Pichia pastoris-expressed dengue 3 envelope-based virus-like particles elicit predominantly domain III-focused high titer neutralizing antibodies

Abstract

Dengue poses a serious public health risk to nearly half the global population. It causes ~400 million infections annually and is considered to be one of the fastest spreading vector-borne diseases. Four distinct serotypes of dengue viruses (DENV-1, -2, -3, and -4) cause dengue disease, which may be either mild or extremely severe. Antibody-dependent enhancement (ADE), by pre-existing cross-reactive antibodies, is considered to be the major mechanism underlying severe disease. This mandates that a preventive vaccine must confer simultaneous and durable immunity to each of the four prevalent DENV serotypes. Recently, we used Pichia pastoris, to express recombinant DENV-2 E ectodomain, and found that it assembled into virus-like particles (VLPs), in the absence of prM, implicated in the elicitation of ADE-mediating antibodies. These VLPs elicited predominantly type-specific neutralizing antibodies that conferred significant protection against lethal DENV-2 challenge, in a mouse model. The current work is an extension of this approach to develop prM-lacking DENV-3 E VLPs. Our data reveal that P. pastoris-produced DENV-3 E VLPs not only preserve the antigenic integrity of the major neutralizing epitopes, but also elicit potent DENV-3 virus-neutralizing antibodies. Further, these neutralizing antibodies appear to be exclusively directed toward domain III of the DENV-3 E VLPs. Significantly, they also lack discernible ADE potential toward heterotypic DENVs. Taken together with the high productivity of the P. pastoris expression system, this approach could potentially pave the way toward developing a DENV E-based, inexpensive, safe, and efficacious tetravalent sub-unit vaccine, for use in resource-poor dengue endemic countries.

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

“…Thus, DENV-4 E expressing biomass was prepared by inducing the cells with 0.75% methanol every 12 hours (which makes it 1.5% methanol per 24 hours), for a period of 3 days (Supplemental Figure 2). These observations of association of DENV-4 E in pellet fraction and optimal expression at 1.5% methanol for 3 days were in consonance with that observed for DENV-1 E, 2 E, and 3 E. [31][32][33] Purification and characterization of recombinant DENV-4 E protein.…”

“…31 Briefly, presence of antibodies against the four DENVs, DENV-Es, and EDIIIs (all eight recombinant proteins developed in-house) was evaluated by indirect ELISA and their ability to recognize virus-infected BHK-21 cells was evaluated by IFA. Fluorescence-activated cell sorting (FACS)-based neutralization test (FNT) 35 was used to determine the neutralizing antibody titer against each DENV serotype, as reported previously for DENV-1 E, 2 E, and 3 E. [31][32][33] To evaluate the antibodies that are generated against EDIII of DENV-4 E, DENV-4 E immune serum was depleted of anti-EDIII antibodies and residual neutralizing antibodies were scored by FNT, according to optimized protocol. 33 Briefly, DENV-4 E immune serum was preincubated separately with EDIII-4-MBP and MBP proteins immobilized on amylose resin and the depleted sera were evaluated for residual neutralization titer against DENV-4 by FNT on Vero cells.…”

“…Ability of DENV-4 E to assemble into VLPs was evaluated by DLS and EM, and was found to assemble into particles of 40 nm ( Figure 2B), which is consistent with our previous results for other recombinant DENV-Es. [31][32][33] Like the other three DENV-Es, DENV-4 E VLPs were also found to be glycosylated as assessed by protein blot and ELISA using Con A-HRPO ( Figure 2C). Similar to our previous reports for E proteins of DENV-1, 2, and 3 serotypes, the N-terminal amino acid sequence analysis of DENV-4 E protein confirmed that the N-terminal 34 aa residues of prM signal sequence had been efficiently cleaved off (data not shown).…”

“…The generation of serotype-specific neutralizing antibody response is consistent with the immune response pattern generated by the DENV-E VLPs corresponding to the remaining three serotypes, reported earlier. [31][32][33] The DENV-4 E VLP immune serum ( Figure 4B, solid curve) ceased to neutralize DENV-4 once it was depleted of EDIII-4 antibodies by incubation with EDIII-4-MBP ( Figure 4B, dotted curve). On the contrary, there was no significant reduction in the titers of anti-DENV-4 E VLP antiserum when subjected to depletion of antibodies on MBP ( Figure 4B, dashed curve).…”

Recent advances in the production of recombinant subunit vaccines inPichia pastoris

“…P. pastoris-produced DENV-3 envelopebased VLPs not only preserved the antigenic integrity of the major neutralizing epitopes, but also were capable of eliciting neutralizing antibodies against DENV-3. 74 HPV L1 major capsid protein, which self-assembles into VLPs when expressed heterologously, is the main component of the currently available vaccines against cervical cancer. 75 However, the commercial HPV vaccines are expensive and still inaccessible to the majority of the population especially in economically disadvantaged regions.…”