Virus-like particles derived from Pichia pastoris-expressed dengue virus type 1 glycoprotein elicit homotypic virus-neutralizing envelope domain III-directed antibodies
- Ankur Poddar†1Email author,
- Viswanathan Ramasamy†1, 2,
- Rahul Shukla1,
- Ravi Kant Rajpoot1,
- Upasana Arora1,
- Swatantra K. Jain2, 3,
- Sathyamangalam Swaminathan4Email author and
- Navin Khanna1Email author
© The Author(s). 2016
Received: 21 March 2016
Accepted: 8 June 2016
Published: 14 June 2016
Four antigenically distinct serotypes (1–4) of dengue viruses (DENVs) cause dengue disease. Antibodies to any one DENV serotype have the potential to predispose an individual to more severe disease upon infection with a different DENV serotype. A dengue vaccine must elicit homotypic neutralizing antibodies to all four DENV serotypes to avoid the risk of such antibody-dependent enhancement in the vaccine recipient. This is a formidable challenge as evident from the lack of protective efficacy against DENV-2 by a tetravalent live attenuated dengue vaccine that has completed phase III trials recently. These trial data underscore the need to explore non-replicating subunit vaccine alternatives. Recently, using the methylotrophic yeast Pichia pastoris, we showed that DENV-2 and DENV-3 envelope (E) glycoproteins, expressed in absence of prM, implicated in causing severe dengue disease, self-assemble into virus-like particles (VLPs), which elicit predominantly virus-neutralizing antibodies and confer significant protection against lethal DENV challenge in an animal model. The current study extends this work to a third DENV serotype.
We cloned and expressed DENV-1 E antigen in P. pastoris, and purified it to near homogeneity. Recombinant DENV-1 E underwent post-translational processing, namely, signal peptide cleavage and glycosylation. Purified DENV-1 E self-assembled into stable VLPs, based on electron microscopy and dynamic light scattering analysis. Epitope mapping with monoclonal antibodies revealed that the VLPs retained the overall antigenic integrity of the virion particles despite the absence of prM. Subtle changes accompanied the efficient display of E domain III (EDIII), which contains type-specific neutralizing epitopes. These VLPs were immunogenic, eliciting predominantly homotypic EDIII-directed DENV-1-specific neutralizing antibodies.
This work demonstrates the inherent potential of P. pastoris-expressed DENV-1 E glycoprotein to self-assemble into VLPs eliciting predominantly homotypic neutralizing antibodies. This work justifies an investigation of the last remaining serotype, namely, DENV-4, to assess if it also shares the desirable vaccine potential manifested by the remaining three DENV serotypes. Such efforts could make it possible to envisage the development of a tetravalent dengue vaccine based on VLPs of P. pastoris-expressed E glycoproteins of the four DENV serotypes.
Dengue disease is a global public health threat caused by four distinct serotypes of dengue viruses (DENV-1, −2, −3 and −4) spread primarily by Aedes aegypti mosquito . According to a report in the year 2013, the number of annual global dengue infections was estimated to be ~400 million, with ~96 million clinically apparent infections . DENV is a positive sense RNA virus with ~11 kilo base (kb) genome, which encodes three structural and seven non-structural proteins . Dengue disease can vary from mild dengue fever to severe, life-threatening syndromes, dengue hemorrhagic fever and dengue shock syndrome [1, 4, 5]. DENV infection, which results in lifelong homotypic immunity, affords only transient heterotypic immunity . In fact, heterotypic antibodies are implicated in promoting DENV uptake during a secondary infection with a different serotype, through Fc receptor pathway and contributing to increased viral load, leading to more severe disease [1, 7]. To preclude the possibility of such antibody-mediated enhancement (ADE) of dengue disease, it is believed that a safe dengue vaccine must be ‘tetravalent’, affording simultaneous type-specific (homotypic) protection against each of the four DENV serotypes. This requirement poses a major hurdle to dengue vaccine development . Many live attenuated vaccine candidates are in clinical development. Of these, Sanofi’s Chimeric Yellow Fever Dengue-Tetravalent Dengue Vaccine (CYD-TDV) has recently completed Phase III trials [9, 10] and is currently being introduced in some dengue-endemic countries . However, this vaccine candidate has certain limitations. It needs to be administered in 3 doses over a one year period. CYD-TDV is not as effective in dengue-naïve individuals, as in those with a history of prior dengue exposure. Its efficacy against DENV-2 is very low, despite its apparent capacity to induce serotype 2-specific neutralizing antibodies [9, 10]. Efforts to understand this, using a mouse model of ADE, strongly suggest that while homotypic neutralizing antibodies do not cause ADE, heterotypic neutralizing antibodies do, at certain concentrations . This underscores the requirement for a dengue vaccine to elicit homotypic neutralizing antibodies to each of the four prevalent DENV serotypes to be both safe and efficacious.
