Vaccination with a chikungunya virus-like particle vaccine exacerbates disease in aged mice

Abstract

Introduction: Chikungunya virus (CHIKV) is a re-emerging pathogen responsible for causing outbreaks of febrile disease accompanied with debilitating joint pain. Symptoms typically persist for two weeks, but more severe and chronic chikungunya illnesses have been reported, especially in the elderly. Currently, there are no licensed vaccines or antivirals against CHIKV available. In this study, we combined a CHIK virus-like particle (VLP) vaccine with different adjuvants to enhance immunogenicity and protection in both, adult and aged mice.

Methods: CHIK VLP-based vaccines were tested in 6-8-week-old (adult) and 18-24-month-old (aged) female C57BL/6J mice. Formulations contained CHIK VLP alone or adjuvants: QuilA, R848, or Imject Alum. Mice were vaccinated three times via intramuscular injections. CHIKV-specific antibody responses were characterized by IgG subclass using ELISA, and by microneutralization assays. In addition, CHIKV infections were characterized in vaccinated and non-vaccinated adult mice and compared to aged mice.

Results: In adult mice, CHIKV infection of the right hind foot induced significant swelling, which peaked by day 7 post-infection at approximately 170% of initial size. Viral titers peaked at 2.53 × 1010 CCID50/ml on day 2 post-infection. Mice vaccinated with CHIK VLP-based vaccines developed robust anti-CHIKV-specific IgG antibody responses that were capable of neutralizing CHIKV in vitro. CHIK VLP alone or CHIK plus QuilA administered by IM injections protected 100% of mice against CHIKV. In contrast, the antibody responses elicited by the VLP-based vaccines were attenuated in aged mice, with negligible neutralizing antibody titers detected. Unvaccinated, aged mice were resistant to CHIKV infection, while vaccination with CHIKV VLPs exacerbated disease.

Conclusions: Unadjuvanted CHIK VLP vaccination elicits immune responses that protect 100% of adult mice against CHIKV infection. However, an improved vaccine/adjuvant combination is still necessary to enhance the protective immunity against CHIKV in the aged.

Fig 1. Chikungunya E and VLP design and verification.

A] Schematic representation of CHIK gene insertion into bacmid. B] PCR verification of C-E; truncated, N-terminal his-tagged E1, and truncated, N-terminal his-tagged E2 clones. C] Expression of CHIK VLPs were verified by Western blot analysis using anti-E Chk48 monoclonal antibody. D] Soluble, his-tagged E1 was purified via chromatography using NiNTA agarose beads and the different fractions were analyzed via SDS-PAGE with PageBlue protein staining solution. Purified, soluble E1 was detected by western blot using anti-E1 polyclonal sera generated in C57BL/6J mice. E] Soluble, his-tagged E2 was purified via chromatography using NiNTA agarose beads and the different fractions were analyzed via SDS-PAGE with PageBlue protein staining solution. Purified, soluble E2 was detected by western blot using anti-E2 polyclonal sera generated in C57BL/6J mice.

Fig 2. Chikungunya antigen-specific, IgG antibody responses in adult vs aged mice.

Vaccine responses in adult and aged C57BL/6J mice were evaluated 8 weeks [three vaccine doses] post-initial vaccination with each VLP-based vaccine formulation delivered via intramuscular [IM] injections, or PBS. ELISAs were performed to evaluate anti-VLP responses: A] total IgG, B] IgG1, C] IgG2c, and D] IgG3. Anti-E1 responses are shown as E] total IgG, F] IgG1, G] IgG2c, and H] IgG3. Anti-E2 responses are shown as I] IgG, J] IgG1, K] IgG2c, and L] IgG3. Mean ± standard error [SEM] are reported. One-way, two-tailed ANOVA, followed by Tukey post-hoc tests were performed: # significantly different from adult PBS mice, $ significantly different from aged PBS mice, *p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Fig 3. In vitro neutralization of CHIK LR2006-OPY1 by mouse immune sera.

The ability of diluted sera from vaccinated mice to neutralize CHIKV LR2006-OPY1 infection of Vero cells was evaluated in vitro. The reciprocal serum dilution of the lowest dilution that prevented cytopathic effect in Vero cells is reported as the neutralization titer for individual mice, by group. Group neutralization titers are shown as mean ± S.E.M. The dotted line indicates the limit of detection of the assay. Kruskall-Wallis group analysis was performed for neutralization assay results, followed by Dunn’s multiple comparisons test [* p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001].

Fig 4. CHIKV-associated disease in adults vs aged mice.

Adult and aged C57BL/6J mice were infected with 10⁵ CCID₅₀ per mouse of CHIKV LR2006-OPY1 via subcutaneous injection of the right hind footpad and monitored for 14 days. A, D] Weights normalized to day 0 measurements are shown by vaccine formulation and age. B, E] Right foot size normalized to day 0 measurements are shown by vaccine formulation and age. C, F] Normalized left foot sizes are shown. The mean ± S.E.M are reported. The grey area loosely represents measurement variabilities observed in the non-infected left foot size of adult mice.

Fig 5. CHIK viral loads in adult vs aged mice.

Infectious viral loads were determined from sera collected on days 2, 4, and 6 post-infection. A] Viral loads in mice vaccinated with VLP in comparison to control groups and B] Viral loads in mice vaccinated with VLP plus QuilA in comparison to control groups. The mean ± S.E.M are reported. One-way, two-tailed ANOVA, followed by Tukey post-hoc tests were performed: ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Fig 6. Baseline inflammation in the aging.

Pools of naïve sera from adult or aged mice were tested for inflammatory cytokines: A] TNF-α, B] IL-6, and C] IL-1β. Two tailed t-tests were performed between adult and aged groups and p < 0.05 was considered significant (* p < 0.05, *** p < 0.001). D] Vero cells were infected with 200 CCID₅₀ in the presence of TNF-α at range of 5–80 ρg/ml. ^a Wells were counted if there was at least 95% CPE.