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Pichia pastoris-expressed dengue 2 envelope forms virus-like particles without pre-membrane protein and induces high titer neutralizing antibodies - PubMed

  • ️Tue Jan 01 2013

. 2013 May 23;8(5):e64595.

doi: 10.1371/journal.pone.0064595. Print 2013.

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Pichia pastoris-expressed dengue 2 envelope forms virus-like particles without pre-membrane protein and induces high titer neutralizing antibodies

Shailendra Mani et al. PLoS One. 2013.

Abstract

Dengue is a mosquito-borne viral disease with a global prevalence. It is caused by four closely-related dengue viruses (DENVs 1-4). A dengue vaccine that can protect against all four viruses is an unmet public health need. Live attenuated vaccine development efforts have encountered unexpected interactions between the vaccine viruses, raising safety concerns. This has emphasized the need to explore non-replicating dengue vaccine options. Virus-like particles (VLPs) which can elicit robust immunity in the absence of infection offer potential promise for the development of non-replicating dengue vaccine alternatives. We have used the methylotrophic yeast Pichia pastoris to develop DENV envelope (E) protein-based VLPs. We designed a synthetic codon-optimized gene, encoding the N-terminal 395 amino acid residues of the DENV-2 E protein. It also included 5' pre-membrane-derived signal peptide-encoding sequences to ensure proper translational processing, and 3' 6× His tag-encoding sequences to facilitate purification of the expressed protein. This gene was integrated into the genome of P. pastoris host and expressed under the alcohol oxidase 1 promoter by methanol induction. Recombinant DENV-2 protein, which was present in the insoluble membrane fraction, was extracted and purified using Ni(2+)-affinity chromatography under denaturing conditions. Amino terminal sequencing and detection of glycosylation indicated that DENV-2 E had undergone proper post-translational processing. Electron microscopy revealed the presence of discrete VLPs in the purified protein preparation after dialysis. The E protein present in these VLPs was recognized by two different conformation-sensitive monoclonal antibodies. Low doses of DENV-2 E VLPs formulated in alum were immunogenic in inbred and outbred mice eliciting virus neutralizing titers >1,1200 in flow cytometry based assays and protected AG129 mice against lethal challenge (p<0.05). The formation of immunogenic DENV-2 E VLPs in the absence of pre-membrane protein highlights the potential of P. pastoris in developing non-replicating, safe, efficacious and affordable dengue vaccine.

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Conflict of interest statement

Competing Interests: Two of the authors (T. Barman and R. Sood) are employed by Ranbaxy Research Laboratories. This does not alter our adherence to all the PLOS ONE policies on sharing data and materials.

Figures

Figure 1
Figure 1. Design of the DENV-2 E antigen.

(A) Schematic representation of the DENV-2 polyprotein, showing the parts of prM and E included in designing the E antigen for expression in P. pastoris. (B) Design of the DENV-2 E antigen consisting of the 395 aa residue E ectodomain, preceded by the C-terminal 34 aa residues of prM. The grey box denotes the pentaglycine linker peptide joining the C-terminus of E ectodomain to the polyhistidine tag (6×H). (C) The predicted aa sequence of the DENV-2 E antigen shown in ‘B’. The color scheme corresponds to that shown in ‘B’. The first two aa residues (MV) were introduced due to the insertion of the initiator codon in a Kozak consensus context. The downward arrows in ‘B’ and ‘C’ denote the signal cleavage site.

Figure 2
Figure 2. Expression of DENV-2 E in P. pastoris.

(A) Map of the DENV-2 expression construct for integration into P. pastoris genome. The DENV-2 E gene is flanked by the AOX1 promoter (5’ AOX1) and transcription terminator (TT) at its 5′ and 3′ ends, respectively. The construct contains an E. coli origin of replication (ori) and the selection marker zeocin (Zeo), which is functional in both E. coli as well as P. pastoris. (B) Localization of the recombinant DENV-2 antigen expression in induced P. pastoris. Aliquots of un-induced (UI) and induced (I) cultures were lysed and separated into soluble (S) and membrane-enriched pellet (P) fractions, run on SDS-polyacrylamide gel and subjected to Western blot analysis using mAb 24A12. Pre-stained protein markers were analyzed in lane ‘M’. Their sizes (in kDa) are indicated to the left. The arrow on the right indicates the position of the recombinant DENV-2 E antigen. (C) Ni-NTA His-Sorb ELISA analysis of S and P fractions obtained from UI (blue bars) and I (red bars) cell lysates described in ‘B’.

