Dengue E Protein Domain III-Based DNA Immunisation Induces Strong Antibody Responses to All Four Viral Serotypes

Abstract

Dengue virus (DENV) infection is a major emerging disease widely distributed throughout the tropical and subtropical regions of the world affecting several millions of people. Despite constants efforts, no specific treatment or effective vaccine is yet available. Here we show a novel design of a DNA immunisation strategy that resulted in the induction of strong antibody responses with high neutralisation titres in mice against all four viral serotypes. The immunogenic molecule is an engineered version of the domain III (DIII) of the virus E protein fused to the dimerising CH3 domain of the IgG immunoglobulin H chain. The DIII sequences were also codon-optimised for expression in mammalian cells. While DIII alone is very poorly secreted, the codon-optimised fusion protein is rightly expressed, folded and secreted at high levels, thus inducing strong antibody responses. Mice were immunised using gene-gun technology, an efficient way of intradermal delivery of the plasmid DNA, and the vaccine was able to induce neutralising titres against all serotypes. Additionally, all sera showed reactivity to a recombinant DIII version and the recombinant E protein produced and secreted from mammalian cells in a mono-biotinylated form when tested in a conformational ELISA. Sera were also highly reactive to infective viral particles in a virus-capture ELISA and specific for each serotype as revealed by the low cross-reactive and cross-neutralising activities. The serotype specific sera did not induce antibody dependent enhancement of infection (ADE) in non-homologous virus serotypes. A tetravalent immunisation protocol in mice showed induction of neutralising antibodies against all four dengue serotypes as well.

Fig 1. Optimised expression and secretion of DIII.

(A) Scheme of constructs encoding DIII or DIII fused to the dimerising γCH3 domain. In both cases, sec indicates a signal leader peptide. The SV5 tag was included to facilitate detection. (B) Western blot (anti-SV5) of total cellular extracts (E) and supernatants (S) of HEK293T/17 cells transfected with the indicated constructs. (C) Western blot of supernatants (SN) of HEK293T/17 cells transfected with the same amounts of plasmid DNA, showing different secretion levels of the four DIII-CH3 proteins. (D) As in C, supernatants of HEK293T/17 cells transfected with DIII constructs with viral (V) or codon-optimised (CO) nucleotide sequences.

Fig 2. Antibody responses by conformational ELISA.

(A) ELISA reactivity of pooled sera performed on plates coated with the distinct DIII serotypes (left panels) and individual sera of gene-gun immunised mice (OD₄₅₀ at a 1:2700 dilution) compared to the pool (right panels). Ctrl.: negative control sera. Reactivity against 3sE and 4sE proteins, coated at the same molar concentration as DIII, is also shown in anti-3DIII and anti-4DIII left panels. (B) Plot of titres of the four different serotypes pools, determined on DIII and sE (for serotypes 3 and 4).

Fig 3. Anti-DIII sera recognise viral E protein.

(A) Immunofluorescence of Vero cells infected with DENV of each serotype, reacted with the serotype-specific anti-DIII pools of sera (top row), the negative control sera (ctrl., middle row) and mAb 4G2 (bottom row). In each case, non-infected cells (n.i., rightmost column) were also used as controls. Bars represent 50 μm. (B) ELISA on whole infective viral particles. Anti-DIII serotype-specific pools diluted to 100 ng/ml and sera from mock-immunised animals were used onto virus particles captured on plates coated with a human serum reactive against all four serotypes. mAb Dengue 1–11 reactive against DENV1 E (at 1 μg/ml) and a Dengue pan-reactive serum against all four serotypes were used as positive controls.

Fig 4. Anti-DIII sera recognise conformational epitopes with high avidity.

(A) Equal amounts of native or denatured biotinylated DIII-εCH4 were captured on avidin-coated plates and reacted with the corresponding homologous anti-DIII sera. (B) Plot of the titres from the curves shown in A. (C) Avidity index of each anti-DIII sera determined on the native homologous DIII-εCH4. Avidity index of mAb 4G2 on 3sE and 4sE is shown as a control.

Fig 5. Cross-reactivity of anti-DIII sera.

(A) ELISA reactivity of the different serotype specific anti-DIII sera on the four DIII-εCH4 (left panels) and on the two secreted sE (3sE and 4sE), (right panels). (B) Cross-reactive titres from the curves shown in A. * indicates titre below control (absence of cross-reacting antibodies).

Fig 6. Virus-neutralising activity of anti-DIII sera.

(A) Plaque reduction neutralisation test (PRNT) of DENV2 on Vero cells with sera from mice immunised with 2DIII-CH3 or mock-immunised. (B) Plaque reduction curves (left panels) using pools of sera from the different groups of animals, relative to negative control sera with PRNT₅₀ titres of each animal serum (right panels) (* indicates PRNT₅₀ titre is higher than 800). (C) PRNT₅₀ titres from curves shown in B.

Fig 7. Comparison of antibody responses of 3DIII and 3sE antigens.

(A) Western blot of total cellular extracts (E) and supernatants (S) of HEK293T/17 cells transfected with plasmid constructs encoding 3DIII^NOp (~16 kDa), 3DIII^NOp-CH3 (~28 kDa), 3DIII-CH3 (~28 kDa) and 3sE (~54 kDa). (B) ELISA of sera derived from mice gene-gun immunised with 3DIII^NOp, 3DIII^NOp-CH3, 3DIII-CH3 or 3sE (immunising antigens indicated in parenthesis) tested on plates coated with biotinylated versions of 3DIII-εCH4 (3DIII), 3sE and 3DI/DII. (C) Antibody titres determined on each of the different coating proteins, from the curves shown in B (* indicates no reactivity detected). (D) Plot of anti-3sE sera reactivity (from 3sE immunised animals) on the three different coating proteins: 3DIII, 3sE and 3DI/DII. Insert: anti-3sE titres for each coating protein. (E) PRNT₅₀ titres of sera from mice immunised with 3DIII^NOp, 3DIII^NOp-CH3, 3DIII-CH3 or 3sE (immunising antigens indicated in parenthesis) tested on DENV3. (F) Avidity index of antibodies derived from animals gene-gun immunised with 3DIII^NOp, 3DIII^NOp-CH3, 3DIII-CH3 or 3sE (immunising antigens indicated in parenthesis) tested on 3sE-coated plates.

Fig 8. Comparison of antibody-dependent enhancement responses of sera in monocytes.

(A-D) Each serotype-specific anti-DIII pool and control sera were diluted two-fold and incubated with (A) DENV1, (B) DENV2, (C) DENV3 and (D) DENV4 before infecting THP-1 cells for 72h. After which, the culture supernatant was quantified for DENV using plaque assay. Dashed line indicates DENV infection alone of THP-1 cells without the addition of any serum sample.

Fig 9. Tetravalent formulation.

(A) ELISA (left panels) and PRNT₅₀ (right panels) of pools of sera from animals gene-gun immunised with a single DIII-CH3 construct or with the tetravalent formulation. Filled and open symbols indicate monovalent and tetravalent immunisations, respectively. In right panels, curves correspond to the tetravalent vaccine and the PRNT₅₀ titres from the monovalent immunisations (determined in Fig 6) are shown for comparison. (B) ELISA titres (expressed as anti-DIII antibody concentrations) from monovalent and tetravalent immunisations, determined on each serotype. (C) Avidity index of sera from monovalent and tetravalent immunisations, determined on the different DIII serotype antigens.