Dengue-specific subviral nanoparticles: design, creation and characterization
- Niyati Khetarpal†1,
- Ankur Poddar†1,
- Satish K Nemani†1, 2,
- Nisha Dhar1,
- Aravind Patil1,
- Priyanka Negi1,
- Ashiya Perween1,
- Ramaswamy Viswanathan1,
- Heinrich Lünsdorf3,
- Poornima Tyagi1,
- Rajendra Raut1,
- Upasana Arora1,
- Swatantra K Jain4,
- Ursula Rinas2, 3,
- Sathyamangalam Swaminathan1Email author and
- Navin Khanna1Email author
© Khetarpal et al.; licensee BioMed Central Ltd. 2013
Received: 7 March 2013
Accepted: 23 May 2013
Published: 25 May 2013
Dengue is today the most significant of arboviral diseases. Novel tools are necessary to effectively address the problem of dengue. Virus-like particles (VLP) offer a versatile nanoscale platform for developing tools with potential biomedical applications. From the perspective of a potentially useful dengue-specific tool, the dengue virus envelope protein domain III (EDIII), endowed with serotype-specificity, host receptor recognition and the capacity to elicit virus-neutralizing antibodies, is an attractive candidate.
We have developed a strategy to co-express and co-purify Hepatitis B virus surface (S) antigen in two forms: independently and as a fusion with EDIII. We characterized these physically and functionally.
The two forms of the S antigen associate into VLPs. The ability of these to display EDIII in a functionally accessible manner is dependent upon the relative levels of the two forms of the S antigen. Mosaic VLPs containing the fused and un-fused components in 1:4 ratio displayed maximal functional competence.
VLPs armed with EDIII may be potentially useful in diagnostic, therapeutic and prophylactic applications.
KeywordsDengue envelope domain III Hepatitis B surface antigen Virus-like particle Bionanoparticles Pichia pastoris
Currently, dengue represents the most important arboviral disease that places nearly half the global population at risk . The mosquito-borne disease is caused by four closely related, yet antigenically distinct, serotypes of dengue viruses (DENV-1, -2, -3 and −4) . All four DENVs and their mosquito vectors are co-prevalent in more than one hundred tropical/sub-tropical countries. Each of the DENVs can cause disease ranging from mild dengue fever to severe dengue hemorrhagic fever and potentially fatal dengue shock syndrome . Tools for diagnosing, treating and ultimately preventing dengue are urgently needed . While increasingly reliable diagnostic tools are becoming available [4–6], antivirals [4, 7, 8] and vaccines [9, 10] for dengue continue to be elusive.
Nanobiotechnology, which seeks to use naturally occurring as well as engineered nanoscale biomaterials to make functional systems, is rapidly emerging as a platform for the development of novel nanotools with potential biomedical applications [11–13]. We are interested in exploring potential applications of this technology to infectious diseases. We have utilized Eu3+-doped polystyrene nanoparticles as very sensitive reporters for detecting Hepatitis B virus  and human immunodeficiency virus  infections. We have also used nanoparticles of biological origin, namely, virus-like particles (VLPs). Many viral capsid proteins possess intrinsic ability to self-assemble into VLPs when expressed in recombinant insect, yeast and mammalian host systems . Recently, we have begun exploring VLP platforms for the display of foreign antigens . The foreign antigen we focus on is derived from the major DENV structural antigen on the virion surface, the envelope protein. Multiple antigenic determinants that are largely serotype-specific map to a C-terminal ~100 amino acid (aa) region of this protein . Further these antigenic determinants tend to elicit DENV-neutralizing antibodies and coincidentally, this C-terminal region, which is known as envelope domain III (EDIII), is also implicated in host receptor recognition . For these reasons, we believe EDIII offers an attractive precursor for developing nanoparticle tools that may be useful in addressing the problem of dengue.
