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Dengue virus envelope protein domain I/II hinge determines long-lived serotype-specific dengue immunity.

ABSTRACT: The four dengue virus (DENV) serotypes, DENV-1, -2, -3, and -4, are endemic throughout tropical and subtropical regions of the world, with an estimated 390 million acute infections annually. Infection confers long-term protective immunity against the infecting serotype, but secondary infection with a different serotype carries a greater risk of potentially fatal severe dengue disease, including dengue hemorrhagic fever and dengue shock syndrome. The single most effective measure to control this threat to global health is a tetravalent DENV vaccine. To date, attempts to develop a protective vaccine have progressed slowly, partly because the targets of type-specific human neutralizing antibodies (NAbs), which are critical for long-term protection, remain poorly defined, impeding our understanding of natural immunity and hindering effective vaccine development. Here, we show that the envelope glycoprotein domain I/II hinge of DENV-3 and DENV-4 is the primary target of the long-term type-specific NAb response in humans. Transplantation of a DENV-4 hinge into a recombinant DENV-3 virus showed that the hinge determines the serotype-specific neutralizing potency of primary human and nonhuman primate DENV immune sera and that the hinge region both induces NAbs and is targeted by protective NAbs in rhesus macaques. These results suggest that the success of live dengue vaccines may depend on their ability to stimulate NAbs that target the envelope glycoprotein domain I/II hinge region. More broadly, this study shows that complex conformational antibody epitopes can be transplanted between live viruses, opening up similar possibilities for improving the breadth and specificity of vaccines for influenza, HIV, hepatitis C virus, and other clinically important viral pathogens.

SUBMITTER: Messer WB

PROVIDER: S-EPMC3918811 | biostudies-literature | 2014 Feb

REPOSITORIES: biostudies-literature

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Dengue virus envelope protein domain I/II hinge determines long-lived serotype-specific dengue immunity.

Messer William B WB de Alwis Ruklanthi R Yount Boyd L BL Royal Scott R SR Huynh Jeremy P JP Smith Scott A SA Crowe James E JE Doranz Benjamin J BJ Kahle Kristen M KM Pfaff Jennifer M JM White Laura J LJ Sariol Carlos A CA de Silva Aravinda M AM Baric Ralph S RS

Proceedings of the National Academy of Sciences of the United States of America 20140102 5

The four dengue virus (DENV) serotypes, DENV-1, -2, -3, and -4, are endemic throughout tropical and subtropical regions of the world, with an estimated 390 million acute infections annually. Infection confers long-term protective immunity against the infecting serotype, but secondary infection with a different serotype carries a greater risk of potentially fatal severe dengue disease, including dengue hemorrhagic fever and dengue shock syndrome. The single most effective measure to control this ...[more]

PMID: 24385585

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Project description:The DENV envelope (E) and pre-membrane (prM) glycoproteins are primary targets of serologic immunity after infection and vaccination. Of these, serotype-specific (TS) antibodies typically target E domains, while serotype cross-reactive (CR) antibodies typically the target prM protein and conserved E regions. To identify and quantify E-domain TS neutralizing antibody responses in polyclonal sera, we developed a panel of chimeric DENV4/2 viruses that incorporate DENV2 envelope domain I, II, and III (DENV4/2-EDI, EDII, EDIII) into the DENV4 E glycoprotein. Chimeric DENV4/2 viruses were recovered, replicated efficiently, and displayed similar maturation states as parental viruses. The recovery of viable DENV4/2-EDII recombinants required the inclusion of chimeric DENV4/2 prM that maintained critical interactions with chimeric E. To assess structural integrity and epitope display of chimeric viruses, we examined neutralization of mature virions by monoclonal antibodies (mAbs) and heterotypic polyclonal sera. The ED-chimeric virions preserved epitopes of TS and envelope-dimer-epitope CR mAbs and had similar sensitivity to CR polyclonal responses as parental strains. Primary sera from natural infection and human challenge target a region centered on EDIII and secondarily target EDII and EDI. Sera from natural infection had a unique neutralization pattern compared to sera from human challenge, which included greater frequency and higher titer of responses against DENV EDII. In summary, DENV4/2 E recombinant viruses delineate the subdomain targets of TS antibodies after vaccination and primary infection, which may provide new correlates of protection or identify epitopes of neutralizing monoclonal antibodies.IMPORTANCEThe four dengue virus (DENV) serotypes infect several hundred million people each year. Although primary infection is generally mild, subsequent infection by differing serotypes increases the risk for symptomatic disease ranging from fever to life-threatening shock. Despite the availability of licensed vaccines, a comprehensive understanding of antibodies that target the viral envelope protein and protect from infection remains incomplete. In this manuscript, we develop a panel of recombinant viruses that graft each envelope domain of DENV2 onto the DENV4 envelope glycoprotein, revealing protein interactions important for virus viability. Furthermore, we map neutralizing antibody responses after primary DENV2 natural infection and a human challenge model to distinct domains on the viral envelope protein. The panel of recombinant viruses provides a new tool for dissecting the E domain-specific targeting of protective antibody responses, informing future DENV vaccine design.

