Zika virus protection by a single low-dose nucleoside-modified mRNA vaccination

Abstract

Zika virus (ZIKV) has recently emerged as a pandemic associated with severe neuropathology in newborns and adults. There are no ZIKV-specific treatments or preventatives. Therefore, the development of a safe and effective vaccine is a high priority. Messenger RNA (mRNA) has emerged as a versatile and highly effective platform to deliver vaccine antigens and therapeutic proteins. Here we demonstrate that a single low-dose intradermal immunization with lipid-nanoparticle-encapsulated nucleoside-modified mRNA (mRNA-LNP) encoding the pre-membrane and envelope glycoproteins of a strain from the ZIKV outbreak in 2013 elicited potent and durable neutralizing antibody responses in mice and non-human primates. Immunization with 30 μg of nucleoside-modified ZIKV mRNA-LNP protected mice against ZIKV challenges at 2 weeks or 5 months after vaccination, and a single dose of 50 μg was sufficient to protect non-human primates against a challenge at 5 weeks after vaccination. These data demonstrate that nucleoside-modified mRNA-LNP elicits rapid and durable protective immunity and therefore represents a new and promising vaccine candidate for the global fight against ZIKV.

Extended Data Figure 1. Design and characterization of ZIKV prM-E mRNA

(a) The ZIKV mRNA encodes the signal peptide (SP) from MHC class II and prM and E glycoproteins from ZIKV H/PF/2013. (b) mRNA was transfected into 293T cells (n=3), human DC (n=3), or murine DC (n=2). E protein expression in cell lysate and supernatant was probed by Western blot, using firefly luciferase-encoding mRNA-transfected cells as a negative control. (c) ZIKV mRNA supernatant from transfected 293T cells was characterized by ultracentrifugation in the presence and absence of 0.5% Triton X-100, followed by Western blot of input (IN), pellet (P), and final supernatant (S) fractions (n=3).

Extended Data Figure 2. Nucleoside-modified ZIKV mRNA-LNP immunization elicits polyfunctional ZIKV E-specific CD4⁺ T cell responses

C57BL/6 mice were immunized with 30 μg of nucleoside-modified ZIKV prM-E mRNA-LNPs (n=8) or control poly(C) RNA-LNPs (n=4). At week 2, antigen-specific CD4⁺ T cells were detected by intracellular cytokine staining. Bar graph shows mean frequencies of combinations of cytokines produced by CD4⁺ T cells. Error bars indicate the SEM, and asterisk indicates a significant difference (p<0.05) by Student’s t-test.

Extended Data Figure 3. ZIKV E-specific IgG concentration in mice

Sera from (a) C57BL/6 mice (n=4 control; n=8 ZIKV mRNA-LNP) or (b) BALB/c mice (n=5 control; n=10 ZIKV mRNA-LNP) were assayed by ELISA, and estimates of ZIKV E-specific IgG concentrations were calculated using murine mAb NR-4747 as a standard. Points represent individual mice; horizontal lines indicate the mean. Responses in vaccine and control groups were compared at each time point by Mann-Whitney test: p<0.01 for all comparisons.

Extended Data Figure 4. Neutralizing antibody responses against ZIKV MR-766 in macaques immunized with ZIKV prM-E mRNA-LNPs

Sera from immunized macaques were evaluated for neutralization of ZIKV MR-766 using (a) the PRNT assay or (b) the RVP assay at the indicated time points. Shaded area indicates values below the limit of detection and horizontal bars indicate the mean. Immune responses in dose groups were compared by Kruskal-Wallis test: p>0.05 for all comparisons.

Extended Data Figure 5. Neutralization curve for a human anti-ZIKV neutralizing mAb

ZIKV MR-766 was neutralized by Ab3594, a human ZIKV-neutralizing monoclonal antibody, as a positive control in the PRNT assay. Shown is a representative curve (n=4). Mean EC₅₀ = 0.026 μg/ml, SD=5.4.

Figure 1. Nucleoside-modified ZIKV mRNA-LNP immunization elicits ZIKV-specific T helper and neutralizing antibody responses

a–d, C57BL/6 mice were immunized i.d. with 30 μg of nucleoside-modified ZIKV prM-E mRNA-LNPs (n=8) or control poly(C) RNA-LNPs (n=4). (a) At week 2, splenic antigen-specific CD4⁺ T cells were detected by intracellular cytokine staining. The antibody response was monitored by (b) ELISA, (c) PRNT using ZIKV MR-766, and (d) RVP using ZIKV H/PF/2013. e–g, BALB/c mice were immunized similarly with ZIKV mRNA-LNPs (n=10) or poly(C) RNA-LNPs (n=5) and monitored by (e) ELISA, (f) PRNT using MR-766, and (g) RVP using H/PF/2013. Points represent individual mice; horizontal lines show the mean; shaded area indicates values below the limit of detection. The controls in d and g are from the week 8 time point. Asterisk indicates p<0.05 in unpaired t-test; antibody responses in vaccine and control groups were compared at each time point by Mann-Whitney test: p<0.01 for all comparisons.

Figure 2. A single immunization of nucleoside-modified ZIKV prM-E mRNA-LNPs provides rapid and durable protection from ZIKV challenge in mice

BALB/c mice immunized i.d. with 30 μg of ZIKV prM-E mRNA-LNPs or control poly(C) RNA-LNPs were challenged i.v. with 200 PFU ZIKV PRVABC59 at (a) 2 weeks (n=9 per group) or (b) 20 weeks (n=5 control mice; n=10 ZIKV mRNA-LNP mice) post-vaccination, and plasma viral loads were measured by qRT-PCR for ZIKV capsid RNA. ‡ symbol indicates two overlapping curves. Shaded area indicates values below the limit of detection (200 copies/ml), with undetectable curves staggered to show individual mice. Day 3 viremia in vaccine and control groups was compared by Mann-Whitney test: p<0.001 for both challenges.

Figure 3. Nucleoside-modified ZIKV mRNA-LNP immunization elicits potent ZIKV-specific neutralizing antibody responses in non-human primates

Rhesus macaques were immunized with 600 μg (n=4), 200 μg (n=3), or 50 μg (n=3) of ZIKV prM-E mRNA-LNPs, and the antibody response was quantified by (a) ELISA, (b) FRNT using ZIKV MEX I-44, and (c) RVP using ZIKV H/PF/2013. Pre-challenge (weeks 0 to 4) and unchallenged animal data are shown. Points represent individual monkeys; shaded area indicates values below the limit of detection; horizontal lines indicate the mean. Immune responses in dose groups were compared by Kruskal-Wallis test: p>0.05 for all comparisons.

Figure 4. A single immunization of nucleoside-modified ZIKV prM-E mRNA-LNPs protects rhesus macaques from ZIKV challenge at 5 weeks post-immunization

Six unvaccinated control macaques and five vaccinated macaques that received 50 μg (n=3), 200 μg (n=1), or 600 μg (n=1) of ZIKV mRNA-LNPs at week 0 were challenged s.c. with 10⁴ TCID₅₀ of ZIKV PRVABC59 at week 5. Viral loads were measured in plasma by qRT-PCR for ZIKV capsid RNA. Shaded area indicates values below the limit of detection (50 copies/ml), and undetectable values were staggered to show individual animals. Day 3 and 5 viremia in vaccine and control groups was compared by Mann-Whitney test: p<0.001.