Effects of Chikungunya virus immunity on Mayaro virus disease and epidemic potential

Abstract

Mayaro virus (MAYV) causes an acute febrile illness similar to that produced by chikungunya virus (CHIKV), an evolutionary relative in the Semliki Forest virus complex of alphaviruses. MAYV emergence is typically sporadic, but recent isolations and outbreaks indicate that the virus remains a public health concern. Given the close phylogenetic and antigenic relationship between CHIKV and MAYV, and widespread distribution of CHIKV, we hypothesized that prior CHIKV immunity may affect MAYV pathogenesis and/or influence its emergence potential. We pre-exposed immunocompetent C57BL/6 and immunocompromised A129 or IFNAR mice to wild-type CHIKV, two CHIKV vaccines, or a live-attenuated MAYV vaccine, and challenged with MAYV. We observed strong cross-protection against MAYV for mice pre-exposed to wild-type CHIKV, and moderately but significantly reduced cross-protection from CHIKV-vaccinated animals. Immunity to other alphavirus or flavivirus controls provided no protection against MAYV disease or viremia. Mechanistic studies suggested that neutralizing antibodies alone can mediate this protection, with T-cells having no significant effect on diminishing disease. Finally, human sera obtained from naturally acquired CHIKV infection cross-neutralized MAYV at high titers in vitro. Altogether, our data suggest that CHIKV infection can confer cross-protective effects against MAYV, and the resultant reduction in viremia may limit the emergence potential of MAYV.

Figure 1

CHIKV immunity provides cross-protection against MAYV infection and disease in an immunocompetent mouse model. (a) Virus-specific neutralizing antibody titers (solid bars) and MAYV cross-neutralization antibody titers (empty bars) were determined prior to MAYV challenge. Dashed lines indicate the upper and lower limits of detection (1:640 and 1:20, respectively); (b) footpad swelling were determined daily throughout the study; (c) Viremia was measured daily for four days post-infection. All plotted values in (b,c) are mean ± S.D. Data in (b) were analyzed using a repeated measures ANOVA with a Bonferroni multiple comparison post hoc analysis, and data in (d) were analyzed with a one-way ANOVA with a Bonferroni post hoc analysis. There were no significant differences in percent weight change among groups; however, footpad swelling was significantly different between the PBS (i.e. PBS-vaccinated and PBS-challenged) control and all other groups except CHIKV-99659 and MAYY/IRES. Additionally, viremia was significantly different between the PBS control and all other groups except CHIKV-99659 and MAYY/IRES on days one and two, and EILV/CHIKV, CHIKV-99659 and MAYY/IRES on days three and four. Statistically significant values are denoted by *p < 0.05.

Figure 2

CHIKV immunity provides cross-protection against MAYV infection and disease in an immunocompromised mouse model. (a) Virus-specific neutralizing antibody titers (solid bars) and MAYV cross-neutralizing antibody titers (empty bars) were determined prior to MAYV challenge. Dashed line indicates the upper and lower limits of detection (1:640 and 1:20, respectively). (b) Weight change, expressed as percent of original, and (c) footpad swelling were determined daily throughout the study. (d) Viremia was measured daily for four days post-infection and (e) survival was recorded. All plotted values in (b–d) are mean ± S.D. Data in (b–d) were analyzed using a one-way ANOVA with a Bonferroni post hoc analysis, and survival curves (e) were analyzed by Kaplan-Meier survival analysis. Footpad swelling was significantly different between the PBS control (i.e. PBS-vaccinated and PBS-challenged) and EILV/CHIKV groups on days one and two, and between the PBS control and EILV/CHIKV and sham-vaccinated groups on day three, and PBS and sham on day four. Weight change was statistically significant between the PBS control and EILV/CHIKV on days three and four. Viremia and survival were statistically significant for EILV/CHIKV and sham throughout four days post-infection. Statistically significant values are denoted by *p < 0.05.

Figure 3

Passive transfer of CHIKV immune sera does not provide cross-protection against MAYV infection and disease in an immunocompromised mouse model. (a) Virus-specific neutralizing antibody titers (solid bars) and MAYV cross-neutralizing antibody titers (empty bars) were determined prior to MAYV challenge. Dashed line indicates the upper and lower limits of detection (1:640 and 1:20, respectively). (b) Weight change, expressed as percent of original, and (c) footpad swelling were determined daily throughout the study. (d) Viremia was measured daily for four days post-infection and (e) survival was recorded. All plotted values in (b–d) are mean ± S.D. Data in (b-d) were analyzed using a one-way ANOVA with a Bonferroni post hoc analysis, and survival curves (e) were analyzed by Kaplan-Meier survival analysis. When compared to the PBS control (i.e. PBS-vaccinated and PBS-challenged), statistically significant footpad swelling was observed in mice from EILV/CHIKV on days 2–6, CHIKV/IRES on days 1 and 3–6, and ZIKV, CHIKV-99659 and sham on days 3–6. Weight change was statistically significant for EILV/CHIKV on days three and five, and CHIKV-99659 on days three and four, and sham on day four. Viremia (days 1–4) and survival was statistically significant for all groups except MAYV/IRES. Statistically significant values are denoted by * p<0.05.

Figure 4

CHIKV immune T-cells are reactive upon stimulation with MAYV 12A. C57/B6J mice were vaccinated or challenged with CHIKV/IRES, EILV/CHIKV, CHIKV-99659, ZIKV, MAYV/IRES or sham-vaccinated with PBS. Six weeks post-vaccination, mice were sacrificed, splenocytes were isolated and stimulated for 24 hours with MAYV, stained with antibodies for CD3, CD4, CD8, IFN-γ and analyzed using flow cytometry. Number of (a) CD4⁺ IFN-γ⁺ and (b) CD8⁺ IFN-γ⁺ T-cells are plotted as mean ± S.D. Data were analyzed using a one-way ANOVA with a Bonferroni post hoc analysis. Statistically significant values are denoted by *p < 0.05.

Figure 5

T-cell depletion studies reveal no significant role for CD4⁺ and CD8⁺ T-cells in diminishing MAYV disease. (a–c) Weight change, expressed as percent of original and (d–f) footpad swelling were determined daily throughout the study. (g–i) Viremia was measured daily for four days post-infection, (j–l) and survival was recorded. All plotted values in (a–i) are mean ± S.E.M. (a–c) were analyzed using a repeated measures ANOVA with a Bonferroni multiple comparison post hoc analysis, and data in (d–i) were analyzed using a one-way ANOVA with a Bonferroni post hoc analysis, and survival curves (j–l) were analyzed by Kaplan-Meier survival analysis. Statistically significant values are denoted by *p < 0.05.