In these studies, we probed the attenuation, immunogenicity and efficacy profile of the chikungunya vaccine candidate, CHIK 181/25 in adult mice deficient in IFN-α/β and/or IFN-γ receptor signaling. In the absence of a functional IFN-α/β response, A129 mice developed only transient morbidity after administration of the CHIK 181/25 vaccine and then fully recovered. In contrast, wt CHIKV-LR administration to A129 mice lead to rapid mortality (). Therefore, our findings highlight the attenuated nature of the CHIK 181/25 vaccine. Surprisingly, the duration of morbidity was inversly proportional to the vaccine dose. It is possible that the higher vaccine doses induce a more potent innate immune response resulting in a quicker recovery.
The importance of IFN-α/β in controlling infection by the CHIK 181/25 virus is supported by two lines of evidence. First, G129 mice that have a functional IFN-α/β receptor showed no mortality nor morbidity after CHIK 181/25 vaccination. Secondly, A129 heterozygote mice also show no morbidity after CHIK 181/25 virus infection demonstrating that a single copy of the IFN-α/β receptor is capable of further inhibiting CHIK 181/25 virus replication and pathogenesis. These data are consistent with the findings by Couderc et al [17
] highlighting the dependence of wt CHIKV infection in A129 mice on the strength of IFN-α/β receptor signaling pathway. IFN-α/β provides the first line of defense by inducing an anti-viral state that controls viral replication and modulates the adaptive immune response [20
]. In the case of CHIKV, IFN-α/β controls early virus replication via its action on nonhematopoietic cells [21
]. After secretion from the infected cells IFN-α/β binds to its INFAR receptor in an autocrine or paracrine manner to amplify the signal or prime uninfected cells to establish an antiviral state. However, in primate cell lines once viral replication has been established further CHIKV replication becomes resistant to inhibition by IFNs. This is due to the suppressive effect exerted by the CHIKV nonstructural protein 2 which inhibits IFN-induced JAK-STAT signaling and therefore prevents IFN–induced stimulating genes (ISGs) expression [22
]. More recently, it was shown that CHIKV envades innate immune response to infection by widespread shut off of cellular protein synthesis and a targeted block of late synthesis of antiviral mRNA transcripts [23
In contrast, AG129 mice that lack both functional IFN-α/β and/IFN-γ receptors rapidly succumbed to CHIK 181/25 virus infection. Thus, in the absence of IFN-α/β, IFN-γ is able to partially control replication of an attenuated CHIKV. These data suggest that although the IFN-γ response is subordinate to IFN-α/β in terms of antiviral activity, it contributes to the control of viral replication and spread. The pattern of cytokine expressed after infection in these mice is consistent with this hypothesis. In A129 mice, the IFN-γ response was stronger and had an earlier onset relative to the AG129 mice following CHIK 181/25 vaccination. In addition, IL-12 was detectable in A129 mice but not in AG129 mice. IFN-α/β normally regulates the induction of IL-12 and IFN-γ during a viral infection [24
]. However, our findings suggest that the induction of both of these cytokines occurs in the absence of IFN-α/β.
In these studies, levels of proinflammatory cytokines TNF-α and IL-6, which are typically induced in response to an inflammatory stimulus, were low. This is in contrast to the proinflammatory cytokine responses observed following infection of A129 mice by other alphaviruses [25
]. Levels of IL-1β were elevated in AG129 mice as compared to the A129 mice. Interestingly, high levels of IL-1β have similarly been observed in humans and associated with disease severity [28
]. This cytokine is known to be involved in the immunopathogenesis of many arthritic pathologies [29
] but also could participate in viral control [6
]. The A129 model with defective IFN-α/β signaling, may result in abnormalities in the cytokine patterns observed after infection. However, since CHIKV has evolved to blunt the human IFN-α/β signaling cascade [22
] or mobilize the apoptotic machinery to invade host cell defenses [31
] these findings may still be reflective of viral pathogenesis in humans.
We evaluated the efficacy of passively transferred anti-CHIK 181/25 antibodies in AG129 mice that have both IFN-type responses compromised and are susceptible to CHIK 181/25 virus. Passive transfer of anti-CHIK 181/25 antibodies abrogated lethality associated with the CHIK 181/25 virus. These data are consistent with earlier findings demonstrating antibody-mediated protection of IFN-deficient mice against Sindbis virus infection [32
]. The protective effect of passively transferred serum could be attributed to its capacity to directly neutralize CHIKV and/or its capacity to induce other protective immune responses such as antibody-dependent and complement–mediated cellular cytotoxicity. Our findings are in agreement with data demonstrating the protective effect of passively transferred low titer anti-Ross River Virus (RRV) antibodies against RRV challenge [33
] and suggest that low levels of antibody responses might be sufficient in mediating effective protection.
The CHIK 181/25 vaccine elicited a uniformly robust neutralizing antibody response after a single injection and protected A129 immune mice after challenge with wt CHIKV-LR. This protection was shown to be at least partially mediated by antibodies confirming the important role that humoral immunity plays in controlling CHIKV infection [19
]. However, studies with other alphaviruses have demonstrated that cellular immunity can protect mice against fatal disease in the absence of an antibody response [34
]. The contribution of cellular immune responses to protection from wt CHIK-LR virus challenge remains to be addressed in future studies. In addition, after CHIKV-LR challenge, there was no overt organ damage in the CHIK 181/25 vaccinated mice. In contrast, the spleen of PBS injected animals showed large areas of necrosis and lacked any lymphoid follicles with only a few scattered lymphocytes. The organ damage and lack of lymphocytes could be a direct consequence of wt CHIKV infection in this model [19
] or could be an indirect consequence of cytokine dysregulation and immunopathogenesis.
The CHIK 181/25 vaccine conferred long-term protection against wt CHIKV-LR virus challenge in A129 mice (ca, 8 months after a single intradermal injection). Neutralizing antibody responses were detectable prior to challenge following vaccination with only 103
PFU/mouse. The long-term duration of neutralizing anti-CHIK 181/25 antibody responses is consistent with the kinetics of antibody responses observed in humans that received this vaccine. Follow-up of healthy vaccinated volunteers demonstrated persistence of antibodies with in vitro
neutralizing activity after 12 months [15
]. Similarly, 18 months after natural infection anti-CHIKV antibodies are still detectable in 40% of patients [36
These studies highlight the role of IFN-α/β in controlling CHIK 181/25 virus replication and demonstrate the potential of a live, attenuated candidate CHIKV vaccine to elicit neutralizing antibody responses that confer short-and long-term protection against wt CHIKV-LR challenge.