Recombinant Dengue Virus 4 Envelope Glycoprotein Virus-Like Particles Derived from Pichia pastoris are Capable of Eliciting Homotypic Domain III-Directed Neutralizing Antibodies

Abstract

Dengue is a viral pandemic caused by four dengue virus serotypes (DENV-1, 2, 3, and 4) transmitted by Aedes mosquitoes. Reportedly, there has been a 2-fold increase in dengue cases every decade. An efficacious tetravalent vaccine, which can provide long-term immunity against all four serotypes in all target populations, is still unavailable. Despite the progress being made in the live virus-based dengue vaccines, the World Health Organization strongly recommends the development of alternative approaches for safe, affordable, and efficacious dengue vaccine candidates. We have explored virus-like particles (VLPs)-based nonreplicating subunit vaccine approach and have developed recombinant envelope ectodomains of DENV-1, 2, and 3 expressed in Pichia pastoris These self-assembled into VLPs without pre-membrane (prM) protein, which limits the generation of enhancing antibodies, and elicited type-specific neutralizing antibodies against the respective serotype. Encouraged by these results, we have extended this work further by developing P. pastoris-expressed DENV-4 ectodomain (DENV-4 E) in this study, which was found to be glycosylated and assembled into spherical VLPs without prM, and displayed critical neutralizing epitopes on its surface. These VLPs were found to be immunogenic in mice and elicited DENV-4-specific neutralizing antibodies, which were predominantly directed against envelope domain III, implicated in host-receptor recognition and virus entry. These observations underscore the potential of VLP-based nonreplicative vaccine approach as a means to develop a safe, efficacious, and tetravalent dengue subunit vaccine. This work paves the way for the evaluation of a DENV E-based tetravalent dengue vaccine candidate, as an alternative to live virus-based dengue vaccines.

Figure 1.

Expression of DENV-4 E in Pichia pastoris. (A) Schematic representation of DENV-4 E gene cloned in pPICZ-A expression vector with AOX1 promoter (5′ AOX1) upstream and transcription terminator sequence (TT) downstream of it. An Escherichia coli origin of replication (ori) and zeocin resistance (Zeo) marker is also present for efficient replication and transformed clone selection, respectively. (B and C) Evaluation of expression and localization of DENV-4 E in induced P. pastoris. Aliquots of un-induced (UI) and induced (I) cell cultures were subjected to lysis and separated into soluble (S) and membrane-enriched pellet (P) fractions by centrifugation. S and P fractions were evaluated through (B) Western blot and (C) ELISA on Ni-NTA His Sorb plate using DENV-4 E-specific monoclonal antibody (mAb), DENV-4 E88. The blot in panel B is a composite figure, where lane “M” denotes pre-stained markers, the sizes (in kDa) of which are indicated on the left. An arrow on the right of panel B indicates the position of DENV-4 E in the blot. In panel C, the S and P fractions are represented by the grey and black bars, respectively. DENV-4 E = dengue virus serotype 4 ectodomain; ELISA = enzyme-linked immunosorbent assay.

Figure 2.

Purification and characterization of DENV-4 E. (A) DENV-4 E was purified from pellet fraction of lysate of induced Pichia pastoris biomass via Ni²⁺ affinity chromatography. Blue curve and dashed grey line represent the UV-absorbance profile and imidazole step gradient, respectively. The inset represents Coomassie-stained SDS-PAGE analysis of the peak fractions (lanes 1–9) of purified DENV-4 E. Lane M represents low-molecular-weight protein markers, the sizes (in kDa) of which are written on the left of the inset. (B) Formation of virus-like particles by purified DENV-4 E as assessed by DLS representing particle size distribution by intensity. The average particle size is also indicated. EM image of the same is provided in the inset. (C) Glycosylation of DENV-4 E (4E, black bar) is evaluated by ELISA using Con A-HRPO conjugate on Ni-NTA His-Sorb plate. EDIII (III, blue bar) and DENV-4 (V, green bar) is used as negative control and positive control, respectively. In the inset, a protein blot using Con A-HRPO conjugate is shown in which lane M represents low-molecular-weight protein markers, which contain ovalbumin. Low-molecular-weight protein markers serve as positive (through its only glycosylated protein, ovalbumin) and negative (proteins other than ovalbumin) controls in the assay, and the sizes (in kDa) of the protein markers are indicated on the left. Con A-HRPO = Concanavalin A-horseradish peroxidase; DENV-4 E = dengue virus serotype 4 ectodomain; ED = envelope domain; ELISA = enzyme-linked immunosorbent assay.

Figure 3.

DENV-4 E VLPs are immunogenic in mice. (A) BALB/c mice were immunized with DENV-4 E VLPs adsorbed on alhydrogel on days 0, 30, and 90. On day 100, sera were collected and evaluated (at various dilutions) for the presence of antibodies against the four DENVs (first panel), four DENV-Es (second panel), and four EDIIIs (third panel) by ELISA. In each of the three cases, curves in red, green, blue, and black represent DENV serotype 1, 2, 3, and 4, respectively. (B) Recognition of DENV-4-infected BHK-21 cells by PBS-immunized negative control mouse serum (first panel), 4G2 mAb (second panel), and DENV-4 E immune serum (third panel) by IFA. DENV-4 E = dengue virus serotype 4 ectodomain; ED = envelope domain; ELISA = enzyme-linked immunosorbent assay; mAb = monoclonal antibody; VLP = virus-like particle.

Figure 4.

DENV-4 E VLPs elicit EDIII-directed serotype-specific neutralizing antibodies. (A) Immune sera collected 10 days after the final boost from DENV-4 E VLP-immunized BALB/c mice were evaluated for existence of neutralizing antibodies against DENV-1 (DV1, red curve), DENV-2 (DV2, green curve), DENV-3 (DV3, blue curve), and DENV-4 (DV4, black curve) by FNT on Vero cells. (B) The contribution of anti-EDIII antibodies in DENV-4 E immune serum (black solid curve) in neutralizing DENV-4 was evaluated by FNT on Vero cells after depleting it of anti-EDIII antibodies by incubating it with serotype 4 EDIII-MBP (black dotted curve). DENV-4 E immune serum was also incubated with MBP (black dashed curve) to score nonspecific depletion of antibodies (if any). The percentage of Vero cells infected with the DENV is represented on y axis, while log of reciprocal of sera dilution is represented on x axis. FNT₅₀ titers, tabulated on the top of each of the graphs, represent the sera dilution resulting in 50% neutralization of respective DENV, and are calculated from the dotted horizontal line in the graphs representing 50% infection (or 50% neutralization). DENV-4 E = dengue virus serotype 4 ectodomain; ED = envelope domain; FNT = fluorescence-activated cell sorting (FACS)-based neutralization test; MBP = maltose-binding protein; VLP = virus-like particle.