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. 2018 Jun 5;8:8643. doi: 10.1038/s41598-018-26904-5

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© The Author(s) 2018

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Schematic representation of different P. pastoris-expressed E-based tetravalent VLP dengue subunit vaccine candidates. The picture depicts three approaches to obtaining tetravalent dengue VLP subunit vaccine. (a) The DENV E genes are expressed separately to obtain four serotype-specific monovalent VLPs (1, 2, 3, 4), which elicit predominantly homotypic nAb responses^16–19. Mixing these four together will result in a tetravalent VLP formulation (M-VLP mix). (b) The E genes of two DENV serotypes are co-expressed in a single P. pastoris host to obtain bivalent mVLPs, which elicit predominantly homotypic nAb responses specific to the two DENV serotypes they are derived from²⁰. Mixing two different kinds of such bivalent mVLPs (1 + 2 and 3 + 4), together representing all four DENV serotypes, constitutes a second approach to a tetravalent VLP formulation (B-mVLP mix). (c) The E genes of all four DENV serotypes can be co-expressed in a single tetravalent P. pastoris host to obtain T-mVLPs containing all four E proteins (1 + 2 + 3 + 4). The T-mVLPs represent the third approach. Shown on top are the monovalent (a), bivalent (b), and tetravalent (c) expression cassettes (EC). P and T in each EC denote the AOX1 promoter and terminator, respectively. The E genes of DENV-1, DENV-2, DENV-3 and DENV-4 are shown in magenta, green, blue and black, respectively. The same colour scheme is followed for the corresponding mRNAs, E proteins (in the VLPs) and the nAb responses (FNT₅₀ histograms) to each of the four DENV serotypes. The VLPs, shown below the mRNAs, in panels a, b and c, are monovalent, bivalent and tetravalent, respectively. The DENV serotypes represented in each VLP species is shown by the Arabic numeral below the VLPs. The schematic FNT₅₀ histogram at the bottom depicts the tetravalent nAb response predicted to be elicited by all three VLP candidates.