Using a non-replicating subunit vaccine approach, we showed recently that it is possible to elicit predominantly homotypic neutralizing antibody titers using P. pastoris-expressed envelope (E) glycoproteins of DENV-2 and DENV-3 in mice [13, 14]. The DENV-E protein is the major surface exposed glycoprotein of about ~500 amino acid (aa) residues, which is organized into three discrete domains, EDI, EDII and EDIII . Of these, EDIII is critical from a vaccine perspective, as it is not only involved in host receptor recognition and virus entry into susceptible cells, but also in the induction of potent type-specific virus neutralizing antibodies [16, 17]. Interestingly, our work showed that the N-terminal 80 % of the E glycoprotein (ectodomain) of DENV-2  and DENV-3 , which encompasses EDIII, produced using P. pastoris assembled into discrete virus-like particles (VLPs). These VLPs served as efficient display platforms for EDIII and elicited potent, homotypic virus-neutralizing antibodies [13, 14], capable of conferring significant protection against live virus challenge in an animal model . Unlike dengue virion particles which contain E along with another structural protein, prM, implicated in the induction of ADE-mediating antibodies [18, 19], these VLPs contain only the E glycoprotein. Thus, the P. pastoris-derived DENV E VLPs, lacking the ADE-associated prM protein, offer a significant safety advantage. This has provided us the rationale to explore the feasibility of developing DENV E VLPs corresponding to the remaining two serotypes as well, so that we may eventually develop a tetravalent DENV E VLP-based vaccine candidate.
In this paper, we specifically focus on expressing and characterizing DENV-1 E glycoprotein using P. pastoris to address the following specific questions: (i) Will DENV-1 E glycoprotein, expressed in P. pastoris, also possess VLP-forming potential? (ii) Would these VLPs preserve the antigenic integrity of the critical virus-neutralizing epitopes? (iii) Would the VLPs be immunogenic, and if so, would the antibodies elicited be homotypic? The work described in this paper demonstrates the feasibility of creating P. pastoris-expressed DENV-1 E-based VLPs that are capable of eliciting EDIII-directed type-specific neutralizing antibodies and moves a step closer to developing a tetravalent dengue VLP vaccine candidate.
Recombinant DENV-1 E undergoes proper processing and glycosylation in P. pastoris and self- assembles into stable VLPs
DENV-1 E VLPs display key viral epitopes on their surface
Analysis of antigenic integrity of DENV-1 E VLPs a
Type-specific murine mAbs
Absorbance, 450 nm
Cross-reactive murine & human mAbs
Absorbance, 450 nm
EDIII, not LR
EDIII, AS, LR
DENV-1 E VLPs elicit EDIII-directed type-specific virus-neutralizing antibodies in mice
Dengue has recently been recognized as one of the fastest spreading vector-borne diseases . Since, a preventive dengue vaccine has been an unmet need for very long has spurred the introduction of a live attenuated vaccine (CYD-TDV) recently in a couple of dengue-endemic countries, despite sub-optimal efficacy in phase III clinical trials [9, 10]. Attempts to understand the basis of lack of vaccine efficacy, have revealed that neutralizing antibody titers elicited by CYD-TDV are directed predominantly towards one serotype , presumably stemming from viral interference [31–33]. This taken in the context of another recent study, which demonstrated that neutralizing antibodies need to be homotypic to preclude the possibility of ADE , strongly suggests that it is important for a safe and effective dengue vaccine to elicit type-specific neutralizing antibodies to each of the four prevalent DENV serotypes. This situation warrants a continued search for alternate non-replicating vaccine candidates that may hopefully eliminate interference and the associated potential safety concerns .