Recombinant Hepatitis B virus surface (S) antigen is well-documented to self-assemble into 20–22 nm VLPs [19–21] and is the main component of commercial Hepatitis B vaccines [22, 23]. In this study, we sought to exploit the S antigen VLP as a carrier for DENV-2 EDIII. To this end we fused EDIII to the amino-terminus of S to create a fusion antigen, herein referred to as ES antigen. We used the methylotrophic yeast Pichia pastoris, which we have found earlier to express S antigen VLPs to very high levels , to express the ES antigen. We found that for DENV-2 EDIII to be displayed on the VLP surface in way that made it accessible to DENV-2-specific antibody and to the host cell receptor, it was necessary to co-express the ES fusion antigen with un-fused S antigen. We describe our strategy to co-express ES and S antigens in P. pastoris, their co-purification and structural as well as functional characterization of the resultant mosaic ES,S VLPs.
Strategy to co-express ES fusion antigen gene in the background of 0–4 copies of S gene
Creation of P. pastoris clones designed to co-express ES,S0-4 antigens
The ES,S0-4 antigens are predominantly associated with the P fraction of induced cell lysates
Co-purification of the ES and S antigens from induced P. pastoris cells
As a prelude to purification, we sought to optimize the conditions of induction. To this end, methanol concentration and induction duration were varied with respect to each other, followed by analysis of ES antigen in the P fractions by immunoblotting and ELISA. Based on these results (Additional file 1: Figure S3), we carried out induction at 1% methanol for 3 days. As the ES antigen was predominantly associated with the P fraction, we decided to adapt a protocol recently developed in our laboratory that exploits a similar behaviour of the S antigen for its purification . Thus, the P fraction from induced cells which served as the starting material was solubilized in the presence of detergent and urea, subjected to polyethylene glycol 6000 (PEG 6000) precipitation, tangential flow filtration (TFF) using a 300 kDa cut-off membrane, and finally chromatography on Phenyl Sepharose. The peak fractions were pooled and aliquots analysed by silver stained SDS-PAGE as shown in Figure 3B. The data reveal that both the ES and S antigens expressed by each of the ES,S1-4 clones co-purified through the multistep purification protocol. A densitometric scan revealed >95% purity for each. That the co-purified proteins were indeed ES and S was confirmed by their identification in a Western blot analysis using mAbs 5S (Figure 3C) and 24A12 (data not shown). Importantly, both the silver stained gel and the immunoblot analyses revealed the copy number effect in S antigen levels. To explore if purification could be achieved in the absence of urea, we used an alternate protocol in which the starting material was the soluble fraction obtained from total cell lysate prepared in the presence of 0.6% Tween 20 . This resulted in low yields (data not shown), commensurate with the low concentrations in the S fractions (Additional file 1: Figure S2).
Co-expressed ES and S antigens form VLPs
S antigen content of VLPs contributes to optimal EDIII accessibility
Taken together, the competitive ELISA and virus binding-blocking experiments support the notion that mosaic VLPs containing the ES and S antigens in 1:4 ratios display EDIII on the surface in a manner that is compatible with its optimal interaction with antibodies and the host cell receptors.
The magnitude of dengue as a public health problem is accentuated by the lack of drugs and vaccines [3, 4, 9]. The emergence of nanotechnology and its increased recent focus on bionanomaterials has opened up new interdisciplinary avenues exploring novel biomedical applications [11–13]. Our interest is in exploring this area with the ultimate goal of developing nanotools that could help address the dengue problem. In this context, our interest is in the use of bionanomaterials which have the intrinsic advantages of biocompatibility and biodegradability . In contrast to naturally occurring viral nanoparticles, which are biohazardous, their genome-free counterparts, the VLPs, are being increasingly preferred for developing biomedical nanotools [11–13].