Figure 3
Figure 3. Purification and characterization of recombinant DENV-2 E antigen.

(A) Ni2+ affinity chromatographic purification of DENV-2 E antigen from the P fraction of induced P. pastoris lysate. The continuous blue and the dashed black curves represent the profiles of UV absorbance (at 280 nm) and the imidazole step gradient, respectively, during chromatography. (B) Coomassie-stained SDS-polyacrylamide gel analysis of the purified protein. (C) Immunoblot analysis of the purified protein using mAb 24A12. (D) Immunoblot analysis of the purified protein using penta-His mAb. (E) Protein blot using Con A-HRPO conjugate. Controls analyzed in parallel include DENV-2 (lane ‘V’), purified EDIII-2 protein (lane ‘III’) and ovalbumin (lane ‘O’). In panels B-E: lanes ‘E’ denote the purified DENV-2 E protein (pooled peak material shown in panel ‘A’). Protein markers (whose sizes, in kDa, are shown to the left of each panel) were run in lanes ‘M’. The arrow to the right of each panel indicates the position of the recombinant DENV-2 E antigen.

Figure 4
Figure 4. Electron microscopic analysis of purified DENV-2 E protein.

(A) Freshly purified DENV-2 E antigen was negatively-stained with uranyl acetate and examined under EM. (B) EM analysis was carried out after incubating the purified antigen at 37°C for 2 weeks.

Figure 5
Figure 5. Evaluation of antibodies elicited by recombinant DENV-2 E antigen VLPs.

(A) Pooled sera from DENV-2 E immunized (solid, blue curve) and mock-immunized (black, dashed curve) Balb/C mice were tested in an indirect ELISA using DENV-2 E protein as the coating antigen. (B) The Balb/C anti-DENV-2 E antiserum (panel A) was tested in ELISAs using recombinant monovalent EDIII-1 (black), EDIII-2 ( blue), EDIII-3 (red) or EDIII-4 (green) antigens. Mock-immunized Balb/C serum was tested against EDIII-1 as coating antigen (black, dashed). (C) Pooled serum from DENV-2 E-immunized Swiss albino mice was tested in ELISAs using either recombinant DENV-2 (solid blue squares) or EDIII-2 (empty blue squares) as the coating antigens. Mock-immunized Swiss albino serum was tested against EDIII-2 as coating antigen (black, dashed). (D) Indirect immunofluorescence analysis of DENV-2-infected BHK cells using mock-immunized serum (i), anti-EDIII-2 antiserum (ii), 4G2 mAb (iii), or anti-DENV-2 E antiserum (iv), as the source of primary antibodies. Bound antibodies were visualized using anti-mouse IgG-FITC conjugate. Antisera in panel (i), (ii) and (iv) were from Balb/C mice.

Figure 6
Figure 6. Characterization of DENV-2-specific antibodies elicited by DENV-2 E VLPs.

(A) Analysis of virus-specific antibody titers in anti-DENV-2 E antisera (blue bars) and mock-immune sera (black bars) in indirect ELISAs using infectious DENVs as coating antigen. (B) Determination of virus-neutralizing antibody titers using FACS neutralization assay. Serial dilutions of anti-DENV-2 E antisera were tested for their capacity to neutralize infectivity of all four DENV serotypes . The vertical axis denotes the serum dilution corresponding to 50% neutralization (FNT50 titre) of virus infectivity. Murine sera used in experiments shown in panels A and B were from Balb/C mice; the Arabic numerals along the x-axis, in both these panels, indicate DENV serotype. (C) Determination of protective efficacy of DENV-2 E VLP immunization. AG129 mice were either mock-immunized (black curve, n = 4) or immunized with DENV-2 E VLPs (blue curve, n = 6) and challenged with a virulent strain of DENV-2. The mice were monitored daily (up to 18 days post challenge) for mortality and the resultant data plotted as Kaplan-Meir survival curves.

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This work was supported by funding to SS and NK from the Department of Biotechnology, Government of India. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.