Screening for immune response against Dengue virus in Vietnamese non-human primates: implications for vaccine developers.

One of the major problems faced for the development of a vaccine against Dengue virus is the lack of a suitable animal model. Although non-human primates do not show overt signs of disease, these animals develop viremia after the infection and are the best model to evaluate vaccine candidates against this pathogen. However, for that purpose, the screening of all animals is mandatory to discard those with previous natural immunity. The most common technique used in the screening is the plaque reduction neutralization test (PRNT). However, most recent studies points to the cell-mediated immunity (CMI) as an important player in the process of controlling Dengue virus (DENV) infections. Here we presented the results from the screening of 55 rhesus monkeys housed in an animal breeding facility at Quang Ninh province, Vietnam. We evaluated the neutralizing antibody response by PRNT and determined the levels of interferon γ (IFNγ)-secretion after the viral stimulation of monkey-peripheral blood mononuclear cells, by enzyme-linked immunosorbent assay (ELISA). We found no correspondence between PRNT and IFNγ-ELISA. In fact, 19 animals were positive only by IFNγ-ELISA. Moreover, to study the protective capacity of the CMI detected, three animals with positive response by IFNγ-ELISA and negative by PRNT were inoculated with an infective preparation of DENV-3 and, as a result, no viremia was detected during 10 days after the challenge. This fact points to the importance of screening non-human primates through a CMI assay together with PRNT. This procedure should discard those false-negative cases which would be protected after the viral challenge in the immunization schedule.

Evaluation in Mice of the Immunogenicity of a Tetravalent Subunit Vaccine Candidate Against Dengue Virus Using Mucosal and Parenteral Immunization Routes.

Our group has developed a subunit vaccine candidate against Dengue virus (DENV) based on two different viral regions, the domain III of the envelope protein and the capsid protein. The chimeric proteins for each serotype (DIIIC1-4), aggregated with the oligodeoxynucleotide 39 M, form the tetravalent formulation named Tetra DIIIC. Tetra DIIIC induces a protective immune response in mice when it is inoculated by intraperitoneal route. However, if children are the main targets for a DENV vaccine, then a needle-free route of administration should be attractive and advantageous. In this study, we evaluated for the first time, in vivo, a vaccine candidate against DENV based on recombinant proteins using the intranasal route. After three doses of Tetra DIIIC in mice, we measured the humoral immune response against the four DENV serotypes and the corresponding recombinant proteins. Moreover, the functionality of these antibodies was evaluated through a plaque reduction neutralization test. Finally, to assess the cellular immune response induced, we measured the IFN-γ-levels secreted by spleen cells after in vitro stimulation with DENV. The results presented in this study indicate that the intranasal immunization with Tetra DIIIC favors the generation of DENV-specific cell-mediated immunity. On the other hand, the immunization using intraperitoneal and intranasal routes, simultaneously, generate functional antibodies (anti-DIIIC and anti-DENV) and an in vitro response of IFN-γ secretion.

Evaluation in mice of the immunogenicity and protective efficacy of a tetravalent subunit vaccine candidate against dengue virus.

A dengue vaccine must induce protective immunity against the four serotypes of the virus. Our group has developed chimeric proteins consisting of the protein P64k from Neisseria meningitidis and the domain III from the four viral envelope proteins. In this study, the immunogenicity of a tetravalent vaccine formulation using aluminum hydroxide as adjuvant was evaluated in mice. After three doses, neutralizing antibody titers were detected against the four viral serotypes, the lowest seroconversion rate being against dengue virus serotype 4. One month after the last dose, immunized animals were challenged with infective virus, and partial but statistically significant protection was found to have been achieved. Based on these results, further studies in mice and non-human primates using this tetravalent formulation in a prime-boost strategy with attenuated viruses are strongly recommended.