Liposomal Delivery of the RNA Genome of a Live-Attenuated Chikungunya Virus Vaccine Candidate Provides Local, but Not Systemic Protection After One Dose

Abstract

Chikungunya virus (CHIKV) is the causative pathogen of chikungunya fever, a mosquito-borne viral disease causing highly debilitating arthralgia that can persist for months and progress to chronic arthritis. Our previous studies have identified the CHIKV live-attenuated vaccine candidate CHIKV-NoLS. Like most live-attenuated vaccines, attenuated replication of CHIKV-NoLS has the potential to limit scalable production. To overcome production limits, as well as other drawbacks of live-attenuated vaccines, we developed an in vivo liposome RNA delivery system to deliver the self-replicating RNA genome of CHIKV-NoLS directly into mice, allowing the recipients' body to produce the live-attenuated vaccine particles. CAF01 liposomes were able to deliver replication-competent CHIKV-NoLS RNA in vitro. Immunodeficient AG129 mice inoculated with liposome-delivered CHIKV-NoLS RNA developed viremia and disease signs representative of this lethal model of CHIKV infection, demonstrating de novo vaccine particle production in vivo. In immunocompetent C57BL/6 mice, liposome-delivered CHIKV-NoLS RNA inoculation was associated with reduced IgM and IgG levels with low antibody CHIKV-neutralizing capacity, compared to vaccination with the original live-attenuated vaccine CHIKV-NoLS. One dose of liposome-delivered CHIKV-NoLS RNA did not provide systemic protection from CHIKV wild-type (WT) challenge but was found to promote an early onset of severe CHIKV-induced footpad swelling. Liposome-delivered CHIKV-NoLS RNA inoculation did, however, provide local protection from CHIKV-WT challenge in the ipsilateral foot after one dose. Results suggest that in the presence of low CHIKV-specific neutralizing antibody levels, local inflammatory responses, likely brought on by liposome adjuvants, have a role in the protection of CHIKV-induced footpad swelling in the ipsilateral foot of mice inoculated with liposome-delivered CHIKV-NoLS RNA. Low IgG and CHIKV-specific neutralizing antibody levels may be responsible for early onset of severe swelling in the feet of CHIKV-WT-challenged mice. These results support previous studies that suggest CHIKV is vulnerable to antibody-mediated enhancement of disease. Further studies using booster regimes aim to demonstrate the potential for liposomes to deliver the self-replicating RNA genome of live-attenuated vaccines and offer a novel immunization strategy.

Keywords: RNA; chikungunya; liposome; live-attenuated; vaccine.

Figure 1

CAF01 RNA formulation and in vitro assays. (A) CHIKV-WT linearized DNA infectious clone (DNA digest) and in vitro transcribed CHIKV-WT RNA (IVT) were run on a 1% agarose gel and visualized using GelRed® under UV light. (B) CHIKV-WT RNA was transfected into Vero cells using either Lipofectamine® or CAF01. Cells were also mock transfected or transfected with RNA alone. At 12 h post transfection, infectivity (percentage and cell count) was measured by flow cytometry. (C) From 29 h post transfection, CHIKV-WT growth kinetics were analyzed by plaque assay. (D) CHIKV-WT and CHIKV-NoLS RNA was transfected into Vero cells using either Lipofectamine® or CAF01. Cells were also mock transfected or transfected with RNA alone. Viral growth kinetics were analyzed by plaque assay. Each symbol represents the mean ± standard error from three independent experiments.

Figure 2

Inoculation with CHIKV-NoLS CAF01 elicits de novo viral particle production and disease in AG129 mice. AG129 mice were inoculated with viable virus/vaccine particles (10⁴ pfu CHIKV-WT or CHIKV-NoLS) or CAF01 delivered RNA (2 μg CHIKV-WT or CHIKV-NoLS RNA complexed with 0.3 mg CAF01) in the ventral/lateral side of the right foot. Mock inoculated mice received 20 μl VPSFM media or VPSFM media containing 0.3 mg CAF01. (A) Serum was collected on days 1–6 post inoculation and virus titrated by plaque assay. Survival curves were constructed (B) as mice were monitored for the development of disease signs (C) and changes in weight (D) every 24 h. Disease signs in inoculated AG129 mice were scored on a scale of 0–4 where 4 is humane endpoint. (E,F) CHIKV-induced footpad swelling was assessed by measuring the height and width of the perimetatarsal area of the foot, using Kincrome digital vernier calipers. Each symbol represents the mean ± standard error from five to six mice. Statistical analysis was performed by non-parametric Mann–Whitney t-test between the groups indicated on the figure legend by the bar and *color. ns, not significant,*P < 0.05, **P < 0.005, ***P < 0.001, and ****P < 0.0001.

