Hum Vaccin Immunother. 2013 Nov;9(11):2326-35

DENV-2 subunit proteins fused to CR2 receptor-binding domain (P28)-induces specific and neutralizing antibodies to the Dengue virus in mice.

García-Machorro J1, López-González M, Barrios-Rojas O, Fernández-Pomares C, Sandoval-Montes C, Santos-Argumedo L, Villegas-Sepúlveda N, Gutiérrez-Castañeda B, Cedillo-Barrón L.

1Department of Molecular Biomedicine Centre for Research and Advanced Studies (CINVESTAV-IPN) Av. IPN # 2508 Col.; San Pedro Zacatenco, D.F. Mexico.



Domain III (DIII) of the dengue virus (DENV) envelope (E) protein induces strong neutralizing type-specific antibodies. In addition, a region near the fusion loop in domain II (DII) induces the production of cross-reactive antibodies with neutralizing potential. Thus, this study aimed to generate DENV-2 recombinant fusion proteins (i.e., rEII*EIII and rEII*EIII/NS1*) either alone or fused to 3 copies of P28, the minimum CR2-binding domain of the complement protein C3d. The 4 recombinant proteins were generated in a Drosophila melanogaster Schneider 2 (S2) cell system. The expression and secretion of the recombinant proteins were confirmed in vitro using immunofluorescence (IF) and western blot (WB) analyses. Human dengue immune serum samples recognized recombinant proteins. The immunogenicity of the 4 proteins in BALB/c mice was analyzed using ELISA, and the results revealed that the induced specific antibody response was higher in the groups of mice immunized with the P28 fusion proteins. Interestingly, although the 4 recombinant proteins were able to elicit high levels of neutralizing antibodies in BALB/c mice; no adjuvant effect was observed in terms of neutralizing antibodies in the groups immunized with proteins containing P28. Thus, ELISA and PRNT50 assays may evaluate different epitopes and responses, where ELISA showed a wider response that did not always correlate with neutralization. Furthermore, the elicited antibodies were able to recognize the immobilized E glycoprotein of DENV. All mice vaccinated with the DENV-2 recombinant proteins showed induction of higher levels of IgG1 antibodies than of IgG2a antibodies.



Dengue virus (DENV) infection is a re-emergent disease and a public health problem (1). In spite of years of effort to develop a dengue vaccine, no licensed vaccine is available to date (2). Substantial evidences indicate that envelop (E) protein, a viral surface protein, is the most immunogenic protein of the DENV. In addition, several studies have identified regions in E protein involved in inducing protective immune response (3).

The long-term goal of our work involves the evaluation of targets to be included in subunit vaccines against the DENV. Previously, we designed a subunit pcDNAEII*EIII/NS1* construct and inoculated it into a mouse model. This was a promising approach because the EII* domain included a region containing a fusogenic peptide and vicinal regions, while the EIII domain included a recognition site for host cell receptors. Moreover, the construct included 75 amino acids from the N-terminus of the NS1* domain, a strongly immunogenic region, fused upstream of the EIII domain. A study showed that the NS1* domain does not cross-react with receptors present on endothelial cells (4).

An illustration of the pcDNA3/EII*EIII/NS1* construct obtained using bioinformatics tools is shown in Fig 1. However, this construct induced a limited immune response in the mouse model (4). To overcome this, we expressed the same proteins in the Drosophila S2 system in the present study. In addition, we used an adjuvant molecule P28, a small peptide from C3d complement, which represent the minimum CD21-binding domain that induces a robust immune response by increasing the magnitude and quality of specific antibodies. P28 interacts with complement receptor CR2 (CD21) expressed on B cells and follicular dendritic cells (Figure 2)(6).

We constructed 4 DENV-2 recombinant plasmids expressing the above proteins (i.e., rEII*EIII and rEII*EIII/NS1*) either alone or fused with 3 copies of P28. Figure 3 shows the 4 plasmids. S2 cells were transfected with these plasmids, and the recombinant proteins were purify from stably transfected cells and were purified Next, we examined whether the recombinant DENV proteins were recognized by immune sera pooled from patients with dengue and healthy donors and we demonstrated that the recombinant dengue proteins are recognized only by human dengue immune serum, but no by healthy donors.

