The second session of the workshop focused on the review of models that are currently used to evaluate vaccines and therapeutics for dengue. The animal models appropriate for evaluating vaccines were reviewed by Stephen Whitehead, Thierry Decelle, Sandra Giannini and Kenneth Eckels. One of the challenges of dengue vaccine development is that vaccines that don’t induce neutralizing antibodies against all 4 serotypes of the virus could theoretically induce enhanced disease in vaccine recipients that are subsequently exposed to wild type infection. Therefore dengue vaccines must produce sufficient neutralizing antibodies against all serotypes to prevent breakthrough infection and disease. Tetravalent live attenuated dengue vaccine candidates are being developed that should induce strong immune responses to all four serotypes with minimal reactogenicity. DENV vaccines should be evaluated pre-clinically in animal models that support viral replication, induce measurable immune responses and that are accessible and easy to use to allow for the evaluation of dozens of different vaccine candidates. Each step of DENV vaccine development requires animal models with different characteristics. For example, to evaluate the attenuation of vaccine strains, wild type viruses need to replicate in the animal host at high titers. The reduction in replication of the vaccine candidate is then a measurable correlate of attenuation. The classical animal model used to measure dengue virus attenuation is non-human primates, since they allow dengue viruses’ replication at high titers (29
). In 2002, a mouse model, the SCID-HuH7, was developed to measurevaccine attenuation. This model consists of SCID mice injected intraperitoneally (IP) with the human hepatoma cell line Huh7, which is then allowed to grow to a visible tumor in a few weeks. All 4 serotypes of wild type (wt) DENV replicated to high titers when injected directly into the tumor while the attenuated strains replicated to significantly lower titers (37
). Since titers of the different viral strains in the SCID-HuH7 mouse model correlated quite well with the ones in NHPs (39
), this model is used by some groups to evaluate vaccine attenuation prior to clinical evaluation. Although this model is limited by the lack of any dengue-like pathology and immune responses, it is very reproducible.
Early-stage evaluation of vaccine immunogenicity is generally conducted in small animal models. Indeed the AG129 mouse model for DENV was first developed as a small animal model for dengue vaccine testing (17
). These mice can be infected and generate neutralizing immune responses against all 4 DENV serotypes. In addition, lethal challenge studies using DENV1 and DENV2 can demonstrate protection. Later-stage preclinical evaluation of vaccine immunogenicity and efficacy is generally performed in NHP models like rhesus macaques and cynomolgus monkeys, as these animals are fully immune-competent and develop measurable viremia and humoral and cellular immune responses at physiologically low inoculation levels of DENV (29
). NHPs are also used by some groups to study how the immune responses against one strain can interfere with the responses against the other 3 viral strains in a tetravalent vaccine formulation (the phenomenon called inter-serotypic interference) and to optimize immunization schedules.
Although NHPs are considered the best model to study the immunogenicity and efficacy of vaccines, these animals have not been able to fully predict vaccine immunogenicity and reactogenicity in humans. In order to evaluate the potential toxicity of live-attenuated dengue vaccines, an animal model has to support replication by all viral serotypes and induce measurable adaptive immune responses. Both NHP and the AG129 mouse models have these characteristics and therefore have been used by different groups to study the reproductive and systemic toxicity of vaccine candidates. Since DENV infection has been sporadically associated with CNS disease in humans, neurovirulence toxicology studies were discussed. The most sensitive system to study dengue neurovirulence appears to be intracranial injection of suckling mice. NHPs have also been used to study neurovirulence but they display very little neuropathology.
Models used to evaluate therapeutics were reviewed by Wouter Schul. Several characteristics were cited during the workshop as being desirable in an animal model to evaluate drug candidates for anti-DENV activity. The model should be affordable, easy to acquire and scalable to allow for the evaluation of large numbers of compounds. It should support replication with all 4 strains of DENV, display simple markers of protection such as reduction of viremia and/or death and should be relevant to human disease (i.e
., display components of both DF/DHF). The AG129 mouse model has several of these characteristics and is currently being used by drug developers to evaluate the in vivo
efficacy of promising DENV therapeutics (43
). One of the drawbacks of this model for evaluating therapeutics is that viremia is transient making it difficult to evaluate the effect of drugs at different times post-infection. Also, since evidence of DF/DHF is observed only after infection with the mouse-adapted DENV2 strain, this model cannot yet be utilized to evaluate the effect of antiviral drugs on disease caused by the other DENV strains. Additional work needs to be done to develop better models for in vivo drug testing.
The last presentation by Lewis Markoff reviewed dengue animal models from the regulatory perspective. Different animal models should be employed to answer particular scientific questions about the vaccines or drugs being evaluated, such as vaccine attenuation, efficacy and safety. None of the dengue animal models currently available are sufficiently comprehensive to be used by themselves in the regulatory process but in combination they should be able to support the clinical evaluation of products in humans.