A bacterium-like particle vaccine displaying Zika virus prM-E induces systemic immune responses in mice

The emergence of Zika virus (ZIKV) infection, which is an unexpectedly associated with congenital defects, has prompted the development of safe and effective vaccines. The gram-positive enhancer matrix-protein anchor (GEM-PA) display system has emerged as a versatile and highly effective platform for delivering target proteins for vaccines. In this article, we developed a bacterium-like particle vaccine ZI-Δ-PA-GEM based on the GEM-PA system. The fusion protein ZI-Δ-PA, which contains the prM-E-Δ protein of ZIKV (with a stem-transmembrane region deletion) and the protein anchor PA3, was expressed. The fusion protein was successfully displayed on the GEM surface, forming ZI-Δ-PA-GEM. Moreover, when BALB/c mice were immunized intramuscularly with ZI-Δ-PA-GEM combined with 201 VG and poly(I:C) adjuvants, durable ZIKV-specific IgG and protective neutralizing antibody responses were induced. Potent B cell/DC activation was also be stimulated early after immunization. Remarkably, splenocyte proliferation, the secretion of multiple cytokines, T/B cell activation and central memory T cell responses were elicited. These data indicate that ZI-Δ-PA-GEM is a promising bacterium-like particle vaccine candidate for ZIKV. Author summary Because Zika virus (ZIKV) infection is considered as an example of “disease X”, the development of a safe and effective ZIKV vaccine is essential. The gram-positive enhancer matrix-protein anchor (GEM-PA) display system has been used in many vaccine studies due to its advantages. In this study, prM-E-△ protein of ZIKV (with a stem-transmembrane region deletion) and the protein anchor PA3 was fusion expressed, termed ZI-△-PA. Then the fusion protein ZI-△-PA could be displayed on the surface of GEM, forming ZI-△-PA-GEM. The author evaluated the immunogenicity of ZI-△-PA-GEM with the 201 VG and poly(I:C) adjuvants. The study demonstrates that ZI-△-PA-GEM induced mice to produce neutralizing antibody and specific cellular immune responses. The author believe that the bacterium-like particle vaccine ZI-△-PA-GEM has the potential to be used as the ZIKV vaccine.

emergency of international concern'. Moreover, ZIKV disease was listed as a "Disease 60 X" by the WHO in the "Research and Development (R&D) Blueprint for Action to 61 Prevent Epidemics" [5]. As of July 2019, cases of ZIKV had been reported worldwide, 62 including 87 countries and territories, according to a WHO report [6]. To date, no 63 accepted therapies or vaccines are available for ZIKV, and the WHO has made ZIKV 64 vaccine development a top priority [7]. Substantial progress has been made on 65 developing vaccines, which are essential to control the spread of ZIKV. Moreover, new 66 vaccine search efforts are needed because of the complexity of ZIKV immunity and 67 pathogenesis [8][9][10]. Neutralizing antibody epitopes exist on the surface of the ZIKV E 68 protein [11,12], and precursor membrane (prM) protein complexes with the E protein 69 are usually ideal candidates for vaccine development.

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The gram-positive enhancer matrix-protein anchor (GEM-PA) is a new bacterial 71 surface display system composed of GEM particles and a protein anchor (PA) [13]. 72 First, the foreign protein and PA are expressed as a fusion. Then, the fusion protein 73 "foreign protein-PA" is displayed on the GEM surface by means of the GEM-PA 74 surface display system [14]. The GEM surface display system has the following 75 advantages. (1) Simple purification of a foreign protein by low-speed centrifugation is 76 convenient and efficient. (2) Lactococcus lactis (L. lactis) is globally recognized as a 77 safe probiotic [15,16]. After treatment, GEM has no safety risk as a carrier due to the 78 absence of nucleic acids and proteins. (3) The displayed protein can effectively 79 stimulate a stronger immune response [17]. In this study, the GEM-PA display system 80 was used to display ZIKV prM-E glycoproteins on the GEM surface and study their 81 immunogenicity. 84 The recombinant baculoviruses were grown in Spodoptera frugiperda clone 9 (Sf9) 85 suspension cells, which were maintained in SFM II 900 medium (Thermo Fisher

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Several gene fragments containing prM-E of ZIKV (accession: KX601168.1) were 97 constructed (strategies shown in Fig 1A). The gene fragment "ZI-△-PA" was obtained 98 by overlap PCR, in which the sequences from the N-terminus to the C-terminus were 99 prM-E of ZIKV (with deletion of the stem-transmembrane (ST-TM) region), a linker 100 and protein anchor 3 (PA3, containing three lysin motifs). In ZI-JE-△-PA, the signal 101 peptide of ZI-△-PA was replaced with the signal peptide of Japanese encephalitis virus 102 (JEV). In ZI-GP-△-PA, the signal peptide of ZI-△-PA was replaced with the gp67 103 signal peptide. Signal peptide replacement was performed by PCR. All of the sequences 104 were optimized for insect cells and synthesized by Sangon Biotech (Shanghai, China).

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The above-mentioned target genes were cloned into the baculovirus transfer vector 106 pFastBac Dual under the pH (Not I + Hind III) and p10 (Sma I + Nhe I) promoters. (1:200) was used as the primary antibody, and an HRP-conjugated goat anti-mouse 121 antibody (1:250) was used as the secondary antibody. Blue peroxidase substrate was 122 added and incubated for 3 h at RT. Blue-stained foci in the wells of the highest dilution 123 that contained a reasonable number of foci were counted, and the titer was determined.   After washing once with sterile distilled PBS, the bacteria were boiled for 30 min with 147 10% trichloroacetic acid (TCA) to obtain GEM particles. After washing three times 148 with PBS, the number of GEM particles was counted, diluted to one unit (1 U, 2.5×10 9 149 particles per 1U) and stored at -70°C.       (Fig. 3A). ZIKV E-protein-287 specific IgG could be detected in the serum for at least 8 weeks (Fig. 3B). The serum at 288 week 6 was subjected to a VNA, and the results (Fig. 3C) showed that the mean PRNT 50 289 titers were all above 10, which is the threshold for protection. 290 We next examined the quality of this humoral response by IgG2a and IgG1 291 subisotype-specific ZIKV-E ELISA at week 6 ( Fig. 3D). IgG2a/IgG1 ratios lower than 292 1.0 indicated that ZI-D/ZI-E induced a Th2-biased response.  Next, the activation of DCs was analyzed by detecting the proportion of CD11c + 317 MHC II + cells (Fig. 4B). On the 3 rd day, there was slight activation of DCs, but there 318 was no significant difference among the groups. On the 6 th and 9 th days after the first 319 immunization, the immune groups had an increased percentage of CD11c + MHC II + 320 double-positive cells. On the 9 th day, the activation levels of some groups decreased,  were detected in the immune groups (Fig. 5B). Next, we tested the levels of multiple 340 cytokines in the supernatants of stimulated splenocytes from all groups. The levels of 341 the cytokines Th1 (IFN-γ) and Th2 (IL-4, IL-6 and IL-10) were significantly higher in 342 the ZI-△-PA-GEM group than in the control group (Fig. 5C). The tumor necrosis factor 343 (TNF)-α levels were also increased slightly, but the difference was not significant. In   induce potent E-specific IgG antibodies, as well as NAb responses. In addition, the 419 antibody subtype was Th2 biased, which might be advantageous for protection in some 420 ways and has been reported for vaccines [24,[31][32][33][34].

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High levels of T cell activation were also detected, especially with ZI-△-PA-GEM.