In 2019, the United States Food and Drug Administration accorded restricted approval to Sanofi Pasteur's Dengvaxia, a live attenuated vaccine (LAV) for dengue fever, a mosquito-borne viral disease, caused by four antigenically distinct dengue virus serotypes (DENV 1-4). The reason for this limited approval is the concern that this vaccine sensitized some of the dengue-naïve recipients to severe dengue fever. Recent knowledge about the nature of the immune response elicited by DENV viruses suggests that all LAVs have inherent capacity to predominantly elicit antibodies (Abs) against the pre-membrane (prM) and fusion loop epitope (FLE) of DENV. These antibodies are generally cross-reactive among DENV serotypes carrying a higher risk of promoting Antibody-Dependent Enhancement (ADE). ADE is a phenomenon in which suboptimal neutralizing or non-neutralizing cross-reactive antibodies bind to virus and facilitate Fcγ receptor mediated enhanced entry into host cells, followed by its replication, and thus increasing the cellular viral load. On the other hand, antibody responses directed against the host-cell receptor binding domain of DENV envelope domain-III (EDIII), exhibit a higher degree of type-specificity with lower potential of ADE. The challenges associated with whole DENV-based vaccine strategies necessitate re-focusing our attention toward the designed dengue vaccine candidates, capable of inducing predominantly type-specific immune responses. If the designed vaccines elicited predominantly EDIII-directed serotype specific antibodies in the absence of prM and FLE antibodies, this could avoid the ADE phenomenon largely associated with the prM and FLE antibodies. The generation of type-specific antibodies to each of the four DENV serotypes by the designed vaccines could avoid the immune evasion mechanisms of DENVs. For the enhanced vaccine safety, all dengue vaccine candidates should be assessed for the extent of type-specific (minimal ADE) vs. cross-reactive (ADE promoting) neutralizing antibodies. The type-specific EDIII antibodies may be more directly related to protection from disease in the absence of ADE promoted by the cross-reactive antibodies.

Background: Pichia pastoris is an established eukaryotic host for the production of recombinant proteins. Most often, protein production is under the control of the strong methanol-inducible aox1 promoter. However, detailed information about the physiological alterations in P. pastoris accompanying the shift from growth on glycerol to methanol-induced protein production under industrial relevant conditions is missing. Here, we provide an analysis of the physiological response of P. pastoris GS115 to methanol-induced high-level production of the Hepatitis B virus surface antigen (HBsAg). High product titers and the retention of the protein in the endoplasmic reticulum (ER) are supposedly of major impact on the host physiology. For a more detailed understanding of the cellular response to methanol-induced HBsAg production, the time-dependent changes in the yeast proteome and ultrastructural cell morphology were analyzed during the production process. Results: The shift from growth on glycerol to growth and HBsAg production on methanol was accompanied by a drastic change in the yeast proteome. In particular, enzymes from the methanol dissimilation pathway started to dominate the proteome while enzymes from the methanol assimilation pathway, e.g. the transketolase DAS1, increased only moderately. The majority of methanol was metabolized via the energy generating dissimilatory pathway leading to a corresponding increase in mitochondrial size and numbers. The methanol-metabolism related generation of reactive oxygen species induced a pronounced oxidative stress response (e.g. strong increase of the peroxiredoxin PMP20). Moreover, the accumulation of HBsAg in the ER resulted in the induction of the unfolded protein response (e.g. strong increase of the ER-resident disulfide isomerase, PDI) and the ER associated degradation (ERAD) pathway (e.g. increase of two cytosolic chaperones and members of the AAA ATPase superfamily) indicating that potential degradation of HBsAg could proceed via the ERAD pathway and through the proteasome. However, the amount of HBsAg did not show any significant decline during the cultivation revealing its general protection from proteolytic degradation. During the methanol fed-batch phase, induction of vacuolar proteases (e.g. strong increase of APR1) and constitutive autophagic processes were observed. Vacuolar enclosures were mainly found around peroxisomes and not close to HBsAg deposits and, thus, were most likely provoked by peroxisomal components damaged by reactive oxygen species generated by methanol oxidation. Conclusions: In the methanol fed-batch phase P. pastoris is exposed to dual stress; stress resulting from methanol degradation and stress resulting from the production of the recombinant protein leading to the induction of oxidative stress and unfolded protein response pathways, respectively. Finally, the modest increase of methanol assimilatory enzymes compared to the strong increase of methanol dissimilatory enzymes suggests here a potential to increase methanol incorporation into biomass/product through metabolic enhancement of the methanol assimilatory pathway.

