Casamino acids facilitate the secretion of recombinant dengue virus serotype-3 envelope domain III in Pichia pastoris

Background: Dengue is a viral disease spread to humans by mosquitoes. Notably, there are four serotypes of Dengue Viruses (DENV) that places ∼40% of the global population at risk of infection. However, lack of a suitable drug or a preventive vaccine exacerbates the matter further. Envelope Domain-III (EDIII) antigen of Dengue Virus (DENV) has garnered much attention as a promising vaccine candidate for dengue, in addition to its use as a diagnostic intermediate. Hence developing a method for efficient production of high quality recombinant EDIII is important for research and industrial purpose. Results: In this work, a Pichia pastoris system was optimized for the secretory over-expression of DENV serotype-3 EDIII under the control of methanol inducible AOX1 promoter. Temperature alone had a significant impact upon the amount of secretory EDIII, with 2.5-fold increase upon reducing the induction temperature from 30 to 20 °C. However surprisingly, supplementation of culture media with Casamino Acids (CA), further augmented secretory EDIII titer, with a concomitant drop of intracellular EDIII levels at both temperatures. Though, reduction in intracellular retention of EDIII was more prominent at 20°C than 30°C. This suggests that CA supplementation facilitates overexpressing P. pastoris cells to secrete more EDIII by reducing the proportion retained intracellularly. Moreover, a bell-shaped correlation was observed between CA concentration and secretory EDIII titer. The maximum EDIII expression level of 187 mg/L was achieved under shake flask conditions with induction at 20°C in the presence of 1% CA. The overall increase in EDIII titer was ∼9-fold compared to un-optimized conditions. Notably, mouse immune-sera, generated using this purified EDIII antigen, efficiently neutralized the DENV. Conclusions: The strategy described herein could enable fulfilling the mounting demand for recombinant EDIII as well as lay direction to future studies on secretory expression of recombinant proteins in P. pastoris with CA as a media supplement

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

Virus-like Particles displaying envelope domain III of dengue virus type 2 induce virus-specific antibody response in mice

Objective: Currently, dengue represents one of the most significant arboviral disease worldwide, for which a vaccine is not yet available. Persistent challenges in live viral dengue vaccines have sparked a keen interest in exploring non-replicating dengue vaccines. We have examined the feasibility of using the methylotrophic yeast Pichia pastoris to develop a chimeric vaccine candidate displaying the dengue virus type-2 (DENV-2) Envelope Domain III (EDIII), implicated in host receptor binding and in the induction of virus-neutralizing antibodies, on the surface of non-infectious Virus-like Particles (VLP)-based on the Hepatitis B virus core antigen (HBcAg). Methods: We designed a fusion antigen by inserting DENV-2 EDIII into c/e1 loop of HBcAg. A codon-optimized gene encoding this fusion antigen was integrated into the genome of P. pastoris, under the control of the Alcohol Oxidase 1 promoter. The antigen was expressed by methanol induction and purified to near homogeneity by Ni²⁺ affinity chromatography. The purified antigen was characterized physically and functionally to evaluate its ability to assemble into VLPs and elicit DENV-2-specific antibodies in mice. Results: This fusion antigen was expressed successfully to high yields and purified to near homogeneity. Electron microscopy and competitive ELISA analyses showed that it formed VLPs in which the EDIII moiety was accessible to different EDIII-specific antibodies. These VLPs were immunogenic in mice, stimulating the production of antibodies that could specifically recognize DENV-2 and neutralize its infectivity. However, virus-neutralizing antibody titers were modest. Conclusions: Our data show: (i) insertion of EDIII into the c/e1 loop of HBcAg does not compromise particle assembly and (ii) the chimeric VLPs elicit a specific humoral response against DENV-2. The strategy of displaying dengue virus EDIII using a VLP platform will need further optimization before it may be developed into a viable alternative option

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

Legal aspects of the criminal offence of poaching

The purpose of this thesis is to provide a detailed analysis of a relatively narrow issue belonging to the criminal law, namely the criminal offence of poaching. The key part of this work examines the merits of § 304 of the Act no. 40/2009, The Criminal Code, and focuses also on its systematic inclusion in the head VIII, called Crimes against the Environment, constituting a part of the special section of the aforementioned code. I attempt to gather the existing conclusions of the doctrine as well as the jurisprudence and to present them in an organized way. I also endeavour to critically assess a number of selected passages and to add my own reflections. In addition, several practical examples are briefly highlighted in order to enrich this work. This thesis will be divided into four main chapters - the Introduction, the Poaching legislation de lege lata, the Poaching legislation de lege ferenda and the Conclusion. Regarding the introduction, the reasons which led me to choose and critically analyse this socially negative phenomenon are shortly outlined. Furthermore, I explain my view of poaching as a long-lasting problem in our society. With respect to the chapter dealing with the poaching legislation de lege lata, it forms the main part of this thesis. Firstly, I aim to consistently categorize..

Sinococuline, a bioactive compound of Cocculus hirsutus has potent anti-dengue activity

Abstract Dengue virus (DENV) infection has increased worldwide, with over 400 million infections annually, and has become a serious public health concern. Several drug candidates, new and repurposed, have failed to meet the primary efficacy endpoints. We have recently shown that Aqueous Extract of the stem of Cocculus hirsutus (AQCH) was effective in vitro and in vivo against DENV and was safe in humans. We now report that an active ingredient of AQCH, Sinococuline, protects against the antibody-mediated secondary-DENV infection in the AG129 mouse model. DENV infection markers were assessed, viz. serum viremia and vital organs pathologies-viral load, proinflammatory cytokines and intestinal vascular leakage. The treatment with Sinococuline at 2.0 mg/kg/day; BID (twice a day), was the most effective in protecting the severely DENV-infected AG129 mice. Also, this dose effectively reduced serum viremia and tissue-viral load and inhibited the elevated expression levels of proinflammatory cytokines (TNF-α and IL-6) in several vital organs. Based on these findings, it could be explored further for pre-clinical and clinical developments for the treatment of dengue