Project description:Dengue is a viral disease spread to humans by mosquitoes. Notably, there are four serotypes of dengue viruses (DENV) that places ~40 % of the global population at risk of infection. However, lack of a suitable drug or a preventive vaccine exacerbates the matter further. Envelope domain-III (EDIII) antigen of dengue virus (DENV) has garnered much attention as a promising vaccine candidate for dengue, in addition to its use as a diagnostic intermediate. Hence developing a method for efficient production of high quality recombinant EDIII is important for research and industrial purpose.In this work, a Pichia pastoris system was optimized for the secretory over-expression of DENV serotype-3 EDIII under the control of methanol inducible AOX1 promoter. Temperature alone had a significant impact upon the amount of secretory EDIII, with 2.5-fold increase upon reducing the induction temperature from 30 to 20 °C. However surprisingly, supplementation of culture media with Casamino acids (CA), further augmented secretory EDIII titer, with a concomitant drop of intracellular EDIII levels at both temperatures. Though, reduction in intracellular retention of EDIII was more prominent at 20 °C than 30 °C. This suggests that CA supplementation facilitates overexpressing P. pastoris cells to secrete more EDIII by reducing the proportion retained intracellularly. Moreover, a bell-shaped correlation was observed between CA concentration and secretory EDIII titer. The maximum EDIII expression level of 187 mg/L was achieved under shake flask conditions with induction at 20 °C in the presence of 1 % CA. The overall increase in EDIII titer was ~9-fold compared to un-optimized conditions. Notably, mouse immune-sera, generated using this purified EDIII antigen, efficiently neutralized the DENV.The strategy described herein could enable fulfilling the mounting demand for recombinant EDIII as well as lay direction to future studies on secretory expression of recombinant proteins in P. pastoris with CA as a media supplement.

Project description:We describe the preclinical development of a dengue virus vaccine targeting the dengue virus serotype 2 (DENV2) envelope domain III (EDIII). This study provides proof-of-principle that a dengue EDIII protein scaffold/DNA vaccine can protect against dengue challenge. The dengue vaccine (EDIII-E2) is composed of both a protein particle and a DNA expression plasmid delivered simultaneously via intramuscular injection (protein) and gene gun (DNA) into rhesus macaques. The protein component can contain a maximum of 60 copies of EDIII presented on a multimeric scaffold of Geobacillus stearothermophilus E2 proteins. The DNA component is composed of the EDIII portion of the envelope gene cloned into an expression plasmid. The EDIII-E2 vaccine elicited robust antibody responses to DENV2, with neutralizing antibody responses detectable following the first boost and reaching titers of greater than 1:100,000 following the second and final boost. Vaccinated and naïve groups of macaques were challenged with DENV2. All vaccinated macaques were protected from detectable viremia by infectious assay, while naïve animals had detectable viremia for 2-7 days post-challenge. All naïve macaques had detectable viral RNA from day 2-10 post-challenge. In the EDIII-E2 group, three macaques were negative for viral RNA and three were found to have detectable viral RNA post challenge. Viremia onset was delayed and the duration was shortened relative to naïve controls. The presence of viral RNA post-challenge corresponded to a 10-30-fold boost in neutralization titers 28 days post challenge, whereas no boost was observed in the fully protected animals. Based on these results, we determine that pre-challenge 50% neutralization titers of >1:6000 correlated with sterilizing protection against DENV2 challenge in EDIII-E2 vaccinated macaques. Identification of the critical correlate of protection for the EDIII-E2 platform in the robust non-human primate model lays the groundwork for further development of a tetravalent EDIII-E2 dengue vaccine.

Dataset Information

Dengue virus envelope protein domain I/II hinge determines long-lived serotype-specific dengue immunity.

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Dengue virus envelope protein domain I/II hinge determines long-lived serotype-specific dengue immunity.

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