The S antigen of Hepatitis B represents the classic example of a viral protein which can independently assemble into nanoparticles [19–21] and is the basis of a highly successful VLP vaccine [22, 23]. We have sought to endow DENV specificity to these S VLPs, as a first step towards developing bionanoparticles with potential dengue-specific applications. To confer DENV specificity we chose a ~100 aa domain known as EDIII, for reasons mentioned already, and genetically linked it to the N-terminus of the S antigen, and expressed it using P. pastoris. We observed that the resultant ES antigen did not form VLPs efficiently. Moreover, it did not display EDIII optimally based on specific assays to test its functionality. In order to obtain VLPs with functionally competent EDIII, we developed a strategy to co-express un-fused S antigen at calibrated levels using defined S gene copy numbers. The resultant mosaic VLPs contain two components, ES antigen and un-fused S antigen. Both ES and S antigens tend to be membrane associated, by virtue of the latter’s intrinsic hydrophobicity [21, 24, 25]. This necessitated their co-purification starting from the membrane fraction using a protocol we developed recently for S antigen purification . A noteworthy feature of this purification scheme is that it eliminates CsCl centrifugation, a bottleneck in downstream processing. Based on a variety of criteria including co-purification through 300 kDa cut-off TFF, CsCl gradient sedimentation analysis, EM and sandwich ELISA’s, we found that ES and S antigens associate together to form mosaic VLPs. Using competitive ELISA and bind blocking assays, we demonstrated that mosaic VLPs containing 4 copies of S antigen per copy of the ES antigen displayed EDIII moiety optimally.
The data have significant implications from the perspective of dengue-specific nanoparticulate tools. As EDIII can induce virus-neutralizing antibodies [26, 27], the repeat-pattern architecture of the mosaic VLP can potentially augment its vaccine potential. This notion is backed by the fact that vaccines based on the VLP platform are already available for Hepatitis B and human papilloma virus infections . Strategies to load VLPs with drugs and target viral nanoparticles to tumors are being actively investigated . Thus, if one can envisage charging these mosaic VLPs with a drug against dengue, these VLPs through their surface displayed EDIII can be targeted for drug delivery to DENV-susceptible cells through host receptor recognition. For vaccine and drug-delivery applications, the non-replicating, infectious viral genome-free VLPs offer the advantage of in-built safety. Additionally, as the EDIII is useful in serotype identification , these mosaic VLPs could also serve in diagnostic serotyping of DENV infections.
This work shows that DENV EDIII fused to the S antigen, co-expressed with un-fused S antigen forms mosaic VLPs. VLPs containing the fused and un-fused components in 1:4 ratio displayed EDIII optimally. These EDIII-displaying VLPs have potential vaccine, drug-delivery and diagnostic applications. As these VLPs can be produced in P. pastoris, which is capable of high productivity in simple media, they can be expected to be inexpensive, a significant advantage for resource-poor regions where dengue is endemic. Finally, the approach developed here could serve as the basis for a common bionanoparticle platform for other infections as well.
The genes ES (1 kb) and S (0.7 kb), codon-optimized for P. pastoris expression were synthesized by Geneart AG (Regensburg, Germany). DENV-2 (NGC strain) stock was from previously reported work . DENV-2 EDIII-specific monoclonal antibody (mAb) 24A12  and S antigen-specific mAb 5S  were in-house reagents.
A panel of four ES (1 copy) expression vectors, co-expressing 0, 1, 2 and 4 copies of the S antigen was created using a head-to-tail in vitro multimerization method . The ES gene was cloned into the unique Eco RI site of pAO815 to generate the expression plasmid pAO-ES,S0. To provide for co-expression of S antigen, 1, 2 and 4 copies of an S gene expression cassette were inserted sequentially to generate pAO-ES,S1, pAO-ES,S2 and pAO-ES,S4, respectively. Each of the four constructs above was integrated into the genome of P. pastoris (GS115) as described before .
Typically, yeast cultures were grown at 30°C to log phase in buffered glycerol-containing medium (BMGY) and switched to buffered 1% methanol-containing medium (BMMY) for induction for 72 hours. Total (T) lysates for analytical experiments were prepared from methanol-induced cells using glass beads in a detergent-containing buffer as described . A portion of the total lysate was separated by centrifugation into S- and the membrane-enriched P-fractions. Suitable dilutions of T, S and P (urea-solubilized) fractions in urea-free buffer were used for sandwich ELISA and immunoblotting experiments.
