The New Insights Tetravalent dengue vaccine based on chimeric Yellow Fever 17D/Dengue viruses.

The New Insights Tetravalent dengue vaccine based on chimeric Yellow Fever 17D/Dengue

Widodo Judarwanto, Children Grow Up Clinic Jakarta Indonesia

Dengue virus is the most widespread geographically of the arboviruses and a major public health threat in the tropics and subtropics. Scientific advances in recent years have provided new insights about the pathogenesis of more severe disease and novel approaches into the development of antiviral compounds and dengue vaccines. Phylogenetic studies show an association between specific subtypes (within serotypes) and severity of dengue. The lack of association between maternal antibodies and development of severe dengue in infants in a recent study has called for the rethinking or refinement of the current antibody-dependent enhancement theory of dengue hemorrhagic syndrome in infancy. Such studies should stimulate new directions of research into mechanisms responsible for the development of severe dengue. The life cycle of dengue virus readily shows that virus entry and replication can be targeted by small molecules. Dengue infection is an important public health issue worldwide. The ChimeriVax-Dengue (CYD) vaccine uses yellow fever (YF) 17D vaccine as a live vector.

Dengue is a common arthropod-borne flaviviral infection in the tropics, for which there is no vaccine or specific antiviral drug. The infection is often associated with serious complications such as dengue hemorrhagic fever (DHF) or dengue shock syndrome (DSS).

Advances in a mouse model (AG 129 mice) have made it easier to test such antiviral compounds. The efforts to find specific dengue inhibitors are intensifying and the tools to evaluate the efficacy of new drugs are now in place for rapid translation into trials in humans. Furthermore, several dengue vaccine candidates are in development, of which the chimeric dengue/yellow fever vaccine has now entered phase 3 trials. Until the availability of a licensed vaccine, disease surveillance and vector population control remain the mainstay of dengue prevention.

A tetravalent dengue vaccine based on four live, attenuated, chimeric viruses (CYD1-4), constructed by replacing the genes coding for premembrane (prM) and envelope (E) proteins of the yellow fever (YF)-17D vaccine strain with those of the four serotypes of dengue virus, is in clinical phase III evaluation.

The study was to evaluate if the antibodies elicited after immunization with a tetravalent dengue vaccine, based on chimeric yellow fever 17D/dengue viruses, can neutralize a large range of dengue viruses (DENV). A panel of 82 DENVs was developed from viruses collected primarily during the last decade in 30 countries and included the four serotypes and the majority of existing genotypes. Viruses were isolated and minimally amplified before evaluation against a tetravalent polyclonal serum generated during vaccine preclinical evaluation in monkey, a model in which protection efficacy of this vaccine has been previously demonstrated.

Neutralization was observed across all the DENV serotypes, genotypes, geographical origins and isolation years. These data indicate that antibodies elicited after immunization with this dengue vaccine candidate should widely protect against infection with contemporary DENV lineages circulating in endemic countries.

Immune responses in human dendritic cells upon infection Dengue-2 and yellow fever 17DD

Flaviviruses cause severe acute febrile and haemorrhagic infections, including dengue and yellow fever and the pathogenesis of these infections is caused by an exacerbated immune response. Dendritic cells (DCs) are targets for dengue virus (DENV) and yellow fever virus (YF) replication and are the first cell population to interact with these viruses during a natural infection, which leads to an induction of protective immunity in humans. Dendritic cells (DCs) play a key role in initiating immune responses and could be an important primary target of dengue infection. We investigated in vitro the consequences of CYD infection of DCs on their activation/maturation and cytokine production. In CYD-infected DCs, we observed an up-regulation of HLA-DR, CD80, CD86, and CD83. Cells exposed to CYD secreted type I interferons, monocyte chemoattractant protein 1 (MCP-1)/CC chemokine ligand 2 (CCL-2), interleukin-6 (IL-6), and low amounts of tumor necrosis factor-alpha (TNF-alpha), but no IL-10, IL-12, or IL-1alpha. Parental dengue viruses induced a similar array of cytokines, but more TNF-alpha, less IL-6, and less MCP-1/CCL-2 than induced by CYD. Chimeras thus induced DCs maturation and a controlled response accompanied by limited inflammatory cytokine production and consistent expression of anti-viral interferons, in agreement with clinical observations of safety and immunogenicity.

