The succesful development of flavivirus vaccines began years

The succesful development of flavivirus vaccines began 80years ago in 1937 with the yellow fever YFV17D live-attenuated vaccine (Monath, 2008). Since then, >600 million people have been vaccinated, with 98% protection and a >10year persistence of vaccine–induced immunity (Barrett and Teuwen, 2009). However, several severe adverse events associated with vaccine administration have been observed over the last 20years. Thus, a purified, inactivated vaccine has been recently developed and its testing results suggest good immunogenicity and tolerability (Monath et al., 2011). A few weeks ago, two studies showing immunogenicity of a plasmid DNA or adenovirus (serotype 52) expressing virus-like particles in mice and non-human primates were published (Larocca et al., 2016; Abbink et al., 2016). Here, to build on these initial findings to develop an effective ZIKV vaccine, we describe the development of a recombinant adenoviral vector expressing codon-optimized ZIKV E antigen and a subunit recombinant ZIKV E vaccine delivered transcutaneously by carboxymethyl cellulose (CMC) microneedle arrays (MNAs) (Bediz et al., 2014; Korkmaz et al., 2015), investigate their ability to induce neutralizing immune responses, and assess their ability to passively protect against ZIKV challenge in a novel neonatal ZIKV infection mouse model.
Materials & Methods
Results We generated recombinant E1/E3-deleted adenovirus serotype 5-based vectors that encode for the human secretory signal peptide hidden Markov model (SP-HMM) followed by the codon-optimized extracellular portion of the ZIKV BeH815744 E gene fused to the T4 fibritin foldon trimerization domain (ZIKV-Efl). Moreover, the ZIKV-Efl antigen was engineered with a polyhistidine-tag and a Tobacco Etch Virus (TEV) protease cleavage sequence to facilitate downstream purification (Fig. 1). The replication-defective adenovirus 5, designated as Ad5.ZIKV-Efl, was generated by loxP homologous recombination as previously described (Kim et al., 2014; Hardy et al., 1997). Recombinant ZIKV-rEfl protein was purified from the supernatant of a Ad5.ZIKV-Efl-infected HEK 293 cell line using His60 Ni Superflow Resin under native conditions and packaged as a subunit vaccine in an MNA (MNA-ZIKV-Efl). We then tested the ability of Ad5.ZIKV-Efl and MNA-ZIKV-rEfl to elicit a specific anti-ZIKV immune response in vivo. C57BL/6 mice were inoculated s.c. with 1011vp of Ad5.ZIKV-Efl or i.d. with 20μg of MNA-ZIKV-rEfl, or with PBS on day 0 followed by booster immunization on day 14 with the same dose i.n. or i.d., respectively (Fig. 2a). At 0, 2, 4, and 6weeks post prime immunization, sera were obtained from all mice and screened for the presence of ZIKV-specific tranylcypromine using ELISA analysis. ZIKV-specific antibodies were detected as soon as two weeks after the first immunization in the sera of mice vaccinated with Ad5.ZIKV-Efl (P=0.0002), while mice immunized MNA-ZIKV-rEfl showed significant titers at four weeks after the booster immunization (P<0.05) when compared with the sera of mice immunized with PBS (Fig. 2b). Furthermore, qualitative neutralizing activity of ZIKV antibodies was tested in a PRNT 50% assay. The presence of ZIKV-neutralizing antibodies was shown in both Ad5.ZIKV-Efl and MNA-ZIKV-rEfl, although the response in the mice immunized with MNA-ZIKV-rEfl was four- to 16-fold lower than the response achieved in the mice immunized with Ad5.ZIKV-Efl. As expected, no neutralizing antibody responses were observed in the control animal group (Fig. 2c). These findings support our premise that Ad5.ZIKV-Efl- and MNA-ZIKV-rEfl-based ZIKV E antigen vaccines are able to induce neutralizing ZIKV-specific immunity. To further understand how the vaccine induced ZIKV E-specific immunity, neutralizing the ZIKV in vivo and protecting the animal from its pathogenic effects, we developed a passive protection suckling mouse model. Building upon the knowledge (Dick et al., 1952) that day 7- (but not day 14-) old suckling mice are susceptible to ZIKV infection via the i.p. route showing neurological signs, pups were obtained by mating immunized female with nonimmunized male mice at week 3 after booster immunization. Pups were challenged i.p. at seven days after birth with 105pfu of ZIKV DAKAR41542, monitored daily for mortality, and weighed for 15days. The mean time to disease onset (10% weight loss) was slightly earlier in the pups from PBS-immunized mice than in those from MNA-ZIKV-rEfl-immunized mice, although the difference was not significant (7.75 vs. 8.25days, P=0.1598) (Table 1). All pups born to PBS-immunized mice showed more than a 20% body weight loss in the 10days postinfection. However, weight loss in the MNA-ZIKV-rEfl pups was reduced and a significant difference was found from day 12 (P<0.01; P<0.001, day 13–day 15) after challenge when compared to the PBS pups. No weight loss was observed in the pups born to the dams immunized with Ad5.ZIKV-Efl vaccine and no significant difference was measured between the pups of Ad5.ZIKV-Efl-immunized mice and the unchallenged control pups for the entire period. The significant difference started at day 8 (P<0.01; P<0.001, day 9–day 15) after challenge when compared to the PBS pups. (Fig. 3a). The survival rates of pups from two animals in each group were also monitored after challenge with ZIKV DAKAR41542. Survival rates of 100% (10/10) and 50% (3/6) were observed in the pups from Ad5.ZIKV-Efl- and MNA-ZIKV-rEfl-immunized dams, respectively, whereas a 12.5% (1/8) survival rate was seen in pups from PBS-immunized dams (Fig. 3b). The differences between the pups from Ad5.ZIKV-Efl- and those from PBS-immunized dams and between the pups from Ad5.ZIKV-Efl- and those from MNA-ZIKV-immunized dams were statistically significant (P=0.0001 and P=0.0136, respectively). When the pups from MNA-ZIKV-rEfl- and PBS-immunized dams were compared, no significant difference in survival rate was observed (P=0.1493), indicating that the Ad5.ZIKV-Efl vaccine candidates were efficient in passively protecting neonatal mice against lethal ZIKV challenge.