For purification, we followed a method reported recently for P. pastoris-expressed S antigen purification starting from the membrane fraction  with some modifications as follows. Induced biomass (100 grams wet weight) was lysed with glass beads (5 cycles) in a Dyno-mill (WAB, Muttenz, Switzerland) and centrifuged to separate out the membrane-enriched P fraction, which was solubilized in a buffer containing 4 M urea and 2% Tween 20. The solubilized P fraction was clarified and subjected to 5% PEG 6000 precipitation overnight. The post PEG supernatant was clarified by centrifugation and 0.45 μ filtration and subjected to TFF across a 300 kDa membrane using 12 L of TFF buffer with step-wise reduction in urea (4 M to 0 M). The TFF retentate was chromatographed on Phenyl 600 M Toyopearl resin (Tosoh Bioscience, Stuttgart, Germany). Bound proteins were eluted using a 0-8 M urea step gradient (with 2 M increase at each step lasting 5 bed volumes) in 20 mM sodium bicarbonate buffer (pH 9.6). Column fractions were analysed by SDS-PAGE, purified peak fractions pooled, and dialyzed against 1× PBS. In some instances, we also carried out purification from the S fraction in the absence of urea as described .
Proteins were characterized by ELISA, immunoblotting, CsCl gradient centrifugation and EM. ES protein was detected by sandwich ELISA using two formats. In one case, DENV-2 EDIII-specific mAb 24A12 was used for antigen capture, followed by revealing it with an S antigen-specific mAb-enzyme conjugate. In the second case, both capture and reveal mAbs were S antigen-specific (from Hepanostika Ultra kit, Biomerieux, Marcy L’Etoile, France). Immunoblot analyses were performed essentially as reported earlier [16, 21] using either mAb 24A12 or 5S mAb. Densitometric analysis of the resultant blots was performed using freely available image analysis software from NIH (ImageJ software). CsCl gradient fractionation was performed as described previously . The presence of VLPs in the purified preparations was visualized by EM as before .
The functional status of EDIII moiety on the VLPs was assessed by its ability to (i) compete for binding to a specific mAb and (ii) inhibit DENV-2 binding to host cell surface receptors. Competitive ELISA was done essentially as reported  using mAb 24A12. Binding blocking assay was performed using Vero cells (American Type Culture Collection, Virginia, USA). Briefly, cells were pre-exposed separately to each of the VLP preparations for 1 hour followed by infection with DENV-2. Culture supernatants were sampled at daily intervals for release of viral NS1 antigen using Dengue NS1 Platelia kit (BioRad Inc., USA) as per the manufacturer’s directions .
Alcohol oxidase 1
Envelope domain III
EDIII of DENV-2 fused to Hepatitis B virus surface antigen
S0-4: ES antigen co-expressed with 0–4 copies of Hepatitis B virus surface antigen
Surface antigen of Hepatitis B virus
- S fraction:
- P fraction:
Tangential flow filtration
NiK and AnP are recipients of junior research fellowships from the Council of Scientific & Industrial Research, Government of India. SS and NaK are grateful to the Department of Biotechnology, Government of India for providing funds to perform this work. SS and NaK thank Drs. Harold Margolis, Dennis Trent, George Siber and Cristina Cassetti for their inputs in designing the mosaic VLPs.
- Gubler DJ: The economic burden of dengue. Am J Trop Med Hyg. 2012, 86: 743-744. 10.4269/ajtmh.2012.12-0157.View Article
- Gubler DJ, Kuno G, Markoff L: Flaviviruses.In Fields Virology.5th edition. Edited by: Knipe DM, Howley PM. Philadelphia: Wolters Kluwer and Lippincott Williams & Wilkins; 2007:1153-1252.
- WHO: Dengue and dengue haemorrhagic fever. 2012, Factsheet No117 2012 http://www.who.int/mediacentre/factsheets/fs117/en/.