Our recent work has shown that in the context of dengue, VLPs offer such an alternate option. These VLPs are spherical, non-replicative, lack viral genomic RNA, and are highly immunogenic. We reported earlier that the E glycoproteins of DENV-2 and DENV-3, when expressed in P. pastoris, in the absence of the companion structural protein, prM, assemble into discrete VLPs [13, 14]. Several attributes of these VLPs are noteworthy from the perspective of a dengue vaccine candidate. First is the observation that prM, a minor structural protein necessary for virion maturation which is documented to elicit antibodies implicated in ADE [18, 19], is not required for VLP formation. Second, these VLPs were shown to preserve the neutralizing epitopes of the cognate DENV serotype. Interestingly, these VLPs displayed EDIII efficiently. This is significant as EDIII is implicated in host receptor recognition and contains multiple type-specific neutralizing epitopes [16, 17]. Third, though these VLPs elicited antibodies against all the DENV serotypes, they neutralized only DENV-1, mediated by anti-EDIII antibodies. This is of significance as only cross-reactive virus-neutralizing antibodies appear to be associated with ADE . Fourth, in an animal challenge model, DENV-2 E VLPs afforded statistically significant protection. Collectively, these findings underlie our efforts to develop a tetravalent VLP vaccine candidate based on P. pastoris-expressed DENV E glycoproteins. Towards this objective, we have extended our VLP vaccine work to a third DENV serotype, namely, DENV-1, in this paper.
The design of the DENV-1 E antigen gene was exactly similar to that of DENV-2 and DENV-3 E antigens described previously [13, 14]. Moreover, a multiple sequence alignment of the four DENV-E aa sequences (of the specific genotypes of the four serotypes) revealed 60–80 % similarity (Additional file 1: Figure S3), which is in compliance with the reports in literature . The DENV-1 E antigen was provided with an N-terminal DENV-1 prM-derived signal peptide and a C-terminal 6x His tag and expressed in P. pastoris by methanol induction. As reported in the earlier studies, the DENV-1 E protein was processed similarly by P. pastoris, in that the prM peptide was cleaved off and the mature protein glycosylated. Consistent with the behavior of DENV-2 E and DENV-3 E proteins, the DENV-1 E protein also self-assembled into VLPs, during downstream processing, as evidenced by EM and DLS analyses. These VLPs were stable and appeared to mature to a greater degree of size homogeneity during storage. Probing the surface of these VLPs using several different type-specific and cross-reactive human and murine mAbs demonstrated that several of the epitopes that exist on the DENV-1 virion surface are present on the DENV-1 E VLPs as well. Of note was the observation that EDIII was displayed on the VLP surface with its LR epitope, critical for the induction of type-specific neutralizing antibodies, intact and freely accessible. Consistent with this, we found that the DENV-1 E VLPs elicited homotypic neutralizing antibodies. EDIII antibodies elicited by these VLPs were almost exclusively responsible for neutralizing DENV-1, as evident from abrogation of neutralizing antibody titer in immune sera following antibody depletion using immobilized DENV-1 EDIII antigen. Like the antibodies induced by DENV-3 E , antibodies induced by DENV-1 E VLPs also did not cause heterotypic enhancement of DENV infection at 1:20 sera dilution (data not shown). This underscores the utility of eliciting serotype-specific neutralizing antibodies to preclude heterotypic ADE. The data thus far on P. pastoris-produced DENV-1 E VLPs essentially mirror our earlier findings with DENV-2 and DENV-3 E VLPs and provide the rationale for extending this work to the E glycoprotein of the last remaining serotype, DENV-4.
