Engineering a fidelity-variant live-attenuated vaccine for chikungunya virus

Abstract

Chikungunya virus (CHIKV), which causes a febrile illness characterized by severe and prolonged polyarthralgia/polyarthritis, is responsible for a global disease burden of millions of cases each year with autochthonous transmission in over 100 countries and territories worldwide. There is currently no approved treatment or vaccine for CHIKV. One live-attenuated vaccine (LAV) developed by the United States Army progressed to Phase II human clinical trials but was withdrawn when 8% of volunteers developed joint pain associated with vaccination. Attenuation of the Army's CHIKV LAV strain 181 clone 25 (CHIKV-181/25) relies on two mutations in the envelope 2 (E2) glycoprotein responsible for cell binding and entry, making it particularly prone to reversion, a common concern for replication-competent vaccines. High error rates associated with RNA virus replication have posed a challenge for LAV development where stable incorporation of attenuating elements is necessary for establishing safety in pre-clinical models. Herein, we incorporate two replicase mutations into CHIKV-181/25 which modulate CHIKV replication fidelity combined with additional attenuating features that cannot be eliminated by point mutation. The mutations were stably incorporated in the LAV and did not increase virulence in mice. Two fidelity-variant CHIKV LAVs generated neutralizing antibodies and were protective from CHIKV disease in adult mice. Unexpectedly, our fidelity-variant candidates were more mutable than CHIKV-181/25 and exhibited restricted replication in mice and Aedes mosquitoes, a possible consequence of hypermutation. Our data demonstrate safety and efficacy but highlight a further need to evaluate fidelity-altering phenotypes before use as a LAV given the potential for virulent reversion.

Keywords: Alphaviruses; Live attenuated vaccines; Viral infection.

Fig. 1. CHIKV live-attenuated vaccine design and replication kinetics.

The organization of CHIKV vaccine candidates is shown with point mutations denoted by a red asterisk (a–d). Live-attenuated vaccine growth is demonstrated on vertebrate baby hamster kidney (e) and mosquito Aedes albopictus larval (f) cells at starting MOI = 1. P < 0.0001 (D), 2-way ANOVA of log-transformed values, n = 3 per experiment, two combined experiments. Geometric mean and geometric standard deviation are indicated with data points and error bars (e, f).

Fig. 2. CHIKV live-attenuated vaccine derivatives are avirulent in neonatal mice.

Two-day-old CD-1 IGS suckling mice were inoculated s.c. with 10³ PFU of each derivative CHIKV LAV or WT virus. Normalized weight (a–d) and mortality (e) were monitored for the duration of infection. P < 0.0001 (D), 2-way ANOVA (a–d), log-rank test (e), n = 9–10 per experiment, two combined experiments. Mean and standard deviation are indicated with data points and error bars (a–d).

Fig. 3. Fidelity-variant CHIKV live-attenuated vaccine generates protective immunity in mice.

Adult C57Bl/6J mice were immunized with 10³ PFU CHIKV LAV derivatives then challenged bilaterally s.c. in footpads with 10³ PFU of WT CHIKV after 28 days. A timeline for infection and sample collection is shown (a). Peak viremias (b, c), weight change (d) hindlimb swelling (e), and total swelling area under the curve (f) are shown post immunization or challenge as indicated. Antibody titers are given for 50% (g) and 80% (h) neutralization thresholds on sera collected on day 28 prior to the challenge. P < 0.05 (A), 0.01 (B), 0.001 (C), 0.0001 (D), 2-way ANOVA on untransformed (d–f), and log-transformed (b, c, g–h) values, n = 5 per experiment, two combined experiments. Geometric mean and geometric standard deviation are indicated with lines and error bars (b–d, g–h). Mean and standard deviation are indicated with data points/bars and error bars (d–f). PRNT plaque reduction neutralization test, AUC area under the curve, ND not determined.

Fig. 4. Immunization with a fidelity-variant vaccine restricts CHIKV dissemination.

Adult C57Bl/6J mice were immunized with 10³ PFU (10⁴ PFU where indicated) of CHIKV LAV then challenged bilaterally s.c. in the footpad with 10³ PFU WT CHIKV on day 28. Animals were killed 1-, 2- and 5-days post-challenge, and viral load was measured in the blood (a) and selected replication-competent tissues (b–f). P < 0.05 (A), 0.01 (B), 0.001 (C), 0.0001 (D), 2-way ANOVA on log-transformed values, n = 4, one experiment. Geometric mean and geometric standard deviation are indicated with lines and error bars.

Fig. 5. Fidelity-variant CHIKV vaccine protects mice from pathology associated with the virulent challenge.

Hindlimbs from adult C57Bl/6J mice immunized with 10³ PFU (10⁴ PFU where indicated) CHIKV LAV and challenged bilaterally with 10³ PFU at day 28 with WT CHIKV were collected at days 1, 2, and 5 post-challenge and prepared for hematoxylin and eosin staining of thin sections. Cross-sections were blindly evaluated by a veterinary pathologist and scored according to Supplementary Table 2. Composite group scores are given for each time point (a) and representative images are shown for each group at 5-days post-challenge. DPBS only (b) DPBS + CHIKV WT (c) CHIKV-181/25 (d) CHIKV-181/25-P2.P4 (e) CHIKV-181/25-P2.P4.E3/E1 (f), and CHIKV-181/25-P2.P4.E3/E1 (10⁴ PFU) (g). Arrows highlight inflammatory infiltrates. The rectangle indicates inflammatory cells and debris within a metatarsal joint space (c). Asterisks indicate inflammatory cell infiltrates within the synovial membrane (c) and tendon sheath (f–g). Mean and standard deviation are indicated with lines and error bars (a). E epithelium, SC subcutaneous tissue, M muscle, B bone, T tendon, J joint, bar = 100 µm. P < 0.001 (C) 0.0001 (D), 2-way ANOVA, n = 4/group, one experiment.

Fig. 6. Apparent mutational accumulation following serial passage is unaffected by replicase fidelity-modulating variant.

CHIKV LAV candidates were serially passaged at MOI = 0.1 in BHK cells and the passage-10 virus was deep-sequenced. Reads were aligned to the consensus genome. Statistics including Shannon entropy (a) mutation frequency (b) and non-synonymous mutation frequency (c) were calculated with ViVan. P < 0.0001 (D), ANOVA (a, b), 2-way ANOVA (c), n = 5, one experiment. Mean and standard deviation are indicated by lines and error bars (a, b).

Fig. 7. Fidelity-variant CHIKV live-attenuated vaccine demonstrates reversion potential in vivo.

LAV candidates were passaged 5 times in triplicate at 10⁴ PFU in the brains of 6-day old suckling CD-1 mice. Three combined replicate passaged and single unpassaged LAVs were inoculated i.c. in 2-day old CD-1 mice and weight (a–b) and survival (c) were monitored. Viruses were sequenced using PrimalSeq and non-synonymous mutations were identified with iVar (d). P < 0.0001 (D), log-rank test, n = 15, 1 experiment. Mean and standard deviation are indicated with data points and error bars (a–b). Passage number and biological replicate are indicated in parentheses for individual samples. The red box indicates a known virulent reversion.