- Swaminathan S, Khanna N: Dengue: recent advances in biology and current status of translational research. Current Mol Med. 2009, 9: 152-173. 10.2174/156652409787581592.View Article
- Dussart P, Petit L, Labeau B, Bremand L, Leduc A, Moua D, Matheus S, Baril L: Evaluation of two new commercial tests for the diagnosis of acute dengue virus infection using NS1 antigen detection in human serum. PLoS Negl Trop Dis. 2008, 2: e280-10.1371/journal.pntd.0000280.View Article
- Peeling RW, Artsob H, Pelegrino JL, Buchy P, Cardosa MJ, Devi S, Enria DA, Farrar J, Gubler DJ, Guzman MG, Halstead SB, Hunsperger E, Kliks S, Margolis HS, Nathanson CM, Nguyen VC, Rizzo N, Vázquez S, Yoksan S: Evaluation of diagnostic tests: dengue. Nat Rev Microbiol. 2010, Suppl: S30-S37. 10.1038/nrmicro2459.View Article
- Noble CG, Chen YL, Dong H, Gu F, Lim SP, Schul W, Wang QY, Shi PY: Strategies for development of dengue virus inhibitors. Antiviral Res. 2010, 85: 450-462. 10.1016/j.antiviral.2009.12.011.View Article
- Pastorino B, Nougairède A, Wurtz N, Gould E, de Lamballerie X: Role of host cell factors in flavivirus infection: Implications for pathogenesis and development of antiviral drugs. Antiviral Res. 2010, 87: 281-294. 10.1016/j.antiviral.2010.04.014.View Article
- Swaminathan S, Batra G, Khanna N: Dengue vaccines: state of the art. Expert Opin Ther Patents. 2010, 20: 819-835. 10.1517/13543771003767476.View Article
- Sabchareon A, Wallace D, Sirivichayakul C, Limkittikul K, Chanthavanich P, Suvannadabba S, Jiwariyavej V, Dulyachai W, Pengsaa K, Wartel TA, Moureau A, Saville M, Bouckenooghe A, Viviani S, Tornieporth NG, Lang J: Protective efficacy of the recombinant, live-attenuated, CYD tetravalent dengue vaccine in Thai schoolchildren: a randomised, controlled phase 2b trial. Lancet. 2012, 380: 1559-1567. 10.1016/S0140-6736(12)61428-7.View Article
- Soto CM, Ratna BR: Virus hybrids as nanomaterials for biotechnology. Current Opin Biotechnol. 2010, 21: 426-438. 10.1016/j.copbio.2010.07.004.View Article
- Yildiz I, Shukla S, Steinmetz NF: Applications of viral nanoparticles in medicine. Current Opin Biotechnol. 2011, 22: 901-908. 10.1016/j.copbio.2011.04.020.View Article
- Chackerian B: Virus-like particles: flexible platforms for vaccine development. Expert Rev Vaccines. 2007, 6: 381-390. 10.1586/147605220.127.116.111.View Article
- Myyryläinen T, Talha SM, Swaminathan S, Vainionpää R, Soukka T, Khanna N, Pettersson K: Simultaneous detection of human immunodeficiency virus 1 and hepatitis B virus infections using a dual-label time-resolved fluorometric assay. J Nanobiotechnol. 2010, 8: 27-10.1186/1477-3155-8-27.View Article
- Talha SM, Salminen T, Swaminathan S, Soukka T, Pettersson K, Khanna N: A highly sensitive and specific time resolved fluorometric bridge assay for antibodies to HIV-1 and −2. J Virol Methods. 2011, 173: 24-30. 10.1016/j.jviromet.2011.01.001.View Article
- Arora U, Tyagi P, Swaminathan S, Khanna N: Dengue virus type 2 envelope domain III displaying bio-nanoparticles. J Nanobiotechnol. 2012, 10: 30-10.1186/1477-3155-10-30.View Article
