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This work lays the foundation for developing a single Ad vector encoding …


Biology Articles » Immunobiology » An adenovirus prime/plasmid boost strategy for induction of equipotent immune responses to two dengue virus serotypes » Discussion

Discussion
- An adenovirus prime/plasmid boost strategy for induction of equipotent immune responses to two dengue virus serotypes

This work was driven by the hypothesis that switching from a strategy reliant on mixing four monovalent attenuated RNA viruses to a single tetravalent DNA viral vaccine vector may provide a means of circumventing viral interference and the associated risk of ADE. In this regard, Ad, a DNA virus, is emerging as a promising vaccine platform [46,47]. Recent data on the use of rAd vectors to develop experimental HIV-1 vaccines are encouraging. As a first step towards our objective, we recently showed that a rAd vector harboring the DEN-2 E gene could elicit DEN-2 virus-neutralizing antibodies in mice [45]. Subsequently, using EDIII of DEN-2 virus, in the context of the rAd vector system, we showed that it was able to induce DEN-2 virus-specific neutralizing antibodies and T cell responses as effectively as the complete E protein [42]. In the current study, we have created and analyzed a bivalent rAd vector. The purpose of this work was to obtain proof for the hypothesis that a single rAd-based dengue vaccine vector could elicit a balanced immune response to more than one DEN virus serotype.

We inserted a chimeric, EDIII-based bivalent gene, EDIII-4/2, designed by splicing together cDNAs encoding EDIIIs of DEN-2 and DEN-4 viruses [43], in place of the E1 region of the Ad5 genome to create a bivalent rAd vector. Mice immunized with a single dose of bivalent rAd manifested modest serum titers of antibodies to DEN-2 and DEN-4 viruses. In order to avoid the possibility that anti-Ad antibodies generated after rAd priming may compromise the vector's efficacy during repeat inoculations, we boosted the rAd primed mice with a plasmid encoding the bivalent antigen. DEN virus-specific antibodies in the sera of immunized animals were assessed by several criteria. This showed that these sera contained antibodies that specifically recognized EDIII-2 and EDIII-4, but manifested barely discernible reactivity towards EDIII-1 and EDIII-3. The critical question that we addressed at this point was, do these antibodies possess the capacity to neutralize, specifically, the infectivity of DEN-2 and DEN-4 viruses? Using the rAd vector prime/plasmid boost strategy, DEN-2 and DEN-4 virus-specific PRNT50 titers in the sera of immunized mice were each ~1:80. Using monkeys, which do not manifest dengue disease, protective efficacy is measured in terms of reduction in viremia in animals challenged with DEN virus. In the monkey model, it has been reported that PRNT50 titers in the range of 1:20 to 1:80, in animals immunized with either pox virus vector based-[48] or yellow fever virus-based [27] experimental dengue vaccines, confer protection against live DEN-2 virus challenge. In the mouse model, again which does not manifest any dengue disease, protective efficacy is measured in terms of survival after lethal DEN virus challenge. As the volume of serum that one can obtain from mice is quite small, PRNT assays are usually done using pooled sera. Consequently, the precise neutralizing antibody titers in individual animals prior to challenge is not known. Thus, the relationship between neutralizing antibody titers and protective efficacy has not been clearly delineated in murine systems. However, in the absence of a good animal model for dengue, neutralizing antibody titers, determined by PRNT titers are widely accepted as surrogate markers of protective immunity. PRNT50 titers = 1:10 are considered as indicative of protective immunity [49,50].

Consistent with the design of the bivalent antigen, the neutralizing antibodies it elicited specifically blocked the infectivity of DEN-2 and DEN-4, but not that of DEN-1 and DEN-3 viruses. This essentially corroborates our previous findings using a bacterially expressed recombinant EDIII-4/2 bivalent antigen [43]. Further, the observed immunogenicity of the EDIII-4/2 bivalent antigen either without [43] or with the GFP tag (this study) suggests that fusion of the reporter has not adversely affected its potential to elicit DEN-2 and DEN-4 virus-specific neutralizing antibodies. The E. coli-expressed antigen which is insoluble needs to be empirically re-folded subjecting antigen preparations to variability. Further, protein antigens are not efficient at inducing cell-based immunity (CMI). Given that the structural integrity of EDIII is essential for its immunogenicity, the gene-based vaccines (this study), which eliminate this variability are far more desirable, with the added advantage that these can also provoke potent CMI.

Our previous [42,43] and current work suggests that EDIII is serotype-specific and that it must be possible to evoke a tetravalent response using a chimeric antigen containing EDIIIs of all four DEN virus serotypes linked to each other. While most recombinant approaches to dengue vaccines deal primarily with monovalent vaccines, recently a couple of tetravalent plasmid vaccine candidates, both based on the full-length E protein genes, have been reported. One is based on mixing four monovalent plasmid vaccines [12], and the other describes a plasmid encoding a single chimeric E gene generated by DNA shuffling [11,14]. While both these are reported to elicit neutralizing antibodies, it is difficult to correlate our data on DEN-2 and DEN-4 PRNT titers with these studies as they have employed homologous plasmid prime/plasmid boost immunizations aside from differences in immunization schedules and neutralization assay protocols. Experiments are underway to investigate the role of the DEN-2 and DEN-4 virus-specific antibodies induced by rAd-Bg immunization in affecting the replication of DEN-1 and DEN-3 viruses.


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