Enhancement of Dengue-2 E Protein Expression by the Expression of the Precursor Membrane Protein (Prm) of the Dengue-3 Virus

Abstract

Several attempts to develop dengue recombinant subunit vaccines have failed due to insufficient levels of expression and incorrect folding of the E protein. In order to verify the importance of the precursor membrane protein to the level of E protein expression, we constructed two recombinant plasmids by cloning the full-length sequence of the prM gene from the dengue-2 and dengue-3 virus strains into a plasmid that expresses a truncated version of the E protein of the dengue-2 virus. Next, we evaluated these two constructs for their effect on the levels of E protein expression in vitro. Our results showed that both plasmids provided a correct expression of the E protein as E protein expression was detected in transfected Vero cells as demonstrated by indirect immunofluorescence and immunoblotting. Densitometry analysis of western blots of the cell extracts showed a 67.02% higher expression of E protein in the cells transfected with pCID2EtD3prM, indicating that the prM sequence of the dengue-3 virus may be more effective in assisting with the correct processing of the E protein. The results of this study may be used to increase the in vitro production of the E protein antigen for use in vaccines and for diagnostic purposes.

DENV-3 precursor membrane (prM) glycoprotein enhances E protein immunogenicity and confers protection against DENV-2 infections in a murine model

“…This is probably due to increased expression of the E protein, corroborating the results obtained in vitro . 20 Despite high values found in ELISA assay Figure 1, neutralizing antibodies titers were low Figure 2. These results were observed to an inactivated H7 Influenza virus vaccine, which was able to confer protection and can be related to the non-neutralizing antibodies production.…”

“…Moreover, it is known that much of the research involving plasmid constructs expressing DENV-3 prM/E protein showed better results than those in which the plasmids contained the DENV-2 prM/E expression cassette. 20,25,34 These observations suggest that prM/DENV-3 protein maybe have a greater capacity as chaperone than prM/ DENV-2, since it is known that prM plays a fundamental role in the synthesis, processing, and correct conformation of E protein. 35,36 In order to investigate this possibility, two plasmids were constructed by insertion of the prM/DENV-2 and prM/ DENV-3 genes into the plasmid vector pCID2Et, previously constructed to express the truncated E/DENV-2 protein.…”

“…18,19 Our work group has previously reported the expression of the truncated envelope (E) protein in VERO cells by two constructions which have a prM of the two Dengue virus serotype (DENV-2 and DENV-3) genes upstream of the E gene. 20 Higher yield was obtained by the vaccine candidate which expresses the prM from the DENV-3 serotype (around 67% more) (data not shown), which suggests that this protein is a better chaperone than the polymorphic prM from DENV-2.…”

“…These cassettes for DENV-2 and DENV-3 were called pCID2EtprMD2 and pCID2EtprMD3, respectively. 20,21 The DENV-2 strain New Guinea C (ATCC™ VR-1584™) and the DENV-3 strain H87 (ATCC™ VR-1256_FD™) were propagated in C6/36 cells. In the experiments were used Swiss mice, which were fed ad libitum and maintained under standard conditions.…”