Virus-like particles derived from Pichia pastoris-expressed dengue virus type 1 glycoprotein elicit homotypic virus-neutralizing envelope domain III-directed antibodies

Background: 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. Results: 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. Conclusions: 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

Recombinant dengue virus 4 envelope glycoprotein Virus-like Particles derived from pichia pastoris are capable of eliciting homotypic domain III-directed neutralizing antibodies

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

Pichia pastoris-Expressed Bivalent Virus-Like Particulate Vaccine Induces Domain III-Focused Bivalent Neutralizing Antibodies without Antibody-Dependent Enhancement in Vivo

Dengue, a significant public health problem in several countries around the world, is caused by four different serotypes of mosquito-borne dengue viruses (DENV-1, -2, -3, and -4). Antibodies to any one DENV serotype which can protect against homotypic re-infection, do not offer heterotypic cross-protection. In fact, cross-reactive antibodies may augment heterotypic DENV infection through antibody-dependent enhancement (ADE). A recently launched live attenuated vaccine (LAV) for dengue, which consists of a mixture of four chimeric yellow-fever/dengue vaccine viruses, may be linked to the induction of disease-enhancing antibodies. This is likely related to viral interference among the replicating viral strains, resulting in an unbalanced immune response, as well as to the fact that the LAV encodes prM, a DENV protein documented to elicit ADE-mediating antibodies. This makes it imperative to explore the feasibility of alternate ADE risk-free vaccine candidates. Our quest for a non-replicating vaccine centered on the DENV envelope (E) protein which mediates virus entry into the host cell and serves as an important target of the immune response. Serotype-specific neutralizing epitopes and the host receptor recognition function map to E domain III (EDIII). Recently, we found that Pichia pastoris-expressed DENV E protein, of all four serotypes, self-assembled into virus-like particles (VLPs) in the absence of prM. Significantly, these VLPs displayed EDIII and elicited EDIII-focused DENV-neutralizing antibodies in mice. We now report the creation and characterization of a novel non-replicating recombinant particulate vaccine candidate, produced by co-expressing the E proteins of DENV-1 and DENV-2 in P. pastoris. The two E proteins co-assembled into bivalent mosaic VLPs (mVLPs) designated as mE1E2bv VLPs. The mVLP, which preserved the serotype-specific antigenic integrity of its two component proteins, elicited predominantly EDIII-focused homotypic virus-neutralizing antibodies in BALB/c mice, demonstrating its efficacy. In an in vivo ADE model, mE1E2bv VLP-induced antibodies lacked discernible ADE potential, compared to the cross-reactive monoclonal antibody 4G2, as evidenced by significant reduction in the levels of IL-6 and TNF-α, suggesting inherent safety. The results obtained with these bivalent mVLPs suggest the feasibility of incorporating the E proteins of DENV-3 and DENV-4 to create a tetravalent mVLP vaccine

Next generation designer virus-like particle vaccines for dengue

Introduction: A safe and efficacious vaccine for dengue continues to be an unmet public health need. The recent licensing of a dengue vaccine (Dengvaxia) developed by Sanofi has brought to the fore the safety issue of vaccine-induced infection enhancement. Areas covered: This article focuses on two new yeast-produced tetravalent dengue envelope domain III-displaying virus-like particulate vaccine candidates reported in early 2018 and reviews the rationale underlying their design, and pre-clinical data which suggest that these may offer promising alternate options. Expert commentary: These are the only vaccine candidates so far to have demonstrated the induction of primarily serotype-specific neutralizing antibodies to all dengue virus serotypes in experimental animals. Interestingly, these antibodies lack infection-enhancing potential when evaluated using the AG129 mouse model

Additional file 1: Figure S1. of Virus-like particles derived from Pichia pastoris-expressed dengue virus type 1 glycoprotein elicit homotypic virus-neutralizing envelope domain III-directed antibodies

Cloning and expression of DENV-1 E gene into shuttle vector pPICZA. Figure S2. Purification and characterization of recombinant DENV-1 E antigen. Figure S3. Sequence alignment of P. pastoris optimized DENV-1, 2, 3, 4 E showing similarities and differences in the amino acid sequences between four dengue serotypes. (DOC 1608 kb

Dengue-specific subviral nanoparticles: design, creation and characterization

Abstract Background 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. Methods 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. Results 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. Conclusions VLPs armed with EDIII may be potentially useful in diagnostic, therapeutic and prophylactic applications

Dengue-specific subviral nanoparticles: design, creation and characterization.

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