Novel attenuated Chikungunya vaccine candidates elicit protective immunity in C57BL/6 mice

Abstract

Chikungunya virus (CHIKV) is a reemerging mosquito-borne alphavirus that has caused severe epidemics in Africa and Asia and occasionally in Europe. As of today, there is no licensed vaccine available to prevent CHIKV infection. Here we describe the development and evaluation of novel CHIKV vaccine candidates that were attenuated by deleting a large part of the gene encoding nsP3 or the entire gene encoding 6K and were administered as viral particles or infectious genomes launched by DNA. The resulting attenuated mutants were genetically stable and elicited high magnitudes of binding and neutralizing antibodies as well as strong T cell responses after a single immunization in C57BL/6 mice. Subsequent challenge with a high dose of CHIKV demonstrated that the induced antibody responses protected the animals from viremia and joint swelling. The protective antibody response was long-lived, and a second homologous immunization further enhanced immune responses. In summary, this report demonstrates a straightforward means of constructing stable and efficient attenuated CHIKV vaccine candidates that can be administered either as viral particles or as infectious genomes launched by DNA.

Importance: Similar to other infectious diseases, the best means of preventing CHIKV infection would be by vaccination using an attenuated vaccine platform which preferably raises protective immunity after a single immunization. However, the attenuated CHIKV vaccine candidates developed to date rely on a small number of attenuating point mutations and are at risk of being unstable or even sensitive to reversion. We report here the construction and preclinical evaluation of novel CHIKV vaccine candidates that have been attenuated by introducing large deletions. The resulting mutants proved to be genetically stable, attenuated, highly immunogenic, and able to confer durable immunity after a single immunization. Moreover, these mutants can be administered either as viral particles or as DNA-launched infectious genomes, enabling evaluation of the most feasible vaccine modality for a certain setting. These CHIKV mutants could represent stable and efficient vaccine candidates against CHIKV.

FIG 1

CHIKV vaccine candidates. (A) Schematic figure of the design and characteristics of CHIKV and the vaccine candidates. CHIKV and the Δ5nsP3 and Δ6K mutants were administered either as viral particles or as DNA-launched infectious genomes. Δ5nsP3 has a 183-bp deletion in the 3′ part of the sequence encoding nsP3 (amino acids 1656 to 1717 in the P1234 polyprotein), and Δ6K is devoid of 6K (amino acids 749 to 809 in the structural polyprotein). Locations for deletions are marked with arrows. (B) Plaque morphology of CHIKV and the Δ5nsP3 and Δ6K mutants.

FIG 2

Setup of mouse challenge model. (A) Outline for the challenge study. Female C57BL/6 mice (5 to 6 weeks old) were infected with 10⁴, 10⁵, or 10⁶ PFU CHIKV subcutaneously (s.c.) in the flank and challenged 7 weeks later with 10⁶ PFU CHIKV in the feet. (B) Peak viremia titer in serum collected at day 1 to day 3 postimmunization and day 1 to day 3 postchallenge. (C) Peak foot swelling of each mouse (mean of height times breadth of both hind feet relative to day 0) at day 4 to day 8 postimmunization and day 4 to day 8 postchallenge. (D) Endpoint IgG titers at 1 week before and 3 weeks after challenge. Bars show mean values (n = 5). A Kruskal-Wallis test followed by Dunn's posttest was used to compare the responses of the different groups. One, two, and three asterisks (*, **, and ***) indicate statistical differences from naive mice (PBS) of P < 0.05, P < 0.01, and P < 0.001, respectively.

FIG 3

Pathogenicity of vaccine candidates. C57BL/6 mice were immunized with CHIKV or mutants thereof. (A) Peak viremia at day 1 to day 3 after a single immunization with 10⁵ PFU viral particles or 10 μg DNA. (B) Peak foot swelling of each mouse (mean of height times breadth of both hind feet relative to day 0) at day 4 to day 8 after a single immunization with 10⁵ PFU viral particles in feet. Bars show mean values (n = 5). A Kruskal-Wallis test followed by Dunn's posttest was used to compare the responses of the different groups. One asterisk and three asterisks (* and ***) indicate statistical differences from CHIKV of P < 0.05 and P < 0.001, respectively.

FIG 4

Immunogenicity of vaccine candidates. C57BL/6 mice were immunized once with either 10⁵ PFU viral particles or 10 μg DNA. (A to C) Total IgG titers (A), IgG1 (black dots) and IgG2c (green dots) titers (B), and 50% neutralization titers (NT50) (C) on serum collected prior to challenge. (D) CD8 T cell responses measured by an IFN-γ ELISpot assay performed on splenocytes at 7 to 10 days postimmunization. The dotted line represents the cutoff (25 spot-forming units [SFU]/10⁶ splenocytes). Bars show mean values (n = 5). A Kruskal-Wallis test followed by Dunn's posttest was used to compare the responses of the different groups. One, two, and three asterisks (*, **, and ***) indicate statistical differences from CHIKV of P < 0.05, P < 0.01, and P < 0.001, respectively.

FIG 5

Challenge studies and correlates of protection. (A and B) Peak viremia titer at day 1 to day 3 (A) and peak foot swelling of each mouse (mean of height times breadth of both feet) at day 4 to day 8 (B) postchallenge with 10⁶ PFU CHIKV in feet of mice previously immunized with 10⁵ PFU viral particles or 10 μg DNA. (C and D) Correlation between endpoint IgG titers before challenge and either peak viremia (C) or peak foot swelling (D) after challenge. Bars show mean values (n = 5). A Kruskal-Wallis test followed by Dunn's posttest was used to compare the responses of the different groups, and a Spearman rank test was used to examine the correlation between the magnitude of immune responses prior to challenge and the level of viremia and foot swelling postchallenge. One asterisk and two asterisks (* and **) indicate statistical differences from naive mice (PBS) of P < 0.05 and P < 0.01, respectively.

FIG 6

Duration of immunity. Two sets of C57BL/6 mice were injected once with 10⁵ PFU Δ5nsP3 (red squares) or PBS (black squares) subcutaneously and challenged with 10⁶ PFU CHIKV in the feet at week 8 or week 20. (A) Endpoint IgG titers prior to challenge. (B) Peak viremia at day 1 to day 3 postchallenge. (C) Peak foot swelling of each mouse (mean of height times breadth of both feet) at day 4 to day 8 postchallenge. Bars show mean values (n = 5). A two-tailed Mann-Whitney test was used to analyze differences between the two sets of mice. NS, not significant.

FIG 7

Homologous prime-boost immunizations. C57BL/6 mice were immunized with 10⁵ PFU viral particles or 10 μg DNA, with a 3-week immunization interval. Mice were challenged with 10⁶ PFU CHIKV in feet at 7 weeks after the second immunization. (A and B) Endpoint IgG titers (A) and 50% neutralization titers (NT50) (B) prior to challenge. (C and D) Peak viremia at day 1 to day 3 (C) and peak foot swelling of each mouse (mean of height times breadth of both feet) at day 4 to day 8 (D) after challenge. Bars show mean values (n = 5). A Kruskal-Wallis test followed by Dunn's posttest was used to compare the responses of the different groups. Three asterisks (***) indicate a statistical difference from naive mice (PBS) of P < 0.001.