- Sukupolvi-Petty S, Austin SK, Purtha WE, Oliphant T, Nybakken GE, Schlesinger JJ, Roehrig JT, Gromowski GD, Barrett AD, Fremont DH, Diamond MS: Type- and subcomplex-specific neutralizing antibodies against domain III of dengue virus type 2 envelope protein recognize adjacent epitopes. J Virol. 2007, 81: 12816-12826. 10.1128/JVI.00432-07.View Article
- Crill WD, Roehrig RT: Monoclonal antibodies that bind to domain III of dengue virus E glycoprotein are the most efficient blockers of virus adsorption to Vero cells. J Virol. 2001, 75: 7769-7773. 10.1128/JVI.75.16.7769-7773.2001.View Article
- Vassileva A, Chugh DA, Swaminathan S, Khanna N: Effect of copy number on the expression levels of Hepatitis B surface antigen in the methylotrophic yeast Pichia pastoris. Protein Exp Purif. 2001, 21: 71-80. 10.1006/prep.2000.1335.View Article
- Gurramkonda C, Adnan A, Gäbel T, Lünsdorf H, Ross A, Nemani SK, Swaminathan S, Khanna N, Rinas U: Simple high-cell density fed-batch technique for high-level recombinant protein production with Pichia pastoris: Application to intracellular production of Hepatitis B surface antigen. Microb Cell Factories. 2009, 8: 13-10.1186/1475-2859-8-13.View Article
- Patil A, Khanna N: Novel membrane extraction procedure for the purification of hepatitis B surface antigen from Pichia pastoris. J Chromatog B. 2012, 898: 7-14.View Article
- McAleer WJ, Buynak EB, Maigetter RZ, Wampler DE, Miller WJ, Hilleman M: Human hepatitis B vaccine from recombinant yeast. Nature. 1984, 307: 178-180. 10.1038/307178a0.View Article
- Shouval D: Hepatitis B vaccines. J Hepatol. 2003, 39: S70-S76.View Article
- Biemans R, Thines D, Petre-Parent B, de Wilde M, Rutgers T, Cabeźon T: Immunoelectron microscopic detection of the Hepatitis B virus major surface protein in dilated perinuclear membranes of yeast cells. DNA Cell Biol. 1992, 11: 621-626. 10.1089/dna.1992.11.621.View Article
- Lünsdorf H, Gurramkonda C, Adnan A, Khanna N, Rinas U: Virus-like particle production with yeast: ultrastructural and immunocytochemical insights into Pichia pastoris producing high levels of hepatitis B surface antigen. Microb Cell Fact. 2011, 10: 48-10.1186/1475-2859-10-48.View Article
- Batra G, Raut R, Dahiya S, Kamran N, Swaminathan S, Khanna N: Pichia pastoris-expressed dengue virus type 2 envelope domain III elicits virus-neutralizing antibodies. J Virol Methods. 2010, 167: 10-16. 10.1016/j.jviromet.2010.03.002.View Article
- Ludolfs D, Schilling S, Altenschmidt J, Schmitz H: Serological differentiation of infections with dengue virus serotypes 1 to 4 by using recombinant antigens. J Clin Microbiol. 2002, 40: 4317-4320. 10.1128/JCM.40.11.4317-4320.2002.View Article
- Khanam S, Pilankatta R, Khanna N, Swaminathan S: An adenovirus type 5 (AdV5) vector encoding an envelope domain III-based tetravalent antigen elicits immune responses against all four dengue viruses in the presence of prior AdV5 immunity. Vaccine. 2009, 27: 6011-6021. 10.1016/j.vaccine.2009.07.073.View Article
- Korrapati AB, Swaminathan G, Singh A, Khanna N, Swaminathan S: Adenovirus delivered short hairpin RNA targeting a conserved site in the 5’ non-translated region inhibits all four serotypes of dengue viruses. PLoS Negl Trop Dis. 2012, 6: e1735-10.1371/journal.pntd.0001735.View Article
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