Dengue is one of the fastest spreading vector-borne diseases, caused by four antigenically distinct dengue viruses (DENVs). Antibodies against DENVs are responsible for both protection as well as pathogenesis. A vaccine that is safe for and efficacious in all people irrespective of their age and domicile is still an unmet need. It is becoming increasingly apparent that vaccine design must eliminate epitopes implicated in the induction of infection-enhancing antibodies. We report a Pichia pastoris-expressed dengue immunogen, DSV4, based on DENV envelope protein domain III (EDIII), which contains well-characterized serotype-specific and cross-reactive epitopes. In natural infection, <10% of the total neutralizing antibody response is EDIII-directed. Yet, this is a functionally relevant domain which interacts with the host cell surface receptor. DSV4 was designed by in-frame fusion of EDIII of all four DENV serotypes and hepatitis B surface (S) antigen and co-expressed with unfused S antigen to form mosaic virus-like particles (VLPs). These VLPs displayed EDIIIs of all four DENV serotypes based on probing with a battery of serotype-specific anti-EDIII monoclonal antibodies. The DSV4 VLPs were highly immunogenic, inducing potent and durable neutralizing antibodies against all four DENV serotypes encompassing multiple genotypes, in mice and macaques. DSV4-induced murine antibodies suppressed viremia in AG129 mice and conferred protection against lethal DENV-4 virus challenge. Further, neither murine nor macaque anti-DSV4 antibodies promoted mortality or inflammatory cytokine production when passively transferred and tested in an in vivo dengue disease enhancement model of AG129 mice. Directing the immune response to a non-immunodominant but functionally relevant serotype-specific dengue epitope of the four DENV serotypes, displayed on a VLP platform, can help minimize the risk of inducing disease-enhancing antibodies while eliciting effective tetravalent seroconversion. DSV4 has a significant potential to emerge as a safe, efficacious and inexpensive subunit dengue vaccine candidate. Dengue is mosquito-borne viral disease which is currently a global public health problem. It is caused by four different types of dengue viruses. Nearly a 100 million people a year suffer from overt sickness, which may range from mild fever to potentially fatal disease. A virus-based dengue vaccine was launched for the first time in late 2015. Unexpectedly, this vaccine mimics the dengue viruses in that it appears to elicit disease-enhancing antibodies. To reduce such risk, safer vaccines that eliminate viral proteins responsible for undesirable antibodies are needed. We focused our attention on a small domain of the dengue virus surface protein known as envelope domain III (EDIII). Humans make only a small amount of antibodies against EDIII, but these antibodies are effective in blocking dengue virus from entering cells. We used a yeast expression system to display EDIIIs of all four types of dengue viruses on the surface of non-infectious virus-like particles (VLPs). These VLPs elicited antibodies, in mice and monkeys, which blocked all four dengue virus types and their variants from entering cells in culture. Importantly, these antibodies did not enhance dengue infection in a mouse model.

There is currently no vaccine to prevent dengue (DEN) virus infection, which is caused by any one of four closely related serotypes, DEN-1, DEN-2, DEN-3, or DEN-4. A DEN vaccine must be tetravalent, because immunity to a single serotype does not offer cross-protection against the other serotypes. We have developed a novel tetravalent chimeric protein by fusing the receptor-binding envelope domain III (EDIII) of the four DEN virus serotypes. This protein was expressed in the yeast, Pichia pastoris, and purified to near homogeneity in high yields. Antibodies induced in mice by the tetravalent protein, formulated in different adjuvants, neutralized the infectivity of all four serotypes. This, coupled with the high expression potential of the P. pastoris system and easy one-step purification, makes the EDIII-based recombinant protein a potentially promising candidate for the development of a safe, efficacious, and inexpensive, tetravalent DEN vaccine.

Background: The prevalence of diabetes is predicted to rise significantly in the coming decades. A recent analysis projects that by the year 2030 there will be ~366 million diabetics around the world, leading to an increased demand for inexpensive insulin to make this life-saving drug also affordable for resource poor countries.Results: A synthetic insulin precursor (IP)-encoding gene, codon-optimized for expression inP. pastoris, was cloned in frame with theSaccharomyces cerevisiaeα-factor secretory signal and integrated into the genome ofP. pastorisstrain X-33. The strain was grown to high-cell density in a batch procedure using a defined medium with low salt and high glycerol concentrations. Following batch growth, production of IP was carried out at methanol concentrations of 2 g L^-1, which were kept constant throughout the remaining production phase. This robust feeding strategy led to the secretion of ~3 gram IP per liter of culture broth (corresponding to almost 4 gram IP per liter of cell-free culture supernatant). Using immobilized metal ion affinity chromatography (IMAC) as a novel approach for IP purification, 95% of the secreted product was recovered with a purity of 96% from the clarified culture supernatant. Finally, the purified IP was trypsin digested, transpeptidated, deprotected and further purified leading to ~1.5 g of 99% pure recombinant human insulin per liter of culture broth.Conclusions: A simple two-phase cultivation process composed of a glycerol batch and a constant methanol fed-batch phase recently developed for the intracellular production of the Hepatitis B surface antigen was adapted to secretory IP production. Compared to the highest previously reported value, this approach resulted in an ~2 fold enhancement of IP production usingPichiabased expression systems, thus significantly increasing the efficiency of insulin manufacture.

Japanese encephalitis virus (JEV) is the causal agent behind Japanese encephalitis (JE), a potentially severe brain infection that spreads through mosquito bites. JE is predominant over the Asia-Pacific Region and has the potential to spread globally with a higher rate of morbidity and mortality. Efforts have been made to identify and select various target molecules essential in JEV’s progression, but until now, no licensed anti-JEV drug has been available. From a prophylactic point of view, a few licensed JE vaccines are available, but various factors, viz., the high cost and different side effects imposed by them, has narrowed their global use. With an average occurrence of >67,000 cases of JE annually, there is an urgent need to find a suitable antiviral drug to treat patients at the acute phase, as presently only supportive care is available to mitigate infection. This systematic review highlights the current status of efforts put in to develop antivirals against JE and the available vaccines, along with their effectiveness. It also summarizes epidemiology, structure, pathogenesis, and potential drug targets that can be explored to develop a new range of anti-JEV drugs to combat JEV infection globally.