The fusion site of envelope fragments from each serotype of Dengue virus in the P64k protein, influence some parameters of the resulting chimeric constructs
Aı́da ZuluetaLisset HermidaLaura LazoIris ValdésRayner Rodrı́guezCarlos LópezRicardo SilvaDelfina RosarioJorge Martı́nMaría G. GuzmánGerardo Guillén
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Keywords:
Dengue vaccine
Envelope (radar)
Flavivirus
The worldwide expansion of four serotypes of dengue virus (DENV) poses great risk to global public health. Several vaccine candidates are under development. However, none is yet available for humans. In the present study, a novel strategy to produce tetravalent DENV vaccine based on envelope protein domain III (EDIII) was proposed. Tandem EDIIIs of two serotypes (type 1–2 and type 3–4) of DENV connected by a Gly-Ser linker ((Gly4Ser)3) were expressed in E. coli, respectively. Then, the two bivalent recombinant EDIIIs were equally mixed to form the tetravalent vaccine candidate MixBiEDIII, and used to immunize BALB/c mice. The results showed that specific IgG and neutralizing antibodies against all four serotypes of DENV were successfully induced in the MixBiEDIII employing Freund adjuvant immunized mice. Furthermore, in the suckling mouse model, sera from mice immunized with MixBiEDIII provided significant protection against four serotypes of DENV challenge. Our data demonstrated that MixBiEDIII, as a novel form of subunit vaccine candidates, might have the potential to be further developed as a tetravalent dengue vaccine in the near future.
Dengue vaccine
Bivalent (engine)
Flavivirus
Antibody-dependent enhancement
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The proliferative T cell responses to dengue vaccines were studied using the parental strains of dengue vaccines as antigens in 26 dengue immune individuals who resided in Bangkok which is the endemic area of dengue infection. The magnitude of the T cell responses in subjects with flavivirus cross-reactive neutralizing antibody was much higher and the cross-reactivity was broader than in those with dengue serotype-specific neutralizing antibodies, Japanese encephalitis (JE) specific antibodies or dengue cross-reactive antibodies. The T cell response in those with neutralizing antibody against a single serotype or in those who had dengue cross-reactive neutralizing antibody was relatively low, independent of the level or degree of cross-reactivity of the antibody. Evaluation of the proliferative T cell responses in 8 recipients of the monovalent dengue-2 (16681-PDK53) or the tetravalent dengue vaccines demonstrated that both vaccines induced high levels of neutralizing antibody as well as high levels of T cell responses to all serotypes of dengue virus. These results indicate that the evaluated dengue vaccines efficiently induced humoral and cell mediated immunity comparable to natural infection with dengue virus.
Dengue vaccine
Flavivirus
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Original antigenic sin
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ABSTRACT Dengue viruses (DENV) infect 50 to 100 million people each year. The spread of DENV-associated infections is one of the most serious public health problems worldwide, as there is no widely available vaccine or specific therapeutic for DENV infections. To address this, we developed a novel tetravalent dengue vaccine by utilizing virus-like particles (VLPs). We created recombinant DENV1 to -4 (DENV1-4) VLPs by coexpressing precursor membrane (prM) and envelope (E) proteins, with an F108A mutation in the fusion loop structure of E to increase the production of VLPs in mammalian cells. Immunization with DENV1-4 VLPs as individual, monovalent vaccines elicited strong neutralization activity against each DENV serotype in mice. For use as a tetravalent vaccine, DENV1-4 VLPs elicited high levels of neutralization activity against all four serotypes simultaneously. The neutralization antibody responses induced by the VLPs were significantly higher than those with DNA or recombinant E protein immunization. Moreover, antibody-dependent enhancement (ADE) was not observed against any serotype at a 1:10 serum dilution. We also demonstrated that the Zika virus (ZIKV) VLP production level was enhanced by introducing the same F108A mutation into the ZIKV envelope protein. Taken together, these results suggest that our strategy for DENV VLP production is applicable to other flavivirus VLP vaccine development, due to the similarity in viral structures, and they describe the promising development of an effective tetravalent vaccine against the prevalent flavivirus. IMPORTANCE Dengue virus poses one of the most serious public health problems worldwide, and the incidence of diseases caused by the virus has increased dramatically. Despite decades of effort, there is no effective treatment against dengue. A safe and potent vaccine against dengue is still needed. We developed a novel tetravalent dengue vaccine by using virus-like particles (VLPs), which are noninfectious because they lack the viral genome. Previous attempts of other groups to use dengue VLPs resulted in generally poor yields. We found that a critical amino acid mutation in the envelope protein enhances the production of VLPs. Our tetravalent vaccine elicited potent neutralizing antibody responses against all four DENV serotypes. Our findings can also be applied to vaccine development against other flaviviruses, such as Zika virus or West Nile virus.
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Dengue vaccine
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Dengue fever is caused by infection with one of four dengue virus (DENV) serotypes (DENV-1-4), necessitating tetravalent dengue vaccines that can induce protection against all four DENV. Takeda's live attenuated tetravalent dengue vaccine candidate (TDV) comprises an attenuated DENV-2 strain plus chimeric viruses containing the prM and E genes of DENV-1, -3 and -4 cloned into the attenuated DENV-2 'backbone'. In Phase 1 and 2 studies, TDV was well tolerated by children and adults aged 1.5-45 years, irrespective of prior dengue exposure; mild injection-site symptoms were the most common adverse events. TDV induced neutralizing antibody responses and seroconversion to all four DENV as well as cross-reactive T cell-mediated responses that may be necessary for broad protection against dengue fever.
