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Recently, several types of ZIKV vaccine candidates, including inactivated and live attenuated viruses, DNA and RNA vaccines, have been shown to have protective efficacy (Durbin and Wilder-Smith 2017). While these vaccine candidates show great promise, difficulties remain to be overcome regarding their licensing, especially on account of safety. The use of live viruses increases the risk of viral dissemination, and these vaccines increase the risk of side effects due to infection with viral nucleic acids. As alternative ZIKV vaccine platform, ZIKV VLPs have been produced in mammalian cells and plants and have been demonstrated to be potentially safe and effective (Boigard et al. 2017; Yang et al. 2017). VLPs are highly ordered multiprotein structures and carry many characteristics of viruses that can be used in vaccine development. Their good safety profile makes ZIKV VLPs an appropriate choice for immunocompromised individuals and pregnant women, as the World Health Organization has identified women of child-bearing age (including pregnant women) as the primary target population for ZIKV vaccination. Furthermore, VLPs have high immunogenicity and efficacy due to their unique structures. Flavivirus infection usually elicits neutralizing antibodies that bind only to complex quaternary epitopes that are only displayed on intact particles, and not to recombinant monomeric E proteins (de Alwis et al. 2012). ZIKV VLPs display E proteins with proper folding and conformation, which means that they have advantages over subunit vaccines (such as the single E protein vaccine) regarding the induction of neutralizing antibodies.
In this study, we described a new strategy for generating ZIKV VLPs consisting of prM and E proteins in insect Sf9 cells using recombinant baculovirus, and these VLPs can potentially induce strong humoral and cellular immune responses in mice. The VLPs were efficiently isolated using sucrose gradient purification. TEM and IEM revealed that the VLPs appear as rough spherical particles and have similar morphology and antigenicity to native virions. However, the lack of an inner shell of capsid protein and the viral genome leads to a smaller size (30-50 nm) compared with native virions.
An animal experiment was performed to determine the immunogenicity and efficacy of this VLP-based vaccine, and a summary of the results is shown in Table 1. The serum samples from the mice before and after immunization were assayed. The ZIKV-specific IgG and NAb levels obviously increased after immunization with VLPs. However, VLPs seemed to result in lower NAb titers than inactivated ZIKV. This may be partially due to the conformation of VLPs potentially being slightly different compared with the conformation of the authentic virus, as this conformational change may affect the neutralizing capacity and binding capacity to ZIKV. We also speculated that the ZIKV-specific antigens in our VLP vaccine were not as much as those in the inactivated virus vaccine and different vaccination dosage should be administered in further experiment. Moreover, an antibody subclass switch occurred, as demonstrated by the findings regarding the ZIKV-specific IgG subclasses in the serum. The ELISA results showed that in VLP-immunized mice, more IgG2a than IgG1 was initially produced. However, after two booster injections, the levels of both IgG2a and IgG1 isotypes increased and were not significantly different, suggesting that booster immunizations led to memory B-cell activation and promote the Th1 immune response switch to a more balanced Th1/Th2 immune response. Immunization with VLPs also elicited robust memory T cell immune responses, which are important for viral clearance (Del Prete and Romagnani 1994). The production of high levels of Th1 type cytokines (IFN-γ and IL-2) as well as Th2 type cytokines (IL-4 and IL-10) indicates that a mixed Th1/Th2 response was primed successfully by VLPs. All these results indicate that the VLP-based vaccine is highly immunogenic (inducing a wide-ranging and balanced immune response), however, the neutralization antibody titers and cytokine levels were relatively weak compared with those elicited by the inactivated virus. This may be due to the lack of proper protein processing and post translational modifications in the baculovirus-insect system, and the proper folding, especially glycosylation of viral antigens plays an important role in the efficacy of VLP-based vaccines as it is critical for immune recognition (Rudd et al. 1999). However, lepidopteran cells are unable to produce glycoproteins with structurally authentic mammalian N-glycans (Jarvis 2003). Although the immune responses elicited by the VLPs are significantly weaker than those elicited by inactivated ZIKV, VLPs certainly represent a safer method of preventing ZIKV infection compared with other clinical prevention strategies. In addition, the VLP production process involving insect cells is low risk and fast. The protective immunity of the VLPs in vivo and the potential of the VLPs to induce antibody-dependent enhancement of infection (ADE) will be investigated further in a suitable animal model, e.g., A129 (type Ⅰ interferon receptor-deficient) or wild-type mice treated with an anti-IFNAR1 antibody.
Table 1. Immune responses elicited by the VLP vaccine and controls
We propose that ZIKV VLPs produced by insect cells using recombinant baculovirus should be further developed as a safe and effective vaccine candidate to protect humans against ZIKV outbreaks.
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This work was supported by the Science and Technology Basic Work Program (2013FY113500) from the Ministry of Science and Technology of China, and the strategic priority research program of the Chinese Academy of Sciences (ZDRW-ZS-2016-4). We acknowledge the Core Facility and Technical Support of Wuhan Institute of Virology for technical assistance.
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HW and FD designed the experiments. SD and YZ carried out the experiments. SD and TZ analyzed the data. SD, HW and FD wrote the paper. All the authors approved the final manuscript.