Development of MVA-d34 Tetravalent Dengue Vaccine: Design and Immunogenicity

Abstract

Dengue fever, an infectious disease that affects more than 100 million people every year, is a global health problem. Vaccination may be the most effective prevention strategy for the disease. However, the development of vaccines against dengue fever is complicated by the high risk of developing an antibody-dependent increase in infection. This article describes the development of an MVA-d34 vaccine against the dengue virus based on a safe and effective MVA viral vector. The DIII domains of the envelope protein (E) of the dengue virus are used as vaccine antigens, as antibodies against these domains do not cause an enhancement of infection. The use of the DIII domains of each of the four dengue virus serotypes made it possible to generate a humoral response against all four dengue virus serotypes in immunized mice. We also showed that the sera of vaccinated mice present virus-neutralizing activity against dengue serotype 2. Thus, the developed MVA-d34 vaccine is a promising candidate vaccine against dengue fever.

Keywords: MVA; dengue; vaccine.

Figure A1

Map of the functional part of the vector pShuttle-136/137. Blocks labeled Shuttle 1 and Shuttle 2 show the division of the design into fragments, necessary for the synthesis of the sequence. p11 and mH5 promoters from the vaccinia virus drive the expression of RFP and GOI, respectively. The vaccinia virus termination signal (labeled as Term on the scheme) was used to stop the transcription of RFP gene and GOI. The DNA sequence of GOI can be inserted using restriction sites NheI/KpnI.

Figure 1

Multiple sequence alignment of the DIII domain amino acid residues of the dengue virus serotypes 1 (aa 578–676, record ID NP_059433), 2 (aa 578–676, record ID NP_056776), 3 (aa 576–674, record ID YP_001621843), and 4 (aa 577–675, record ID NP_073286) used to obtain the d34 antigen sequence. The color shows the similarity in the sequence.

Figure 2

Schematic of the structure (A) of the antigen d34 and (B) expression cassette located in the MVA genome. Leader—the mouse Igk leader; linker—2x(GGGGS). The light box with the black box is the left homology arm (Left HA), the black box is the short left homology arm (Short HA), and the pink box is the right homology arm (Right HA). Gray boxes represent p11 and mH5 promoters from the vaccinia virus, driving the expression of RFP and d34 antigens, respectively. Vaccinia virus termination signal TTTTTNT (labeled as Term on the scheme) was used for stopping the transcription of RFP and d34 ORFs. More details can be found in Section 2.2.

Figure 3

Western blot analysis of d34 antigen expression in BHK-21 cells after infection with the MVA-wt virus or recombinant rMVA-d34 virus (MOI = 2) using anti-6x-His Tag antibodies for detection. M is a marker of the molecular weight of proteins. K- indicates uninfected BHK-21 cells. The molecular weight of the d34 protein is 48.3 kDa. M-ECL™ Rainbow™ marker.

Figure 4

Analysis of the interaction of the d34 antigen with blood sera using Western blotting. HEK293FT cells were infected with MVA (wt), recombinant MVA-d34 (d34), or mock (K−). The d34 antigen was probed with (A) anti-6x-His Tag antibodies (anti-His); (B) dengue fever-positive human sera (serum 1); (C) human sera from a TBEV-vaccinated individual (serum 2); (D) human sera from a TBEV- and YFV-vaccinated individual (serum 3); and (E) human sera from a YFV-vaccinated individual (serum 4). The numbers on the sides correspond to the protein molecular weight marker (M-PageRuler™ Plus Prestained Protein marker). K cells, HEK. The weight of the d34 protein is 48.3 kDa.

Figure 5

Antibody titer in blood sera of mice immunized with MVA-d34 (N = 5) or wt MVA (N = 5) measured via in-house indirect d34 ELISA. The mean value is shown by a thick line. The asterisk denotes a statistically significant difference between MVA-d34 group and wt MVA control group (p-value < 0.05 according to the Mann-Whitney U test).