Conflict of interest: MHC, HT, BDM, SAD, and AMDS are co-inventors on a patent application (US 62589006, filed 11/21/2017) describing the discovery and use of A9E and G9E human mAbs. BD and ED are employees of Integral Molecular.
BackgroundThe 4 dengue serotypes (DENV) are mosquito-borne pathogens that are associated with severe hemorrhagic disease. DENV particles have a lipid bilayer envelope that anchors two membrane glycoproteins prM and E. Two E-protein monomers form head-to-tail homodimers and three E-dimers align to form “rafts” that cover the viral surface. Some human antibodies that strongly neutralize DENV bind to quaternary structure epitopes displayed on E protein dimers or higher order structures forming the infectious virus. Expression of prM and E in cell culture leads to the formation of DENV virus-like particles (VLPs) which are smaller than wildtype virus particles and replication defective due to the absence of a viral genome. There is no data available that describes the antigenic landscape on the surface of flavivirus VLPs in comparison to the better studied infectious virion.MethodsA large panel of well characterized antibodies that recognize epitope of ranging complexity were used in biochemical analytics to obtain a comparative antigenic surface view of VLPs in respect to virus particles. DENV patient serum depletions were performed the show the potential of VLPs in serological diagnostics.ResultsVLPs were confirmed to be heterogeneous in size morphology and maturation state. Yet, we show that many highly conformational and quaternary structure-dependent antibody epitopes found on virus particles are efficiently displayed on DENV1–4 VLP surfaces as well. Additionally, DENV VLPs can efficiently be used as antigens to deplete DENV patient sera from serotype specific antibody populations.ConclusionsThis study aids in further understanding epitopic landscape of DENV VLPs and presents a comparative antigenic surface view of VLPs in respect to virus particles. We propose the use VLPs as a safe and practical alternative to infectious virus as a vaccine and diagnostic antigen.Electronic supplementary materialThe online version of this article (10.1186/s12985-018-0970-2) contains supplementary material, which is available to authorized users.
The current leading Zika vaccine candidates in clinical testing are based on live or killed virus platforms, which have safety issues, especially in pregnant women. Zika subunit vaccines, however, have shown poor performance in preclinical studies, most likely because the antigens tested do not display critical quaternary structure epitopes present on Zika E protein homodimers that cover the surface of the virus. Here, we produce stable recombinant E protein homodimers that are recognized by strongly neutralizing Zika specific monoclonal antibodies. In mice, the dimeric antigen stimulate strongly neutralizing antibodies that target epitopes that are similar to epitopes recognized by human antibodies following natural Zika virus infection. The monomer antigen stimulates low levels of E-domain III targeting neutralizing antibodies. In a Zika challenge model, only E dimer antigen stimulates protective antibodies, not the monomer. These results highlight the importance of mimicking the highly structured flavivirus surface when designing subunit vaccines.
Dengue virus (DENV) is responsible for the most prevalent and significant arthropod-borne viral infection of humans. The leading DENV vaccines are based on tetravalent live-attenuated virus platforms. In practice, it has been challenging to induce balanced and effective responses to each of the four DENV serotypes because of differences in the replication efficiency and immunogenicity of individual vaccine components. Unlike live-vaccines, tetravalent DENV envelope (E)-protein subunit vaccines are likely to stimulate balanced immune responses because immunogenicity is replication independent. However, E protein subunit vaccines have historically performed poorly, in part, because the antigens utilized were mainly monomers that did not display quaternary structure epitopes found on E-dimers and higher order structures that form the viral envelope. In this study, we compared the immunogenicity of DENV2 E-homodimers and DENV2 E-monomers. The stabilized DENV2 homodimers but not monomers were efficiently recognized by virus specific and flavivirus cross-reactive potent neutralizing antibodies that have been mapped to quaternary structure epitopes displayed on the viral surface. In mice, the dimers stimulated 3-fold higher levels of virus specific neutralizing IgG that recognized epitopes that were different from the epitopes recognized by lower level neutralizing antibodies induced by monomers. The dimer induced a stronger ED-I and ED-II targeted response, while the monomer antigens stimulated an ED-III epitope response and induced fusion loop epitope antibodies that are known to facilitate ADE. This study shows DENV E subunit antigens that have been designed to mimic the structural organization of viral surface are better vaccine antigens than E protein monomers. IMPORTANCE Dengue virus vaccine development is particularly challenging because vaccines have to provide protection against 4 different dengue stereotypes. The leading dengue virus vaccine candidates in clinical testing are all based on live virus vaccine platforms and struggle to induce balanced immunity. Envelope subunit antigens have the potential to overcome these limitations, but have historically performed poorly as vaccine antigens, because the previously tested versions were presented as monomers and not in their natural dimer configuration. This study shows that the authentic presentation of DENV2 E-based subunits has a strong impact on antibody responses, underscoring the importance of mimicking the complex protein structures that are found on DENV virus particle surfaces when designing subunit vaccines.
