An RNA nanoparticle vaccine against Zika virus elicits antibody and CD8+ T cell responses in a mouse model

Abstract

The Zika virus (ZIKV) outbreak in the Americas and South Pacific poses a significant burden on human health because of ZIKV's neurotropic effects in the course of fetal development. Vaccine candidates against ZIKV are coming online, but immunological tools to study anti-ZIKV responses in preclinical models, particularly T cell responses, remain sparse. We deployed RNA nanoparticle technology to create a vaccine candidate that elicited ZIKV E protein-specific IgG responses in C57BL/6 mice as assayed by ELISA. Using this tool, we identified a unique H-2D^b-restricted epitope to which there was a CD8⁺ T cell response in mice immunized with our modified dendrimer-based RNA nanoparticle vaccine. These results demonstrate that this approach can be used to evaluate new candidate antigens and identify immune correlates without the use of live virus.

Figure 1

RNA nanoparticle vaccine design and function. (a) ZIKV polypeptide region encoded in the RNA replicon as the target antigen (numbers refer to amino acid positions), and predicted topology of the expressed truncated protein segment. (b) Immunoblot performed on lysates of replicon-transfected BHK21 cells 72 h post-transfection with an anti-ZIKV E rabbit polyclonal antibody. Asterisks indicate additional high-molecular weight bands possibly corresponding to incompletely processed E protein (*) or a combination of incompletely processed E protein and background band also present in untransfected control cells (**). (c) Immunoblot performed as in (b) but on conditioned supernatant removed from transfected cells. The asterisk indicates a common background band presumably produced by cross-reactivity of the polyclonal antibody used for detection against a culture medium serum component. The membrane was cropped above the 75 kDa marker to eliminate contaminating bovine serum albumin (from the culture medium) bands from the blot. (d) Anti-ZIKV recombinant E protein IgG titers from C57BL/6 mice immunized with the indicated RNA nanoparticle vaccine.

Figure 2

RMA-S cell peptide-binding assay. The binding of the indicated peptides to MHC class I molecules was determined by surface stabilization of H-2Kb or H-2Db molecules on RMA-S cells as measured by flow cytometry. Assays were performed in triplicate. The “−” denotes that no peptide was added, and the “+” denotes the addition of a peptide known to bind to the measured MHC class I molecule (OVA SIINFEKL for H-2K^b, and HPV16 E7 RAHYNIVTF for H-2D^b). Fluorescence intensities were normalized to the no peptide control and data are shown as the means ± SD. ns, not statistically significant from the no-peptide control value according to an unpaired two-tailed t-test (p > 0.05).

Figure 3

Candidate ZIKV E H-2D^b-binding peptide stimulates CD8⁺ T cells from ZIKV-immunized mice. Splenocytes from mice vaccinated with the indicated RNA nanoparticle vaccine were stimulated ex vivo with 0.2 μg/mL of the indicated peptide. After 7 h, intracellular cytokine staining for IFNγ was performed and measured by flow cytometry. Error bars represent SD, and N = 5 animals per group.