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

Abstract

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.

Protective Malaria Vaccine in Mice Based on the Plasmodium vivax Circumsporozoite Protein Fused with the Mumps Nucleocapsid Protein

“…This carrier was selected after taking into account two facts: the internal protein is weakly recognized even in mumps-vaccinated individuals [31], and the P. pastoris secreted expression system was already successfully used for the expression of mumps NLPs [30]. This is described as one of the most powerful systems for recombinant protein expression, yielding proteins with high purity [44] and the ability to express viral particles in the form of VLPs [45,46]. Using the same strategy, we successfully expressed, characterized, and purified two mumps NLPs carrying PvCSP sequences that remained stable and presented the appropriate structural conformation, as evidenced by the mean diameter of the particle corresponding to mumps nucleocapsid [30], confirmed after scanning electron microscopy.…”

Recent Developments in Recombinant Protein–Based Dengue Vaccines

“…This mVLP elicited EDIII specific virus neutralizing antibodies against Dengue virus 1 and 2 in BALB/c mice. The ADE potential of this mVLP was evaluated in AG129 mice which showed that these VLPs did not induce ADE inducing antibodies ( 89 ). Further, a tetravalent VLP (DSV4) was designed by fusion of EDIII of all the four serotypes and HBsAg, which was co-expressed with unfused S antigen to form mVLP.…”

“…Further, it was also reported that the recombinant subunit vaccine needed a low antigen dose for vaccination ( 11 , 135 ). It also reduces the risk of ADE ( 89 ). In addition, recombinant vaccines are believed to offer full protection in a significantly shorter time-frame than that needed for live attenuated vaccines.…”

Virus-Like Particle Systems for Vaccine Development against Viruses in the Flaviviridae Family

“…Due to the significant limitations on the usage of Dengvaxia, many other tetravalent DENV vaccine candidates, including VLP vaccines, are being extensively studied and tested in the hope of finding another effective vaccine. In the past two decades, a number of DENV VLPs have been evaluated as vaccine candidates [69][70][71][72][73][74][75][76][77][78][79][80][81][82][83][84]. Of which, tetravalent DENV VLP vaccines, recently developed and currently in preclinical testing, possess several features that confer safety, immunogenicity, and production advantages that ideally fulfilled the requirements for a successful DENV vaccine development [81][82][83][84].…”