Figure 3

CHIKV-NoLS CAF01 inoculated AG129 mice survive CHIKV-WT challenge and are protected from development of disease. AG129 mice were inoculated with 2 μg CHIKV-NoLS RNA complexed with 0.3 mg CAF01 in the ventral/lateral side of the right foot. Mock inoculated mice received 20 μl VP-SFM media or VP-SFM media containing 0.3 mg CAF01. Mice were challenged with 10⁴ pfu CHIKV-WT subcutaneously in the ventral/lateral side of the right (ipsilateral) foot 30 days post inoculation. Survival curves were constructed (A) as mice were monitored for CHIKV-induced footpad swelling (B), the development of disease signs (C) and changes in weight every 24 h. Disease signs in inoculated AG129 mice were scored on a scale of 0–4 where 4 is humane endpoint. (D) Serum was collected on days 1–5 post challenge and virus titrated by plaque assay. Each symbol represents the mean ± standard error from five to six mice. Statistical analysis was performed by non-parametric Mann–Whitney t-test.

Figure 4

Viremia following CHIKV-NoLS CAF01 inoculation and CAF01 induced footpad swelling in C57BL/6 mice. C57BL/6 mice were inoculated with viable virus particles (10⁴ pfu CHIKV-WT or CHIKV-NoLS), CAF01 delivered RNA (2 μg CHIKV-WT or CHIKV-NoLS RNA complexed with 0.3 mg CAF01) or RNA alone (2 μg CHIKV-WT or CHIKV-NoLS RNA) subcutaneously in the ventral/lateral side of the right foot. Mock inoculated mice received 20 μl VP-SFM media alone or VP-SFM media containing 0.3 mg CAF01. (A) Serum was collected on days 1–5 post inoculation and virus titrated by plaque assay. Mice were monitored daily for signs of CHIKV-induced footpad swelling in the (B) ipsilateral (inoculated) and (C) contralateral (non-inoculated) foot. Each symbol represents the mean ± standard error from five to six mice.

Figure 5

CHIKV-NoLS CAF01 inoculation protects against CHIKV disease in the ipsilateral foot of C57BL/6 mice upon CHIKV-WT challenge. C57BL/6 mice were inoculated with viable virus particles (10⁴ pfu CHIKV-WT or CHIKV-NoLS), CAF01 delivered RNA (2 μg CHIKV-WT or CHIKV-NoLS RNA complexed with 0.3 mg CAF01) or RNA alone (2 μg CHIKV-WT or CHIKV-NoLS RNA) subcutaneously in the ventral/lateral side of the right foot. Mock inoculated mice received 20 μl VP-SFM media or VP-SFM media containing 0.3 mg CAF01. At 30 days post inoculation, mice were challenged with 10⁴ pfu CHIKV-WT in the ventral/lateral side of the (A,B) ipsilateral or (C) contralateral foot and monitored daily for footpad swelling. Each symbol represents the mean ± standard error from five to six mice. Statistical analysis was performed by non-parametric Mann–Whitney t-test between the groups indicated on the figure legend by the bar and *color, and P-value.

Figure 6

Cellular infiltration in challenged C57BL/6 mice. C57BL/6 mice were inoculated with viable virus particles (10⁴ pfu CHIKV-WT or CHIKV-NoLS), CAF01 delivered RNA (2 μg CHIKV-NoLS RNA complexed with 0.3 mg CAF01), or RNA alone (2 μg CHIKV-NoLS RNA) subcutaneously in the ventral/lateral side of the right foot. Mock inoculated mice received 20 μl VP-SFM media alone or VP-SFM media containing 0.3 mg CAF01. At 30 days post inoculation, all groups except Mock unchallenged mice were challenged (Ch) with 10⁴ pfu CHIKV-WT in the ventral/lateral side of the ipsilateral or contralateral foot and monitored daily for footpad swelling. Feet at 6 days post challenge were processed for flow cytometry analysis. The number of neutrophils (A), activated neutrophils (B), inflammatory monocytes (C), macrophages (D), CD4⁺ (E), CD4⁺IFNγ⁺ (F), CD8⁺ (G), and CD8⁺IFNγ⁺ T cells (H) was examined. Each symbol represents the mean ± standard error from five to six mice. Statistical analysis was performed by non-parametric Mann–Whitney t-test. *P < 0.05.

Figure 7

Antibody response in CHIKV-NoLS CAF01 inoculated C57BL/6 mice. C57BL/6 mice were inoculated with viable virus particles (10⁴ pfu CHIKV-WT or CHIKV-NoLS), CAF01 delivered RNA (2 μg CHIKV-WT or CHIKV-NoLS RNA complexed with 0.3 mg CAF01), or RNA alone (2 μg CHIKV-WT or CHIKV-NoLS RNA) subcutaneously in the ventral/lateral side of the right foot. Mock inoculated mice received 20 μl VP-SFM media or VP-SFM media containing 0.3 mg CAF01. Serum was harvested at various times post inoculation and titrated for IgM (A) and IgG (B,C) by ELISA. (D) Neutralizing antibody titers at day 30 post inoculation were examined. Each symbol represents the mean ± standard error from 5 to 6 mice. Statistical significance of IgM and IgG titers was analyzed by one-way ANOVA with Tukey posttests and neutralizing antibody titers by Kruskal–Wallis test with Dunn's posttests (groups indicated on the figure legend by the bar and *color). *P < 0.05, **P < 0.005, and ***P < 0.001. N.A., data not available.