Infectious RNA vaccine protects mice against chikungunya virus infection

Abstract

We describe a novel vaccine platform that can generate protective immunity to chikungunya virus (CHIKV) in C57BL/6J mice after a single immunization by employing an infectious RNA (iRNA), which upon introduction into a host cell launches an infectious attenuated virus. We and others have previously reported that an engineered deletion of 183 nucleotides in the nsP3 gene attenuates chikungunya virus (CHIKV) and reduces in vivo viral replication and viremia after challenge in mice, macaques and man. Here, we demonstrated that in vitro transfection of iRNA carrying the nsP3 deletion generated infectious viruses, and after intramuscular injection, the iRNA induced robust antibody responses in mice. The iRNA was superior at eliciting binding and neutralizing antibody responses as compared to a DNA vaccine encoding the same RNA (iDNA) or a non-propagating RNA replicon (RREP) lacking the capsid encoding gene. Subsequent challenge with a high dose of CHIKV demonstrated that the antibody responses induced by this vaccine candidate protected animals from viremia. The iRNA approach constitutes a novel vaccine platform with the potential to impact the spread of CHIKV. Moreover, we believe that this approach is likely applicable also to other positive-strand viruses.

Figure 1

CHIKV vaccine candidates. Schematic representation of CHIKV genome and the vaccine candidates; iRNA Δ5nsP3 has a 183-bp deletion in the 3′ part of the sequence encoding nsP3. For comparison, we used an iDNA Δ5nsP3 mutant launched as DNA infectious genome and the RNA replicon (RREP). The RREP vaccine candidate is derived from the WT CHIKV infectious clone after deletion of the capsid encoding region which prevents RNA packaging and virion assembly. Deletions are indicated by arrows.

Figure 2

In vitro and in vivo propagation of iRNA vaccine candidate. (a) Plaque assay of CHIKV and virus made from the iRNA encoding the Δ5nsP3 deletion. iRNA was electroporated into BHK-21 cells, supernatants were harvested after 48 h and used to infect a monolayer of BHK-21 cells that was analyzed for plaque formation 48 h post infection. (b) Peak viremia in serum after a single intramuscular injection of 1.25 μg of iRNA vaccine (n = 16) or subcutaneous injection of 10⁶ pfu of WT CHIKV (n = 5). The limit of detection for the plaque assay was 80 pfu/ml. The line indicates the geometric mean of each group.

Figure 3

(a) Immunogenicity of vaccine candidates. C57BL/6J mice were immunized once with indicated doses of candidate vaccines. Total antigen-specific IgG titers were determined by ELISA three weeks post immunization. The line indicates the geometric mean of each group (n = 10 animals per group). (b) Antibody isotype analysis. Sera from immunized were analyzed by ELISA for anti-CHIKV Env p62-E1 protein antibody isotypes IgG2c and IgG1. Results are presented as IgG2c:IgG1 endpoint antibody titer ratios against CHIKV Env p62-E1 protein. A ratio above 1 indicates a Th1 biased response. Non-responders in the binding IgG ELISA were not included in the analysis. For the statistical analysis, we performed Kruskal–Wallis test followed by Dunn’s test for multiple comparisons. One asterisk (*) indicates statistical differences of p < 0.05.

Figure 4

Challenge study. (a) Schematic representation of the experiment schedule. C57BL/6J mice were immunized once with 1.25 μg of either iRNA Δ5nsP3 or iDNA Δ5nsP3 and challenged five weeks later with 10⁶ plaque forming units (pfu) of CHIKV. (b) Anti-CHIKV IgG endpoint titers and (c) 50% neutralization titers (NT₅₀) were determined three weeks after a single immunization with the candidate vaccines. (d) Viremia was determined by plaque assay using serum samples collected at day two post challenge. Filled symbols indicate mice that were protected against challenge, whereas open symbols indicate mice that were not protected against infection with CHIKV. The line indicates the geometric mean of each group (n = 10 animals per group). A two-tailed Mann–Whitney test was used to analyze differences between two groups. One asterisk (*) indicates p < 0.05.