The study the infectivity of DENV2 (strain 16681), a YF vaccine (YF17DD) and a chimeric YF17D/DENV2 vaccine in monocyte-derived DCs in vitro with regard to cell maturation, activation and cytokine production. Higher viral antigen positive cell frequencies were observed for DENV2 when compared with both vaccine viruses. Flavivirus-infected cultures exhibited dendritic cell activation and maturation molecules. CD38 expression on DCs was enhanced for both DENV2 and YF17DD, whereas OX40L expression was decreased as compared to mock-stimulated cells, suggesting that a T helper 1 profile is favoured. Tumor necrosis factor (TNF)-α production in cell cultures was significantly higher in DENV2-infected cultures than in cultures infected with YF17DD or YF17D/DENV. In contrast, the vaccines induced higher IFN-α levels than DENV2. The differential cytokine production indicates that DENV2 results in TNF induction, which discriminates it from vaccine viruses that preferentially stimulate interferon expression. These differential response profiles may influence the pathogenic infection outcome.

Genetic stability

The vaccine’s genetic stability by fully sequencing each vaccine virus throughout the development and manufacturing process. The four viruses displayed complete genetic stability, with no change from premaster seed lots to bulk lots. When pursuing the virus growth beyond bulk lots, a few genetic variations were observed. Usually both the initial nucleotide and the new one persisted, and mutations appeared after a relatively high number of virus duplication cycles (65-200, depending on position). Variations were concentrated in the prM-E and non-structural (NS)4B regions. PrM-E variations had no impact on lysis-plaque size or neurovirulence in mice. None of the variations located in the YF-17D-derived genes corresponded with reversion to the wild-type Yellow Fever sequence. Variations in NS4B likely reflect virus adaptation to Vero cells growth. A low to undetectable viremia has been reported previously [1-3] in vaccinated non-human and human primates. Combined with the data reported here about the genetic stability of the vaccine strains, the probability of in vivo emergence of mutant viruses appears very low.

Cell-mediated immunity in naive or flavivirus-primed subjects.

Three independent, phase 1 clinical trials were conducted in Australia and in USA to assess the safety and immunogenicity of sanofi pasteur dengue vaccine candidates. In this context, Dengue 1-4 and Yellow Fever 17D-204 (YF 17D)-specific CD4 and CD8 cellular responses induced by tetravalent chimeric dengue vaccines (CYD) were analyzed in flavivirus-naive or flavivirus-immune patients. Tetravalent CYD vaccine did not trigger detectable changes in serum pro-inflammatory cytokines, whatever the vaccinees immune status, while inducing significant YF 17D NS3-specific CD8 responses and dengue serotype-specific T helper responses. These responses were dominated by serotype 4 in naive individuals, but a booster vaccination (dose #2) performed 4 months following dose #1 broadened serotype-specific responses. A similar, broader response was seen after primary tetravalent immunization in subjects with pre-existing dengue 1 or 2 immunity caused by prior monovalent live-attenuated dengue vaccination. In all three trials, the profile of induced response was similar, whatever the subjects’ immune status, i.e. an absence of Th2 response, and an IFN-gamma/TNF-alpha ratio dominated by IFN-gamma, for both CD4 and CD8 responses.

The absence of cross-reactivity between YF 17D or Dengue NS3-specific CD8 responses, and allowed the identification of 3 new CD8 epitopes in the YF 17D NS3 antigen. These data are consistent with the previously demonstrated excellent safety of these dengue vaccines in flavivirus-naive and primed individuals.

Attenuation and immunogenicity of recombinant yellow fever 17D-dengue type 2 virus for rhesus monkeys.

