Secretion of dengue virus envelope protein ectodomain from mammalian cells is dependent on domain II serotype and affects the immune response upon DNA vaccination

Abstract

Dengue virus (DENV) is currently among the most important human pathogens and affects millions of people throughout the tropical and subtropical regions of the world. Although it has been a World Health Organization priority for several years, there is still no efficient vaccine available to prevent infection. The envelope glycoprotein (E), exposed on the surface on infective viral particles, is the main target of neutralizing antibodies. For this reason it has been used as the antigen of choice for vaccine development efforts. Here we show a detailed analysis of factors involved in the expression, secretion and folding of E ectodomain from all four DENV serotypes in mammalian cells, and how this affects their ability to induce neutralizing antibody responses in DNA-vaccinated mice. Proper folding of E domain II (DII) is essential for efficient E ectodomain secretion, with DIII playing a significant role in stabilizing soluble dimers. We also show that the level of protein secreted from transfected cells determines the strength and efficiency of antibody responses in the context of DNA vaccination and should be considered a pivotal feature for the development of E-based DNA vaccines against DENV.

Temperature-dependent folding allows stable dimerization of secretory and virus-associated E proteins of Dengue and Zika viruses in mammalian cells

“…We have previously reported a detailed analysis of the secretory profile of recombinant E derived from the different DENV serotypes in mammalian cells and shown that DENV2 E ectodomain (2sE) undergoes different folding depending on temperature 36 . Here we have further investigated the role of temperature and PrM in the production and secretion of sE from DENV and ZIKV in transfected HEK293T cells, using constructs encoding sE alone or sE and the viral proteins PrM or only M (Fig.…”

“…The same set of reagents was used for cytofluorimetric analysis of cell membrane display of sE-cvD ((m)sE-cvD), achieved by fusing the trans-membrane and cytosolic tail of the type-I trans-membrane protein MHC-Iα at the C-terminus of sE, as previously reported 36 . Transfected cells were incubated with the set of mAbs following expression of (m)sE-cvD (Fig.…”

“…We have previously reported that DENV serotypes showing sE poor secretory phenotypes were misfolded at 37 °C but properly folded at 28 °C 36 . Our data indicate that, while proper folding is heavily influenced by temperature, secretion of recombinant E proteins depends on their capacity to form relatively stable dimers as they transit through the ER and the secretory pathway.…”

“…Given the importance of complex quaternary epitopes for inducing potent neutralizing antibodies and based on our previous work regarding the secretory profile of DENV sE from mammalian cells 36 , we set out to analyze dimerization of DENV and ZIKV E protein. Although each of the proteins tested showed a characteristic secretory behavior, we found in all cases that intracellular dimerization of newly synthesized sE was highly dependent on temperature but independent of the presence of PrM.…”

Novel recombinant DNA vaccine candidates for human respiratory syncytial virus: Preclinical evaluation of immunogenicity and protection efficiency

“…22 Removal of the transmembrane domain also provided superior immunogenicity in vivo compared with the full-length F gene. 43,44 Expression of the immuno-dominant epitope of the HRSV M2 protein (residues 82-90) in conjunction with the F or G proteins induced a potent CTL response and a balanced Th1/Th2 response in mice.…”