A major obstacle to the development of dengue virus (DENV) vaccines has been the need to immunise concurrently against each of the four DENV serotypes in order to avoid sensitising recipients to developing severe DENV infections. A problem already encountered with live attenuated tetravalent DENV vaccines has been the difficulty in eliciting adequate immune responses against all four DENV serotypes in human hosts. This could have been due to variations in the antigenicity and/or the replication rates of the four DENV serotypes. Non-replicating DNA vaccines avoid the issue of different replication rates. Currently, only DENV-1 and DENV-2 DNA vaccines have been evaluated. In this study, a number of DNA vaccines for each of the four DENV serotypes were developed and their immunogenicity was evaluated in outbred mice. These vaccines included DNA vaccines encoding the DENV prM-E protein genes derived from the four DENV serotypes (pVAX-DEN1, -DEN2, -DEN3 and -DEN4), and DNA vaccines encoding DENV prM and hybrid-E protein genes derived from multiple DENV serotypes. The hybrid-E protein genes were constructed by substituting either domains I and II, domain III, and/or the stem-anchor region from the E protein of one DENV serotype with the corresponding region from another DENV serotype. A number of superior DNA vaccines against each of the four DENV serotypes were identified based on their ability to elicit high titres (≥40, FFURNT50) of neutralising antibodies against the corresponding DENV in mice. The superior DNA vaccines against DENV-1 were pVAX-DEN1, pVAX-C2M2E211, pVAX-C2M2E122 and pVAX-C2M1E122. The superior DNA vaccine against DENV-2 was pVAX-C2M1E122 and the superior DNA vaccines against DENV-3 were pVAX-DEN3 and pVAX-C2M3E344. The superior DNA vaccines against DENV-4 were pVAX-C2M3E344, pVAX-C2M4E434 and pVAX-C2M4E433. Each of these DNA vaccines could provide effective protection against infection by the corresponding DENV serotypes. This is the first study to describe the development of DNA vaccines against DENV-3 and DENV-4. However, mice immunised with a tetravalent DENV DNA vaccine, composed of a DNA vaccine encoding the prM-E protein genes from each of the four DENV serotypes (pVAX-DEN1-4), elicited high titres of neutralising antibodies against DENV-1 and DENV-3 only. Nevertheless, the results from this study suggested that a tetravalent DENV DNA vaccine, composed of pVAX-DEN1, pVAX-C2M1E122, pVAX-DEN3 and pVAX-C2M4E434, may provide effective concurrent protection against infection by each of the four DENV serotypes. In addition, mice immunised with pVAX-C2M1E122, which encoded a hybrid-E protein gene derived from DENV-1 and DENV-2, elicited high titres of anti-DENV-1 and anti-DENV-2 neutralising antibodies, and mice immunised with pVAX-C2M3E344, which encoded a hybrid-E protein gene derived from DENV-3 and DENV-4, elicited high titres of anti-DENV-3 and anti-DENV-4 neutralising antibodies. This result suggested that the co-immunisation of these two hybrid-E DNA vaccines also may provide effective concurrent protection against infection by each of the four DENV serotypes. Extracellular E proteins, believed to be in the form of recombinant subviral particles (RSPs), were recovered from the tissue culture supernatant of all DNA vaccine-transfected mammalian cells by ultracentrifugation, except for cells transfected with the pVAX-C2M2E122 hybrid-E DNA vaccine. Western blotting with the monoclonal antibody 4G2 (flavivirus cross-reactive) demonstrated that the extracellular E proteins expressed by the DNA vaccines were synthesized and cleaved in a manner similar to that of native DENV E proteins. In addition, mammalian cells transfected with pVAX-DEN1, pVAX-DEN2 or pVAX-DEN3 secreted higher amounts of extracellular E proteins than cells transfected with pVAX-DEN4. The amount of extracellular E protein secreted by pVAX-DEN4-transfected cells increased when the c-region of the prM/E signal peptidase cleavage site was made more polar. In contrast, decreasing the polarity of the c-region of the C/prM signal peptidase cleavage site of pVAX-DEN4 resulted in no detectable extracellular E proteins from pVAX-DEN4-transfected cells. This result suggested that the amount of extracellular E proteins secreted by cells transfected with DNA expressing the DENV prM-E protein genes may be dependent of the efficiency of C/prM and prM/E protein cleavages by host-derived signal peptidases. Mice immunised with the mutated pVAX-DEN4, which was capable of expressing large amounts of extracellular E proteins in vitro, produced significantly higher concentrations of Th1-type anti-DENV-4 antibodies than mice immunised with the unmodified pVAX-DEN4, but failed to produce detectable levels of anti-DENV-4 neutralising antibodies. In contrast, increasing the ratio of CpG-S to CpG-N motifs in the pVAX-DEN2 DNA vaccine by incorporating either an additional CpG-S motif, or an antibiotic resistance gene with a high ratio of CpG-S to CpG-N motifs, resulted in a significant increase in both the concentration of Th1-type anti-DENV-2 antibodies and the titres of anti-DENV-2 neutralising antibodies in immunised mice. This result suggested that increasing the amount of CpG-S motifs in DENV DNA vaccines may present an simple and effective approach to increasing the immunogenicity of the DENV DNA vaccines.