A chimeric yellow fever (YF)-dengue serotype 2 (dengue 2) virus was constructed by replacing the premembrane and envelope genes of the YF 17D virus with those from dengue 2 virus strains of Southeast Asian genotype. The virus grew to high titers in Vero cells and, after passage 2, was used for immunogenicity and attenuation studies in rhesus monkeys. Subcutaneous immunization of naive rhesus monkeys with the 17D-D2 chimeric virus induced a neutralizing antibody response associated with the protection of 6 of 7 monkeys against viremia by wild-type dengue 2 virus.

Neutralizing antibody titers to dengue 2 were significantly lower in YF-immune animals than in YF-naive monkeys and protection against challenge with wild-type dengue 2 virus was observed in only 2 of 11 YF-immune monkeys. An anamnestic response to dengue 2, indicated by a sharp increase of neutralizing antibody titers, was observed in the majority of the monkeys after challenge with wild-type virus. Virus attenuation was demonstrated using the standard monkey neurovirulence test. The 17D-D2 chimera caused significantly fewer histological lesions than the YF 17DD virus. The attenuated phenotype could also be inferred from the limited viremias compared to the YF 17DD vaccine. Overall, these results provide further support for the use of chimeric viruses for the development of a new live tetravalent dengue vaccine.

Viremia and immunogenicity

Chimeric yellow fever (YF)-dengue (DEN) viruses (ChimeriVax-DEN) were reconstructed to correct amino acid substitutions within the envelope genes of original constructs described by Guirakhoo et al. (2001, J. Virol. 75, 7290-7304). Viruses were analyzed and compared to the previous constructs containing mutations in terms of their growth kinetics in Vero cells, neurovirulence in mice, and immunogenicity in monkeys as monovalent or tetravalent formulations. All chimeras grew to high titers [ approximately 7 to 8 log(10), plaque-forming units (PFU)/ml] in Vero cells and were less neurovirulent than YF 17D vaccine in mice.

For monkey experiments, the dose of DEN2 chimera was lowered to 3 log(10) PFU in the tetravalent mixture in an effort to reduce its dominant immunogenicity. The magnitude of viremia in ChimeriVax-DEN immunized monkeys was similar to that of YF-VAX, but significantly lower than those induced by wild-type DEN viruses. All monkeys developed high levels of neutralizing antibodies against homologous (chimeras) or heterologous (wild-type DEN viruses isolated from different geographical regions) viruses after a single dose of monovalent or tetravalent vaccine. Administration of a second dose of tetravalent vaccine 2 months later increased titers to both homologous and heterologous viruses. A dose adjustment for dengue 2 chimera resulted in a more balanced response against dengue 1, 2, and 3 viruses, but a somewhat higher response against chimeric dengue 4 virus. This indicates that further formulations for dose adjustments need to be tested in monkeys to identify an optimal formulation for humans.

Protection of Rhesus monkeys against dengue virus challenge

Rhesus monkeys develop viremia after dengue virus (DENV) inoculation and have been used as an animal model to study DENV infection and DENV vaccine candidates. Sun W, et al.reported that evaluated, in this model, the protective efficacy of a live attenuated tetravalent DENV vaccine (TDV) candidate against parenteral challenge with parental near-wild-type DENV strains. Twenty monkeys were vaccinated with TDV at 0 and 1 month, and 20 unvaccinated monkeys served as controls. Vaccinated animals and their controls were inoculated with 10(3)-10(4) pfu of challenge virus 4.5 months after the second vaccination. Primary vaccination resulted in 95%, 100%, 70%, and 15% seroconversion to DENV serotypes 1, 2, 3, and 4 (DENV-1, -2, -3, and -4), respectively. After the second vaccination, the seropositivity rates were 100%, 100%, 90%, and 70%, respectively. Vaccination with TDV resulted in complete protection against viremia from DENV-2 challenge and in 80%, 80%, and 50% protection against challenge with DENV-1, -3, and -4, respectively. Our results suggest that the TDV can elicit protective immunity against all 4 DENV serotypes. Interference among the 4 vaccine viruses may have resulted in decreased antibody responses to DENV-3 and -4, which would require reformulation or dose optimization to minimize this interference during testing of the vaccine in humans.

