Development of a safe and effective vaccine against dengue is a major public health priority because of the increase in the number of DF and DHF/DSS cases reported in dengue-endemic areas and the global spread of dengue to additional countries. Potential vaccine candidates need to be safe in all age groups and induce long lasting B cell and T cell immune responses against all four DENV serotypes.
Cynomolgus macaques immunized with the tetravalent DENVax formulations showed no clinical signs of disease caused by vaccination. Subcutaneous administration of the tetravalent DENVax resulted in low or undetectable viremia as shown by a highly sensitive qRT-PCR assay. Only DENVax-2 viremia was detected in the immunized animals, and the DENV-2 viremic profile was affected by the ratio of the four DENVax components in the different formulations. When tested in vitro
, DENVax-1, DENVax-2, and DENVax-3 replicated in the monkey-originating Vero cell line with similar kinetics; DENVax-4 replicates somewhat slower and to slightly lower peak titers.21
In non-human primates in vivo
, DENVax-2 demonstrated a significant replication advantage over the other three chimeric DENVax components, particularly in the low titer Formulation 1. When titers of DENVax-3 and DENVax-4 were increased in Formulation 2, DENVax-2 viremia was reduced and delayed (). Although we do not have data for viremia induced by the individual monovalent vaccine candidates, these data are suggestive of in vivo replicative competition between the four viruses (i.e., interference30
Infectious viremia titers associated with these serum samples positive for viral RNA were all low (undetectable to 2.4 log10
PFU/mL). Such low virus titers are unlikely to infect feeding mosquitoes.38
Furthermore, DENV-2 PDK-53 shows reduced replication in mosquito cells21,22
and in mosquitoes.39
The genetic loci affecting PDK-53 viral replication in mosquito cells and mosquitoes have been mapped to the attenuating mutations in the non-coding region and nonstructural genes of the DEN-2 PDK-53 vaccine virus. These three attenuating mutations were retained in all four DENVax viruses. Therefore, unintended transmission of DENVax-2 and the other DENVax recombinants is highly unlikely. The absence of post-boost viremia in all but one animal (monkey CY0181) suggests that the primary vaccine-induced immune responses can efficiently reduce virus replication on secondary exposure.
Detectable virus replication was not a requirement for induction of potent immune responses. Although DENVax-2 replicated to detectable levels in many animals and induced high levels of neutralizing antibodies measured by PRNT, high primary neutralizing antibody responses were also induced in many animals against DENV1 and DENV3 without detectable serum viremia. Responses to the second immunization were most marked for DENVax-3 in Formulation 1, DENVax-3 and DENVax-4 in Formulation 2, and all four viruses in the high dose Formulation 3 (). Overall, all dose levels resulted in substantial primary PRNT titers against DENV-1, -2, and -3 and potent secondary titers against all four serotypes, with the exception of low titers of antibodies against DENV-4 in animals in the low dose Formulation 1 group. Importantly, Formulation 2 provided the most balanced immune responses.
Because of the increased disease severity (DHF/DSS) associated with secondary infections, tetravalent vaccines that can stimulate immunity against all four dengue serotypes are needed.8,9
Low titers of antibodies against DENVax-4 observed with Formulation 1 and the improvement in titers with Formulation 2 suggest that future DENVax formulations should optimize the relative concentration of DENVax-4. We also intend to assess different routes and delivery methods for vaccination to further optimize the immunogenicity of the tetravalent DENVax vaccine in future non-human primate studies and in human clinical trials.
There is limited information on cellular immune responses to candidate dengue vaccines in non-human primates. In this study, macaques immunized with DENVax formulations showed robust numbers of DENV-2–induced IL-2 and IFN-γ–secreting cells by ELISPOT analysis. We did not observe consistent DENV-1, DENV-3, or DENV-4 stimulation of cytokine-secreting cells. These DENV-2 cellular immune responses could be caused by predominance of DENVax-2 replication. Alternatively, because all chimeric viruses contained the nonstructural proteins from the DENV-2 backbone, strong T cell–mediated responses upon stimulation with DENV-2 were expected. Interestingly, the low dose Formulation 1 induced lower levels of IFN-γ–secreting T cells, although the induction of IL-2–secreting T cells were comparable to those induced by Formulations 2 and 3. A comparison of ex vivo induced IFN-γ–secreting T cells in PMBC after two doses of the DENVax formulations were similar to the numbers of IFN-γ–secreting T cells in PBMC after two doses of wt DENV-2 inoculations in dengue-negative animals (Osorio JE and others, unpublished data), an indication of an effective vaccine induced T cell activation. Overall, strong cell-mediated memory responses were seen in animals immunized with DENVax Formulations 2 and 3, a hallmark of effective immunization with a live attenuated vaccine.
Previous studies of dengue vaccine efficacy in non-human primates measured viremia by direct virus isolation.31,32,40,41
In this study, we observed substantial short-term protection from infectious viremia caused by challenge with the wt viruses. Only two animals challenged with wt DENV-1 and one animal challenged with wt DENV-2 had detectable infectious viremia, which was limited in duration and lower in titer than in the non-immunized animals. Using a highly sensitive qRT-PCR assay, we found that several animals with no or limited infectious viremia showed significant levels of viral RNA in serum samples (as high as 6.3 log10
ge/mL); 11 of the 24 vaccinated animals did not have detectable viral RNA after challenge. Transient viral replication may have occurred in some animals after wt virus challenge, but the anamnestic immune response eliminated infectious particles, leaving only viral RNA. Some of the viral RNA detected in serum samples might be residual RNA fragments released from the degraded virus or present in neutralized antibody (virus complexes). We could not detect any correlation between absence of viral RNA and the pre-challenge PRNT titers or cytokine-secreting T cells as detected by ELISPOT.
Eight immunized animals showed no increase in neutralizing antibody responses after challenge. This lack of anamnestic response has previously been viewed as evidence of sterilizing immunity and lack of viral replication. Interestingly, the absence of an anamnestic response does not correlate with absence of viral RNA. Three of eight animals that did not demonstrate anamnestic responses after challenge showed evidence of viral replication by qRT-PCR (). These cases of sterilizing immunity were occasionally observed with recombinant live, attenuated vaccines with a different dengue virus backbone,41
but were not observed using recombinants based on a yellow fever backbone.32
The role of cellular immunity in sterilizing immunity and in protection associated with strong, anamnestic antibody responses remains to be investigated.
In addition to being immunogenic and efficacious, these DENVax formulations were safe and did not induce any injection site reactions or significant effects on body weights, hematologic parameters, and body temperature. These data highlight the safety of tetravalent DENVax formulations and are consistent with previous observations demonstrating the safety and immunogenicity of the attenuated DENV-2 PDK-53 vaccine in phase I and II clinical trials in the United States and Thailand.14–16
In conclusion, these data provide evidence of DENVax safety, immunogenicity, and short-term protection from viremia in non-human primates and support the further evaluation of DENVax formulations in human clinical trials.