WNV continues to present public health threat to the western hemisphere, yet, there is neither effective therapy nor vaccines available for humans. Thus developing a safe and effective vaccine is a priority. Several immunization strategies have been described, some of which utilize vectored vaccines expressing WNV glycoprotein E resulted in 80%-100% protective immunity in animal model [30–34]. In this present study, attempts were made by using recombinant baculovirus approach for WNV vaccine development since baculovirus exhibit low toxoxicity in mammalian cells and can induce interferon production . Two recombinant baculoviruses, Bac-G-prM/E and Bac-prM/E, expressing the major immunogenic protein, glycoprotein E, were constructed,and the humoral and cellular immune responses were determined in mice. WNV-specific humoral (WNV-specific antibodies and neutralizing antibodies) and cellular immunity (WNV-specific IFN-γ and inflammatory cytokines) were induced in mice inoculated with recombinant baculoviruses. These data are in concordance with previous studies that showed robust humoral and cellular immune responses against PRRSV, PCV, or JEV by recombinant baculoviruses .
It has been controversial as to the relative role of the humoral versus cellular immune responses in the WNV-protective immunity. High antibody titers are believed to protect animals against flavivirus infections through direct neutralization of receptor binding, inhibition of viral fusion, and/or complement-mediated viral clearance [35, 36]. Neutralizing antibody titers of >1:10 were reported to be sufficient to afford protection in humans against JE . Schneeweiss et al. also reported that mice immunized once with pT-WNV-E produced neutralizing antibodies titer of 1:16 which can protect 100% of mice from WNV challenge . However, others have argued that cellular immune responses such as robust anti-CD8+ T cell responses are essential for clearing WNV [39, 40]. In our study, the antibody responses were evaluated in mice immunized with recombinant baculoviruses. Majority of mice immunized with two dosages of Bac-G-prM/E or Bac-prM/E developed neutralizing antibodies titers of >1:16 (Table 1). Furthermore, these baculovirus-derived vaccines can also induce anti-JEV cross-neutralizing antibodies although the titers of the antibodies were lower than antibodies against WNV. These results strongly demonstrate that the antibody response induced by WNV protein could partially cross-neutralize JEV, as has been reported in previous studies . It is reasonable to anticipate that immunization with these recombinant baculoviruses expressing WNV antigens will not only provide protection against WNV infection, but also provide partial protection against JEV infection. Cellular immunity such as IFN-γ production and inflammatory cytokines responses were also determined in mice after immunization with these baculoviruses since these cytokines play a critical role in directing the cell-mediated immune responses . Bac-G-prM/E- or Bac-prM/E-immunized mice produced higher levels of IFN-γ than any other group (Figure 4) and promoted the release of inflammatory cytokines, including tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), and interleukin-2 (IL-2) (Figure 5). In this case, secretion of IFN-γ, TNF-α, or IL-2 are indicative of a Th1 response, whereas a Th2 response is characterized by induction of IL-6 . These indicate that the recombinant baculoviruses can stimulate both humoral and cellular immune responses which are essential to control dissemination of infection and to inhibit the entry of virus into brain.
Our results also demonstrate that Bac-G-prM/E exhibited better immunogenicity than Bac-prM/E, as indicated by the higher levels of antibody titers, IFN-γ and inflammatory cytokines production. It is highly probable that the expression of VSV/G was associated with enhancement of the immune responses by augmenting transduction efficiency of baculovirus into mammalian cells . In addition, the VSV/G-modified baculovirus exhibited greater resistance to animal serum inactivation than the unmodified baculovirus , which may contribute to the better immunogenicity of Bac-G-prM/E.
It has been reported that recombinant E-DIII protein induces high neutralizing antibody titers, as well as protection against lethal WNV and partial protection against lethal JEV . Therefore, the recombinant E-DIII protein of WNV expressed in E.coli was used as a positive control in the present study. However, intramuscular injection of mice with E-DIII protein elicited lower levels of neutralization antibody titers than mice immunized with the recombinant baculoviruses even at a low dosage (108 PFU/mouse) (Table 1), although the total IgG level was high (Figure 3). This could be due to differences in experimental designs such as the route of immunization, with or without adjuvant, and mouse strain. In addition, it has been shown that E-DIII protein immunization elicits low level of neutralizing antibodies with relatively high IgG responses , which is consistent with our results. It is noticed that E-DIII protein also induced lower levels of cellular immune response than recombinant baculoviruses, since E protein expressed by recombinant baculovirus contains more T cell epitopes than its domain III , and the baculovirus augment cellular immunity.
Baculovirus has been shown to possess a strong adjuvant activity and to promote humoral and cellular immune responses for foreign antigens, maturation of dendritic cells, and production of inflammatory cytokines and IFN . The transduction of macrophages in vitro by baculovirus led to the induction of significant levels of IL-6 and TNF-α . It has been proposed that baculovirus genome, especially its CpG motifs, could be recognized by macrophages and DCs. Furthermore, baculoviruses enter into the cells through mannose receptor (MR)-mediated endocytosis or phagocytosis, leading to the secretion of inflammatory cytokines through a MyD88/TLR9-dependent signaling pathway [23, 24]. However, Chen et al. reported that recombinant baculoviruses are able to induce a robust secretion of inflammatory cytokines through a TLR3-dependent pathway . In our study, induction of a measurable IFN-γ and inflammatory cytokines, including TNF-α, IL-6, and IL-2 by control baculovirus (Bac-G-EGFP and Bac-EGFP) was observed, demonstrating that this virus can function as a stimulator for production of cell-mediate immunity in an antigen- independent manner (Figure 4). Similar results have been reported previously that injection with control baculovirus is capable of inducing IFN-γ responses [26, 27, 29]. This is probably due to direct interaction of baculovirus with dendritic cells and macrophages in the spleen. Since it has been reported that splenic dendritic cells and macrophages play a central role in baculovirus-induced inflammatory responses in mice . These baculovirus- induced non-specific inflammatory responses raise concerns that whether they will compromise the use of baculovirus vectors for in vivo gene therapy in humans and animals. However, Bac-G-E2 inoculated mice released inflammatory cytokines as early as 6h post-injection, and returned to background levels by 48h post-injection . Furthermore, there was no cytokine-induced acute toxicity or mortality in baculovirus-immunized mice, which suggests that non-specific inflammatory responses induced by baculovirus vectors may be minimized by host. Nevertheless, more investigations are needed to ensure the safety of baculovirus vectors.
The protection is an important index to evaluate the efficiency of vaccine. However, the ability of our recombinant baculoviruses to protect against lethal challenge of WNV was not determined in consideration of the safety problem since WNV has not been found in China. Nevertheless, the neutralizing antibodies and the cellular immune responses induced by recombinant baculoviruses may provide the basis for protection. Immunization with baculovirus expressing JEV antigens have been shown to confer protection against JEV challenge, demonstrating that cellular immunity plays a crucial role against JEV infection . Thus it is well supported that recombinant baculoviruses expressing WNV prM/E proteins could provide protective immunity against WNV challenge.