. and 5% CO2 for 1 hour, with constant rocking at 15-min interval. The inoculum was removed, the cell monolayer washed with RPMI containing 2% FCS, and 1 ml of CMC overlay medium wad added to each well. The plate was incubated at 37° C. and 5% CO2 for 4.5 days until plaques were formed. The remaining cells were finally stained with crystal violet and the unstained plaques were counted.
To obtain DENV1-4 BN-rEDIII proteins for aptamer screening, new expression plasmids were designed by engineering in a biotinylation acceptor peptide (BAP), followed by an enterokinase cleavage site, at the N-terminus of the DENV1-4 envelope DIII gene. The DNA sequence corresponding to the BAP was chemically synthesized (Kaur et al., 2013), whereas the DENV1-4 envelope DIII DNA sequences were derived from the cDNA of DENV1-4, respectively. The BAP sequence with the enterokinase cleavage site was linked upstream of DIII through overlapping extension PCR (OE-PCR) as illustrated in
The DENV1-4 BN-rEDIII proteins were expressed in E. coli BL21 (DE3). After DENV1-4 BN-rEDIII protein expression was confirmed via Western blotting using an anti-His antibody, expression was scaled up to produce large amounts of DENV1-4 BN-rEDIII proteins. Crude protein was extracted from the inclusion bodies and subjected to IMAC affinity purification, refolding, and size exclusion chromatography as explained in Materials and Methods. The representative FPLC-SEC profile for DENV2 BN-rEDIII protein is shown in
DENV2 BN-rEDIII protein immobilized on monomeric avidin-agarose resin was incubated with a library solution of modified aptamers. The resin was then washed repeatedly to remove weakly bound modified aptamers before the modified aptamer: DENV2 BN-rEDIII complexes were eluted from the resin using a biotin solution. The eluted complexes were treated with alkali to remove the side chains and liberate the DNA aptamer backbone for PCR, sequencing, and subsequent cloning to allow determination of the DNA sequence of the bound aptamers. DNA sequences of 136 DENV2 BN-rEDIII modified aptamer candidates were obtained and these modified aptamers were synthesized by a DNA synthesizer. Screening of DENV2 BN-rEDIII modified aptamer candidates was repeated by applying them to DENV2 BN-rEDIII protein immobilized on a CM5 Biacore sensor chip by amine-coupling. The top 10 DENV2 BN-rEDIII modified aptamer candidates were selected for further analysis.
SPR Analysis Using DENV2 rEDIII Protein:
For KD measurement, each of the ten DENV2 BN-rEDIII modified aptamer candidates was biotinylated and immobilized on a Biacore SA chip separately. Their individual KD was determined for various concentrations of DENV2 rEDIII protein in MES buffer at pH 5.5 (see
In order to evaluate the binding of the 10 selected modified aptamers to DENV2 rEDIII protein, DENV2 rEDIII protein coated ELMASA was carried out using biotinylated modified aptamers of various concentrations (0 to 32 nM). It was observed that modified aptamers B002, B006, B027 and B128 bound most efficiently to DENV2 rEDIII protein although modified aptamers B012, B060, B113, B118 and B121 also bound significantly to the DENV2 rEDIII proteins at all concentrations tested. The binding of the modified aptamers against rEDIII protein of DENV1, 3 and 4 were evaluated, and the results were shown in
Binding of the modified aptamers to purified DENV2 rEDIII protein was further confirmed using wildtype virus. DENV2 (1000 PFU/well) was coated on the ELISA plate overnight, followed by incubation with different concentrations of aptamers. It was still observed that modified aptamers B060, B118, B121 and B128 bound significantly to DENV2 as compared with the control (
After establishing that the modified aptamers were able to bind to purified DENV2 rEDIII protein and native envelope DIII protein on wildtype DENV2, their ability to neutralize DENV2 was evaluated. Prior incubation of viruses with different concentrations of modified aptamers, followed by infection of BM cells was carried out. There was a reduction in the number of virus-induced plaques when DENV2 was pretreated with 1 μM of modified aptamer. The results showed that pretreatment with 1 μM of modified aptamers B060 and B118 resulted in more than 60% neutralization, whereas neutralization by the other modified aptamers varied between 40% and 58%. Thus, modified aptamers B060 and B118 had the potential to be developed into therapeutics against DENV2.
Cross Reactivity of DENV2 DIII Modified Aptamers with Other Flavivirus Envelope Protein:
In order to evaluate potential non-specific and cross-reactive binding of the modified aptamers to other flavivirus envelope protein, protein coated ELMASA was performed using the envelope or DIII proteins of West Nile virus (WNV), tick-borne encephalitis virus (TBEV) (ProSpecbio, USA) and Japanese Encephalitis Virus (JEV) (ProSpecbio, USA). No significant binding to the envelope or DIII proteins of all three viruses above was detected at all the modified aptamer concentrations tested (see
TBE-281:
Tick-borne encephalitis is caused by tick-borne encephalitis virus (TBEV), a member of the virus family Flaviviridae. TBE-281 is the E. coli derived recombinant protein comprising residues 95 to 229 of the Tick-borne Encephalitis Virus envelope glycoprotein.
JEV-290:
Japanese encephalitis previously known as Japanese B encephalitis is a virus from the virus family Flaviviridae. It is closely related to WNV and St. Louis encephalitis virus. JEV-290 protein is the 50-kDa full length Japanese Encephalitis virus envelope protein expressed in E. coli and is fused to a 6× histidine tag.
Comparison of Binding for the DENV2 DIII Modified Aptamers of the Present Invention and Other Commercial Aptamer to DENV2 rEDIII Protein.
The functionality of the DENV2 DIII modified aptamers of the present invention was compared to that of commercially available aptamers against DENV2 DIII (D2A) (OTC Biotech, USA). The commercial aptamer was evaluated in a similar manner as the DENV2 DIII modified aptamers. As illustrated in
All references herein mentioned are hereby incorporated by reference.
| Number | Date | Country | Kind |
|---|---|---|---|
| 201308420-7 | Nov 2013 | SG | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/SG2014/000532 | 11/13/2014 | WO | 00 |