Dengue virus (DENV) is the causative agent of dengue fever and dengue hemorrhagic shock syndrome. Dengue vaccine development is challenging because of the need to induce protection against four antigenically distinct DENV serotypes. Recent studies indicate that tetravalent DENV vaccines must induce balanced, serotype-specific neutralizing antibodies to achieve durable protective immunity against all 4 serotypes. With the leading live attenuated tetravalent DENV vaccines, it has been difficult to achieve balanced and type-specific responses to each serotype, most likely because of unbalanced replication of vaccine viral strains. Here we evaluate a tetravalent DENV protein subunit vaccine, based on recombinant envelope protein (rE) adsorbed to the surface of poly (lactic-co-glycolic acid) (PLGA) nanoparticles for immunogenicity in mice. In monovalent and tetravalent formulations, we show that particulate rE induced higher neutralizing antibody titers compared to the soluble rE antigen alone. Importantly, we show the trend that tetravalent rE adsorbed to nanoparticles stimulated a more balanced serotype specific antibody response to each DENV serotype compared to soluble antigens. Our results demonstrate that tetravalent DENV subunit vaccines displayed on nanoparticles have the potential to overcome unbalanced immunity observed for leading live-attenuated vaccine candidates.
8The current leading Zika vaccine candidates in clinical testing are based on live or killed virus 9 platforms, which have safety issues, especially in pregnant women. Zika subunit vaccines, 10 however, have shown poor performance in preclinical studies. We hypothesized that Zika 11Envelope (E) protein subunit vaccines have performed poorly because the antigens tested have 12been recombinant E monomers that do not display critical quaternary structure epitopes present 13on Zika E protein homodimers that cover the surface of the virus. To test this hypothesis, we 14 engineered and produced stable recombinant E protein homodimers. Unlike the E monomer, the 15 dimer was recognized by strongly neutralizing monoclonal antibodies isolated from Zika-immune 16 individuals. In a mouse model of vaccination, the dimeric antigen stimulated strongly neutralizing 17 antibodies that targeted epitopes that were similar to epitopes recognized by human antibodies 18 following natural Zika virus infection. In contrast, the monomer antigen stimulated lower levels of 19 neutralizing antibodies directed to simple epitopes on domain III of E protein. In a mouse model 20 of ZIKV challenge, only E dimer antigen stimulated protective antibodies, not the 21 monomer. These results highlight the importance of mimicking the highly structured flavivirus 22 surface when designing subunit vaccines. The flavivirus field has a long history of using E 23 monomers as vaccine antigens with limited success. These results are applicable to developing 24 second generation subunit vaccines against Zika as well as other medically important flaviviruses 25 such as dengue and yellow fever viruses. 26 130 Figure 3: ZIKV rE D stimulates antibodies that target complex epitopes on the virion. A) Method for 131 depleting EDIII binding antibodies from mouse immune sera. Recombinant ZIKV EDIII (His-tagged) was 132 coupled to nickel beads i then incubated with immune sera ii . Magnetic pull down removes nickel beads and 133 EDIII binding antibodies iii , leaving leavind sera depleted from EDIII binding antibodies iv . B) The level of 134 EDIII-binding antibodies in the serum of mice immunized with the indicated antigens is shown as a 135 percentage of the total level of ZIKV specific IgG, as measured by ELISA of EDIII-depleted and control-136 depleted serum. C) The neutralizing activity of EDIII-depleted and undepleted sera was determined and 137 expressed as the dilution at which 50% of the virus was neutralized (Neut50). D) A blockade of binding 138 (BOB) assay was used to evaluate if mice immunized with rE M or rE D developed antibodies that blocked 139 the binding of A9E, G9E and EDE C10 human mAbs. Data points represent individual mice. Statistical140differences were determined by one-way ANOVA followed by a Tukey's test (p<0.05).
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