Zika virus (ZIKV) and the 4 dengue virus (DENV) serotypes are mosquito-borne Flaviviruses that are associated with severe neuronal and hemorrhagic syndromes. The mature flavivirus infectious virion has 90 envelope (E) protein homo-dimers that pack tightly to form a smooth protein coat with icosahedral symmetry. Human antibodies that strongly neutralize ZIKV and DENVs recognize complex quaternary structure epitopes displayed on E-homo-dimers and higher order structures. The ZIKV and DENV E protein expressed as a soluble protein is mainly a monomer that does not display quaternary epitopes, which may explain the modest success with soluble recombinant E (sRecE) as a vaccine and diagnostic antigen. New strategies are needed to design recombinant immunogens that display these critical immune targets. Here we present two novel methods for building or stabilizing in vitro E-protein homo-dimers that display quaternary epitopes. In the first approach we immobilize sRecE to enable subsequent dimer generation. As an alternate method, we describe the use of human mAbs to stabilize homo-dimers in solution. The ability to produce recombinant E protein dimers displaying quaternary structure epitopes is an important advance with applications in flavivirus diagnostics and vaccine development.
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 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.
The dengue virus (DENV) causes over 350 million infections, resulting in ∼25,000 deaths per year globally. An effective dengue vaccine requires generation of strong and balanced neutralizing antibodies against all four antigenically distinct serotypes of DENV. The leading live-attenuated tetravalent dengue virus vaccine platform has shown partial efficacy, with an unbalanced response across the four serotypes in clinical trials. DENV subunit vaccine platforms are being developed because they provide a strong safety profile and are expected to avoid the unbalanced immunization issues associated with live multivalent vaccines. Subunit vaccines often lack immunogenicity, requiring either a particulate or adjuvanted formulation. Particulate formulations adsorbing monomeric DENV-E antigen to the particle surface incite a strong immune response, but have no control of antigen presentation. Highly neutralizing epitopes are displayed by DENV-E quaternary structures. To control the display of DENV-E and produce quaternary structures, particulate formulations that covalently attach DENV-E to the particle surface are needed. Here we develop a surface attached DENV2-E particulate formulation, as well as analysis tools, using PEG hydrogel nanoparticles created with particle replication in nonwetting templates (PRINT) technology. We found that adding Tween-20 to the conjugation buffer controls DENV-E adsorption to the particle surface during conjugation, improving both protein stability and epitope display. Immunizations with the anionic but not the cationic DENV2-E conjugated particles were able to produce DENV-specific and virus neutralizing antibody in mice. This work optimized the display of DENV-E conjugated to the surface of a nanoparticle through EDC/NHS chemistry, establishing a platform that can be expanded upon in future work to fully control the display of DENV-E.
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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