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Flavivirus
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Background. Protection against dengue requires immunity against all 4 serotypes of dengue virus (DENV). Experimental challenge may be useful in evaluating vaccine-induced immunity. Methods. Ten subjects previously vaccinated with a live attenuated tetravalent dengue vaccine (TDV) and 4 DENV-naive control subjects were challenged by subcutaneous inoculation of either 103 plaque-forming units (PFU) of DENV-1 or 105 PFU of DENV-3. Two additional subjects who did not develop DENV-3 neutralizing antibody (NAb) from TDV were revaccinated with 104 PFU of live attenuated DENV-3 vaccine to evaluate memory response. Results. All 5 TDV recipients were protected against DENV-1 challenge. Of the 5 TDV recipients challenged with DENV-3, 2 were protected. All DENV-3–challenge subjects who developed viremia also developed elevated liver enzyme levels, and 2 had values that were >10 times greater than normal. Of the 2 subjects revaccinated with DENV-3 vaccine, 1 showed a secondary response to DENV-2, while neither showed such response to DENV-3. All 4 control subjects developed dengue fever from challenge. Protection was associated with presence of NAb, although 1 subject was protected despite a lack of measurable NAb at the time of DENV-1 challenge. Conclusions. Vaccination with TDV induced variable protection against subcutaneous challenge. DENV-3 experimental challenge was associated with transient but marked elevations of transaminases.
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Viremia
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Abstract The global incidence of dengue, which is caused by dengue virus (DENV) infection, has grown dramatically in recent decades and secondary infection with heterologous serotype of the virus may cause severe symptoms. Efficacious dengue vaccines should be able to provide long-lasting immunity against all four DENV serotypes simultaneously. In this study, we constructed a novel vaccine platform based on tetravalent dengue virus-like particles (DENV-LPs) in which envelope (E) protein carried a FLAG tag sequence at the position located not only in the exterior loop on the protruding domain but outside of dimerization interface of the protein. We demonstrated an effective strategy to produce the DENV-LPs by transient transfection with expression plasmids for pre-membrane and E proteins of DENV-1 to DENV-4 in mammalian cells and to concentrate and purify them with one-step affinity chromatography. Characteristic features of VLPs such as particle size, shape and density were comparable to flavivirus-like particles reported. The neutralizing activity against all four DENV serotypes was successfully induced by immunization with the purified tetravalent VLPs in mice. Simple, one-step purification systems for VLP vaccine platforms using epitope-tagging strategy should be advantageous for vaccine development not only for dengue but for emerging pandemics in the future.
Dengue vaccine
Flavivirus
Antibody-dependent enhancement
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Flaviviruses include important human pathogens, such as dengue virus and Zika virus. NS5 is a nonstructural protein essential for flavivirus RNA replication with dual MTase and RdRp enzyme activities and thus constitutes a major drug target. Insights into NS5 structure, dynamics, and evolution should inform the development of antiviral inhibitors and vaccine design. We found that NS5 from DENV2 can adopt a conformation resembling that of NS5 from JEV and ZIKV. Replacement of the DENV2 NS5 linker with the JEV and ZIKV NS5 linkers abolished DENV2 replication in cells, without significantly impacting in vitro DENV2 NS5 enzymatic activities. We propose that heterotypic flavivirus NS5 linkers impede DENV2 NS5 protein-protein interactions that are essential for virus replication.
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Flavivirus
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Monoclonal antibodies produced against the four dengue virus serotypes identified four categories of reactions by immunofluorescence: flavivirus group reactive, dengue complex specific, dengue subcomplex specific (DEN-1, DEN-3), and dengue serotype specific. This is the first time that monospecific antibodies have been available for all of these unique antigenic determinants. Hybridoma cell lines producing dengue type-specific antibodies have been deposited in the Hybridoma Cell Bank of the American Type Culture Collection (Rockville, MD) for general distribution.
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Immunofluorescence
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Because infection with any of the 4 Dengue virus serotypes may elicit both protective neutralizing antibodies and nonneutralizing antibodies capable of enhancing subsequent heterotypic Dengue virus infections, the greatest risk for severe dengue occurs during a second, heterotypic Dengue virus infection. It remains unclear whether the replication of live attenuated vaccine viruses will be similarly enhanced when administered to Dengue-immune individuals.We recruited 36 healthy adults who had previously received a monovalent live Dengue virus vaccine 0.6-7.4 years earlier. Participants were assigned to 1 of 4 cohorts and were randomly chosen to receive placebo or a heterotypic vaccine. The level of replication, safety, and immunogenicity of the heterotypic vaccine virus was compared with that of Dengue virus immunologically naive vaccinees.Vaccine virus replication and reactogenicity after monovalent Dengue virus vaccination in naive and heterotypically immune vaccinees was similar. In contrast to naive vaccinees, the antibody response in heterotypically immune vaccinees was broadly neutralizing and mimicked the response observed by natural secondary Dengue virus infection.Enhanced replication of these live attenuated Dengue virus vaccines was minimal in heterotypically immune vaccinees and suggests that the further evaluation of these candidate vaccines in populations with preexisting DENV immunity can proceed safely.
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Antibody-dependent enhancement
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