Outer Membrane Vesicles from Neisseria Meningitidis (Proteossome) Used for Nanostructured Zika Virus Vaccine Production

Abstract

The increase of Zika virus (ZIKV) infections in Brazil in the last two years leaves a prophylactic measures on alert for this new and emerging pathogen. Concerning of our positive experience, we developed a new prototype using Neisseria meningitidis outer membrane vesicles (OMV) on ZIKV cell growth in a fusion of OMV in the envelope of virus particles. The fusion of nanoparticles resulting from outer membrane vesicles of N. meningitidis with infected C6/36 cells line were analyzed by Nano tracking analysis (NTA), zeta potential, differential light scattering (DLS), scan and scanning transmission eletronic microscopy (SEM and STEM) and high resolution mass spectometry (HRMS) for nanostructure characterization. Also, the vaccination effects were viewed by immune response in mice protocols immunization (ELISA and inflammatory chemokines) confirmed by Zika virus soroneutralization test. The results of immunizations in mice showed that antibody production had a titer greater than 1:160 as compared to unvaccinated mice. The immune response of the adjuvant and non-adjuvant formulation activated the cellular immune response TH1 and TH2. In addition, the serum neutralization was able to prevent infection of virus particles in the glial tumor cell model (M059J). This research shows efficient strategies without recombinant technology or DNA vaccines.

Figure 1

Protocol for vaccine production. Schematic representation of OMV (extracted from N. meningitidis strain C2135 in yellow and blue little circles) in fusion process of ZIKV (red circles) replicated in C6/36 cells. The agitation force promoves the fusion of OMV with vírus particle producing the OMV/ZIKV fusion particles (orange and blue circles).

Figure 2

Field emission electronic microscopy. In (A) Scanning Transmission Electronic Microscopy - STEM the OMV nanostructure from N. meningitidis. In (B) STEM of OMV/ZIKVfusioned nanoparticle. The size increasing of the nanostructures showed in (B) when compared with the OMV in (A).

Figure 3

Clustering result for the 85 top features in the PLS-DA VIP scores, shown as a heat map (distance measured by Euclidean and clustering algorithm using ward.D), with a color-coded thermometer (bottom) indicating the relative presence of metabolites on each respective group.

Figure 4

ELISA analysis of mice immunized with OMV/ZIKVfusion (group II). The antibody recognized was compared with non immunized control group (group I). The significant values were obtained until the titers 1:160.

Figure 5

Expression of inflammatory chemokines from mice vaccined splenocytes. In this analysis were perfomed the qRTPCR using specific primers for IL2 (TH1 marker), IL4 (TH2 marker), IL10, INFγ and TGFβ (memory marker). The (*) indicate the significant imune response compared with control group not vaccined.

Figure 6

(A) Soroneutralization expressed in ηg/µg ZIKV particles detected in a total amount of 1 µg of RNA. The values found by qRTPCR expressed were all considered very significant with P < 0,005. (B) Soroneutralization expressed in log of copy number of ZIKV particles detected in a total amount of 1 µg of RNA.