We used groups of 10 BALB/c mice, which were immunized with the four recombinant proteins rEII*EIII/NS1* alone or fused with 3 copies of C3d (P28)3 or rEII*EIII alone or fused with P28. We administered 3 doses of rEII*EIII/NS1*, rEII*EIII/NS1*(P28)3, EII*EIII, and EII*EIII (P28)3. We observed that all the fusion proteins induced a robust immune response, with IgG1-type response being the predominant response, against wild-type DENV in BALB/c mice. Furthermore, the fusion proteins induced an effective neutralizing antibody response similar to that in mice immunized with DENV-2. Our results suggested that P28 conjugated EII*EIII/NS1* not only increased the levels of antibodies against the viral E protein but also accelerated the affinity maturation of these antibodies. In conclusion, a recombinant DENV-2 protein containing relevant wild-type sequences fused with 3 copies of P28 induced an effective Th2 response based on neutralizing antibody.


Acknowledgments: We thank Dr T. Ross for providing the plasmid encoding CR2-binding domain of murine complement C3d (P28). The authors would like to thank Julio García Cordero for his technical assistance. This work was supported by the National Council for Science and Technology (CONACyTGrant FONSEC0115401). Additionally, JGM and MLG received fellowships from CONACyT. LCB, LSA, NVS, are members of the National System of Researchers, SNI.



1.-PAHO Dengue regional information: Number of cases. mid=363. PAHO (2011).

2.- MegretF, HugnotJP, FalconarA, GentryMK, MorensDM, MurrayJM, et al.Use of recombinant fusion proteins and monoclonal antibodies to define linear and discontinuous antigenic sites on the den- gue virus envelope glycoprotein.Virology1992; 187:480-91; PMID:1372140; http://dx.doi. org/10.1016/0042-6822(92)90450-4.

3.- BeltramelloM, WilliamsKL, SimmonsCP, MacagnoA, SimonelliL, QuyenNT, et al.The human immune response to Dengue virus is domi- nated by highly cross-reactive antibodies endowed with neutralizing and enhancing activity.Cell Host Microbe2010; 8:271-83; PMID:20833378; http://

4.-Mellado-SánchezG, García-MachorroJ, Sandoval- MontesC, Gutiérrez-CastañedaB, Rojo- DomínguezA, García-CorderoJ, et al.A plasmid encoding parts of the dengue virus E and NS1 pro- teins induces an immune response in a mouse model. Arch Virol2010; 155:847-56; PMID:20390312;

5.-BowerJF, RossTM. A minimum CR2 bind- ing domain of C3d enhances immunity fol- lowing vaccination.AdvExp Med Biol2006; 586:249-64; PMID:16893077; http://dx.doi. org/10.1007/0-387-34134-X_17

6.-Toapanta FR and Ross TM. Complement mediated activation of adaptive immune response: role of c3d in linking the innate and the adaptive immunity. Immunol Res 2006 36(1-6)197- 2010 PMID:17337780

Diapositiva FIG1Figure 1. Predicted model of EII*EIII/NS1* chimerical protein, obtained with bioinformatic algorithms. The modeling showed three structural domains (EII*, EIII and NS1*) 80 amino acids insertion EII* highlighted in blue, 129 aa insertion EIII highlighted in red, and 73 amino acids insertion NS1* highlighted in yellow.

Diapositiva FIG2Figure 2. Schematic diagram of DENV-2 sequences used for the construction of the recombinant plasmid pEII*EIII/NS1*. Black arrow represents EII* region, gray arrow represents EIII region, and segmented arrow represents NS1* region from the N terminus of NS1 protein.

Diapositiva FIG3Figure 3. C3d enhancement of immune responses. DENV protein fused with P28 interact with B cells through the surface IgM (sIgM) and CR2. Co-ligation of these two receptors activate pathways that cross-talk and conduct to activation of B cell. The scheme was modified from the original review (6).



Multiselect Ultimate Query Plugin by InoPlugs Web Design Vienna | Webdesign Wien and Juwelier SchönmannMultiselect Ultimate Query Plugin by InoPlugs Web Design Vienna | Webdesign Wien and Juwelier Schönmann