We have evaluated the potential of P. pastoris-expressed DENV-1 E glycoprotein-based VLPs as a potential vaccine candidate. We show that these DENV-1 E VLPs generate predominantly EDIII-directed, DENV-1 serotype specific, neutralizing immune antibody responses. The complete absence of prM would be an in-built safety advantage. Multimeric presentation of antigenic epitopes with predominance of EDIII determinants on DENV-1 E VLPs may overcome the problem of low immunogenicity associated with monomeric subunit based vaccine candidates. Stability of VLPs at higher temperature would help in reducing the challenges faced during vaccine storage and administration. The P. pastoris-expressed E glycoproteins of three DENV serotypes so far studied manifest several attributes, desirable from a vaccine standpoint, namely, the capacity to: (i) form immunogenic VLPs, (ii) efficiently display EDIII that contains type-specific neutralizing epitopes, and (iii) induce homotypic neutralizing antibody titers. This, coupled to the high expression potential of the P. pastoris host system strongly suggests that extension of this work to the last remaining serotype, DENV-4, may set the stage for developing a safe, effective and inexpensive VLP dengue vaccine candidate.
DENV-1 E gene, expression plasmid, cell line, virus and other reagents
DENV-1 E gene (~1.3 kb: Genbank accession no: JX292264) codon-optimized for expression in P. pastoris system was custom synthesized by GeneScript, New Jersey, USA. E.coli strain DH5α, P. pastoris strain KM71H and expression plasmid pPICZA were procured from Invitrogen Life Technologies, Carlsbad, USA. Vero and BHK-21 cell lines were purchased from American Type Cell Culture (ATCC), Virginia, USA. WHO reference viral strains DENV-1 (WP 74), DENV-2 (S16803), DENV-3 (CH53489), DENV-4 (TVP-360); E. coli clones expressing MBP (Maltose Binding Protein) and MBP-EDIII-1 (EDIII domain of DENV-1 fused with MBP) fusion proteins were received from Dr. Aravinda de Silva, University of North Carolina (UNC), USA. All mAbs used in this study were the same as before [13, 14]. Concanavalin A (Con A) peroxidase conjugate was purchased from Sigma-Aldrich. Goat anti-mouse and anti-human IgG monoclonal antibody-HRPO conjugates and anti-mouse fluorescein isothiocyanate (FITC) conjugate were procured from Life Technologies, USA and Merck, Germany, respectively. Ni-NTA resin was procured from Qiagen, Hilden, Germany. High binding, polystyrene ELISA plates were purchased from Corning Incorporated, USA. N-linked oligosaccharide profiling was performed at GlycoSolutions Corp., Marlborough, USA.
