Both the purified recombinant protein GP-Fc and the gene vaccine pVR-GP-Fc containing the recombinant protein sequence induced antibodies against GP protein in mice. For the recombinant adjuvant, the recombinant protein induced the production of binding antibodies faster than the genetic vaccine, and the titer was higher. In the first week after immunization of the two vaccines, the endpoint of antibody titration in serum exceeded 103, and both vaccines rapidly induced specific immune production. There was no significant difference in antibody titer induced by the two vaccines 2 weeks after priming. After the third week of boosting, the recombinant protein induced IgG production was faster and stronger, and the serum dilution was 8.8 × 105 after two boosts. Specific antibodies could still be detected afterwards. The ability of the gene vaccine to induce antibody production was slightly worse than that of the recombinant protein. Moreover, at week 8 (i.e., two boosts) the antibody titer reached 3 × 105. Consistent with related studies, the results indicate that genetic vaccines induce humoral immune responses that are weaker than direct antigen immunity, both in strength and speed, compared to recombinant subunits (Figure 3).
The immunofluorescence assay was carried out using the EBOV neutralizing antibody MIL-77 as the primary antibody. The cell surface of the transfected GP expression plasmid pCAGGS-EBOV GP showed obvious fluorescence signals, while the cells transfected with the empty vector did not produce any green signal (Figure 4). The constructed expression plasmid pCAGGS-EBOVGP can express EBOV GP protein in HEK293 cells with correct protein localization and molecular structure, and could stably bind to neutralizing antibodies. This plasmid could be used as a pseudovirus envelope plasmid.
The micro-neutralization test of the prepared pseudovirus pEBOV and neutralizing antibody showed that it could be blocked by the EBOV neutralizing antibody MIL-77, while the Mers-NP monoclonal antibody could not block the virus infection (Table 1). RLU values were analyzed using GraphPad Prism software, t-test, with significant differences (t = 2.55, P = 0.0286 < 0.05).
Mers-NP Monoclonal Antibody
RLU Neutralization percentage (%) RLU Neutralization percentage (%) 20.00 3976.0 81.1 18039.0 14.3 5.00 7182.3 65.9 19116.3 9.2 1.25 10640.7 49.5 17941.0 14.8 0.31 13839.7 34.3 20025.7 4.9 0.08 17387.7 17.4 18523.7 12.0
Table 1. pEBOV Neutralize Verification Test
The neutralization percentage regression equation y = 11.501 ln(x) + 47.038 was used to calculate the corresponding ND50 of the MIL-77 neutralizing antibody. The calculated ND50 was 1.29 μg/mL (Figure 5). The pEBOV prepared by this method could be used in the EBOV neutralization test.
The identified Ebola pseudovirus pEBOV detection system was used to evaluate whether the recombinant protein GP-Fc and the genetic vaccine pVR-modGP-Fc could induce protective antibodies by a serum micro-neutralization test. The results of micro-neutralization experiments showed that both the recombinant protein GP-Fc and the gene vaccine pVR-modGP-Fc induced neutralizing antibodies in BALB/c mice. One-way ANOVA test analysis using GraphPad Prism software revealed a significant difference between the post-immune serum and the negative serum control (F = 13.29, P < 0.01). Both vaccine-induced neutralizing antibody half-inhibitor amounts (ND50) exceeded 1:80, and the percent neutralization at this dilution for the recombinant protein GP-Fc and the genetic vaccine pVR-modGP-Fc was 52.6% and 60.6%, respectively. Although the recombinant protein induced a better binding titer than the genetic vaccine, the neutralization test showed that the genetic vaccine pVR-modGP-Fc induced higher neutralizing titers in BALB/c mice. The test results are presented in Table 2, and the trend of the percentage of neutralization is shown in Figure 6.
Dilution Ratio GP-Fc Recombination Protein pVR-modGP-Fc DNA Vaccine Negative Serum Control RLU Neutralization percentage (%) RLU Neutralization percentage (%) RLU Neutralization percentage (%) 1:40 12953.0 59.5 10358.3 67.6 24736.7 22.6 1:80 15159.3 52.6 12597.3 60.6 27080.7 15.3 1:160 20677.7 35.3 16828.3 47.4 27977.7 12.5 1:320 20472.3 36.0 21543.7 32.6 37679.7 0.0 1:640 22451.0 29.8 21886.0 31.5 33855.0 0.0 1:1, 280 22784.3 28.7 22885.0 28.4 33328.7 0.0
Table 2. Neutralization Test of Vaccinate Mice Serum