Safety and efficacy

To construct chimeric YF/DEN viruses (ChimeriVax-DEN), the premembrane (prM) and envelope (E) genes of yellow fever (YF) 17D virus were replaced with those of each wild-type (WT) dengue (DEN) virus representing serotypes 1 to 4. ChimeriVax-DEN1-4 vaccine viruses were prepared by electroporation of Vero cells with RNA transcripts prepared from viral cDNA. Progeny viruses were subjected to three rounds of plaque purifications to produce the Pre-Master Seed viruses at passage 7 (P7). Three further passages were carried out using U.S. current Good Manufacturing Practices (cGMP) to produce the Vaccine Lot (P10) viruses. Preclinical studies demonstrated that the vaccine candidates are replication competent and genetically stable and do not become more neurovirulent upon 20 passages in Vero cells.

The safety of a tetravalent vaccine was determined and compared to that of YF-VAX in a formal monkey neurovirulence test. Brain lesions produced by the tetravalent ChimeriVax-DEN vaccine were significantly less severe than those observed with YF-VAX. The immunogenicity and protective efficacy of four different tetravalent formulations were evaluated in cynomolgus monkeys following a single-dose subcutaneous vaccination followed by a virulent virus challenge 6 months later. All monkeys developed low levels of viremia postimmunization, and all the monkeys that had received equal concentrations of either a high-dose (5,5,5,5) or a low-dose (3,3,3,3) formulation seroconverted against all four DEN virus serotypes. Twenty-two (92%) of 24 monkeys were protected as determined by lack of viremia post-challenge. This report is the first to demonstrate the safety of a recombinant DEN virus tetravalent vaccine in a formal neurovirulence test, as well as its protective efficacy in a monkey challenge model.

The construction of a chimeric yellow fever-dengue type 2 virus (YF/DEN2) and determined its safety and protective efficacy in rhesus monkeys. The construction of three additional YF/DEN chimeras using premembrane (prM) and envelope (E) genes of wild-type (WT) clinical isolates: DEN1 (strain PUO359, isolated in 1980 in Thailand), DEN3 (strain PaH881/88, isolated in 1988 in Thailand), and DEN4 (strain 1228, isolated in 1978 in Indonesia). These chimeric viruses (YF/DEN1, YF/DEN3, and YF/DEN4) replicated to ~7.5 log(10) PFU/ml in Vero cells, were not neurovirulent in 3- to 4-week-old ICR mice inoculated by the intracerebral route, and were immunogenic in monkeys. All rhesus monkeys inoculated subcutaneously with one dose of these chimeric viruses (as monovalent or tetravalent formulation) developed viremia with magnitudes similar to that of the YF 17D vaccine strain (YF-VAX) but significantly lower than those of their parent WT viruses. Eight of nine monkeys inoculated with monovalent YF/DEN1 -3, or -4 vaccine and six of six monkeys inoculated with tetravalent YF/DEN1-4 vaccine seroconverted after a single dose.

When monkeys were boosted with a tetravalent YF/DEN1-4 dose 6 months later, four of nine monkeys in the monovalent YF/DEN groups developed low levels of viremia, whereas no viremia was detected in any animals previously inoculated with either YF/DEN1-4 vaccine or WT DEN virus. An anamnestic response was observed in all monkeys after the second dose. No statistically significant difference in levels of neutralizing antibodies was observed between YF virus-immune and nonimmune monkeys which received the tetravalent YF/DEN1-4 vaccine or between tetravalent YF/DEN1-4-immune and nonimmune monkeys which received the YF-VAX. However, preimmune monkeys developed either no detectable viremia or a level of viremia lower than that in nonimmune controls. This is the first recombinant tetravalent dengue vaccine successfully evaluated in nonhuman primates


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