DENV-1 E gene cloning, expression, purification and characterization
DENV-1 E gene was cloned at EcoRI and NotI site of pPICZA expression plasmid. The resultant plasmid was linearized with Sac I, electroporated into P. pastoris strain KM71H and expressed under the control of AOX1 promoter as done previously for DENV-2 E  and DENV-3 E . Transformants were selected on zeocin plates and induced with 1.5 % methanol every 24 h for 72 h. DENV-1 E was purified from induced P. pastoris cells using Ni-NTA chromatography under denaturing conditions as described previously . Purified protein was analyzed on Coomassie gel and Western blot using anti-EDIII mAbs. The identity and similarities between the amino acid sequences of DENV-1 (West Pac-74), DENV-2 (New Guinea C), DENV-3 (H87) and DENV-4 (Dominica) derived recombinant E genes with GenBank accession number JX292264, JX292265, JX292266, JX292267 respectively was determined by multiple sequence alignment using Clustal W tool. Further, DENV-1 E protein was evaluated for assembly into VLPs by EM and DLS studies . Briefly, EM studies were performed by coating the purified and dialyzed DENV-1 E (at 5–10 ug/ml) on carbon-formvar grid, followed by negative staining with 1 % uranyl acetate, which were examined under electron microscope. Malvern Zetasizer NanoZ was used to assess the particle size and distribution of purified and dialysed DENV-1 E VLPs by dynamic light scattering. The stability of VLPs after incubation at 37 °C for 14 days was also evaluated by DLS studies as described before . Further, the protein was characterized by MALDI-TOF-Mass spectroscopy to determine the structure of N-linked glycan attached to protein. The integrity of conformational epitopes of DENV-1 E protein was evaluated using a panel of murine and human mAbs by indirect ELISA as reported previously . Further, the reactivity of antibodies in the DENV-1 E VLP immunized mice sera was evaluated by indirect ELISA and IFA as reported previously [13, 14]. BALB/c mice (n = 6) were immunized with 20 μg of DENV-1 E VLP on day 0 and subsequently boosted on days 30 and 90. Mice were bled on days 37 and 100 for seroanalysis. The neutralization efficacy of DENV-1 E sera was evaluated against all the four DENV serotypes on Vero cell line by FACS based neutralization assay as described before [13, 14]. Further, the proportion of EDIII-directed neutralizing antibody titers in DENV-1 E serum was evaluated by pre-incubating it with amylose resin coated with MBP-fused in-frame to DENV-1 EDIII, prior to use in FACS based DENV neutralization assay . FACS data were analysed using FlowJo software.
ADE, Antibody dependent Enhancement; AOX, Alcohol oxidase; CYD-TDV, Chimeric yellow fever dengue-tetravalent dengue vaccine; DENV, Dengue virus; DLS, Dynamic light scattering; E, Envelope; ED, Envelope domain; EM, Electron microscopy; FNT, Flow cytometry based neutralization titer; IFA, Immunofluorescence assay; LAV, Live attenuated vaccine; mAb, monoclonal antibody; MBP, Maltose binding protein; prM, pre-membrane; VLP, Virus like Particle
The authors are grateful to Drs. Harold Margolis, Carole Heileman, Cristina Cassetti, and the Indo-US Vaccine Action Program Committee members for their valuable inputs and support. Access to several DENV mAbs through BEI resources is acknowledged.
The work was funded in part by Department of Biotechnology. Government of India, grant no. BT/PR11807/MED/29/871/2014 to Navin Khanna. Sathyamangalam Swaminathan is supported partly by OPERA funds granted by BITS Pilani. The funders did not have any role in the design, collection, analysis and interpretation of data, as well as in writing of the manuscript and in the decision to submit the manuscript for publication.
Availability of data and materials
The data set supporting the results of this article are included within the article and in Additional file 1.
AP, VR performed cloning, expression, purification, mouse immunization, ELISA, IFA, FNT and antibody depletion assays. RS, RKR carried out EM and DLS studies. SKJ, UA participated in data interpretation. SS and NK conceived and designed the work, analyzed the data and wrote the final manuscript. All authors provided inputs for the initial draft and approved the final version.
The authors declare no conflict of interest. None of the authors has any financial or non-financial competing interests to declare.
Consent for publication
Ethics approval and consent to participate
Animal experiments performed in the present study are in agreement with the animal ethical guidelines of the Committee for the Purpose of Control and Supervision of Experimental Animals (CPCSEA) of Government of India. Protocols adopted for animal experiments were approved by Institutional Animal Ethics Committees (IAEC) of International Centre for Genetic Engineering and Biotechnology, New Delhi and Syngene International Limited, Bangalore (IAEC No. Syngene/IAEC/520/06-2014). This study did not involve human participants.
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