The RVFV MP-12 strain is one of the most immunogenic candidate live-attenuated vaccines for Rift Valley fever in animals and the only RVFV strain that is excluded from the select-agent rule (26
). Single vaccination of pregnant ewes with MP-12 results in the protection of both ewes and newborn lambs that receive the colostrum of immunized ewes (50
), while the residual virulence of MP-12 induces abortion or fetal malformation in pregnant ewes vaccinated early in gestation (23
). The S segment of MP-12 maintains the virulent phenotype (2
), and the M and L segments of MP-12 are most likely responsible for MP-12 attenuation (67
). It was demonstrated that removal of functional NSs is an effective strategy to attenuate wt RVFV, and the resulting RVFV lacking NSs is an attractive candidate live-attenuated vaccine for Rift Valley fever, e.g., the clone 13 strain, which lacks 69% of the NSs ORFs in the wt RVFV 74HB59 strain backbone (14
), or ΔNSs-ΔNSm-rRVFV vaccine, which lacks both NSs and NSm in the wt RVFV ZH501 strain backbone (8
). Thus, we generated rMP12-C13type, which encodes strain C13-like NSs in place of MP-12 NSs with the MP-12 backbone, yet mice vaccinated with rMP12-C13type were not completely protected after wt RVFV challenge. To increase the immunogenicity of MP-12, we incorporated the dominant-negative form of PKR into MP-12. rMP12-mPKRN167 encodes a dominant-negative PKR (the N-terminal 167 amino acids of mouse PKR) in place of NSs, which is analogous to human PKRΔE7. PKRΔE7 is alternatively spliced with a deletion of exon 7 and exhibits dominant-negative functions in human cells, but not in mouse cells (39
). We confirmed that rMP12-mPKRN167 inhibits eIF2α phosphorylation in infected MEFs and increases the accumulation of N proteins ().
Mice vaccinated with rMP12-mPKRN167 were completely protected from wt RVFV challenge after a single subcutaneous immunization. MP-12 and rMP12-mPKRN167 induced similar titers of neutralizing antibodies, while some of the mice vaccinated with MP-12 did not raise antibodies at all. All dead mice lacked neutralizing antibodies before wt RVFV challenge, while one mouse vaccinated with rMP12-mPKRN167 survived wt RVFV challenge without neutralizing antibody (). Thus, neutralizing antibody plays a key role in protection, while some mice may survive wt RVFV challenge without the induction of neutralizing antibodies, as described previously (31
To understand the significance of PKR degradation for MP-12 immunogenicity, we also made rMP12-NSsR173A, which inhibits host transcription, including IFN-β mRNA synthesis, yet does not degrade PKR. rMP12-NSsR173A poorly accumulated viral proteins at draining lymph nodes and did not induce neutralizing antibodies (data not shown). Thus, it is suggested that PKR degradation is indispensable for the strong immunogenicity of MP-12 and that transcription suppression by NSs inhibits the host immune responses. On the other hand, rMP12-C13type and rMP12-mPKRN167, which lack a host transcription suppression function, were highly immunogenic in mice. Thus, the transcription suppression function is not required for MP-12 immunogenicity, and the suppression of PKR in the absence of host transcription maximizes the efficacy of MP-12 vaccine.
We found that MP-12 does not accumulate viral proteins in the draining lymph nodes, while rMP12-C13type and rMP12-mPKRN167, which lack NSs, could accumulate viral proteins. We further confirmed that viral N proteins were localized in the cytoplasm of CD11c-positive and langerin-negative cells (see Fig. S1 in the supplemental material), which were morphologically identical to DCs (). Recent findings suggested that RVFV lacking NSs can replicate in DCs, macrophages, and granulocytes in IFNAR1-deficient mice (19
), and DC-SIGN is one of the receptors of RVFV (42
), supporting the notion that DCs are the major target of rMP12-mPKRN167. Our results also suggest that a lack of NSs-mediated host transcription suppression leads to efficient accumulation of infected DCs. It is known that foreign antigens that are administered via the subcutaneous route are trapped by dermal DCs (dDCs) in the dermis, which later migrate into the local draining lymph node, where antigen presentation to B and T cells takes place through dDCs (33
). Thus, efficient accumulation of viral proteins in DCs in draining lymph nodes may explain the strong immunogenicity of rMP12-mPKRN167 ( and ).
Because MP-12 did not accumulate in the draining lymph nodes at 1, 2, or 3 days p.i. (), we wondered how MP-12 can induce host immune responses. Cytokines and chemokines, including IL-5, IFN-γ, IL-17, GM-CSF, MIP-1β, RANTES, KC, and eotaxin, were significantly increased in sera of mice that were vaccinated with MP-12 (), while some mice that were vaccinated with MP-12 showed detectable viremia (). It is likely that MP-12 can induce cytokines secreted by all three T helper cell subsets in a systemic manner. The most susceptible target cells of wt RVFV in mice are hepatocytes (70
), while the spleen removes blood-borne pathogens and cell debris from circulation (46
). Replication of MP-12 is observed in mouse liver, although the titer is approximately 1,000 times lower than that of wt RVFV (20
). Thus, it might be possible that MP-12 reaches the liver or spleen by escaping host immune responses at the draining lymph nodes, while rMP12-C13type or rMP12-mPKRN167 induces a local immune response in the draining lymph nodes.
Based on our study, MP-12 encoding dominant-negative PKR is a highly efficacious vaccine candidate for Rift Valley fever. To develop such a vaccine, we need to address the following issues: (i) host specificity of dominant-negative PKR and (ii) the safety of dominant-negative PKR. It is known that human PKRΔE7 does not exhibit a dominant-negative function in mouse cells (39
). On the other hand, PKRΔE7 inhibits PKR of Chlorocebus aethiops
(Vero E6 cells) (27
). The phylogenetic analysis of the PKR gene suggests that ruminant PKRs are distantly related to human or murine PKR (66
). Thus, the dominant-negative PKR specific to each host species will need to be evaluated before vaccine development. The safety of dominant-negative PKR should also be addressed. Past studies suggested that increased translation initiation by introducing eIF2α encoding an alanine at serine 51 into spontaneously immortalized NIH 3T3 cells, which are deficient in the INK4 locus (63
), supported the development of tumors, while the same study using 3T3 L1 cells that retain the INK4 locus did not lead to tumor formation (60
). Similarly, the introduction of dominant-negative PKR, PKRΔ6, which lacks exon 6 of human PKR, into NIH 3T3 cells induced tumor formation (36
), while PKR-null mice do not have high frequencies of tumors (1
), suggesting that the suppression of PKR is not the sole factor to induce tumors. On the other hand, PKR is often activated in tumors (35
), and the apoptosis of cancer cells was triggered by adenovirus encoding PKRΔ6 (58
). In this study, we tested the dominant-negative PKR in a live-attenuated vaccine and demonstrated successful improvement of immunogenicity and efficacy. Mice vaccinated with rMP12-mPKRN167 were in good health for at least 180 days after immunization. The mice did not develop viremia after immunization, suggesting rMP12-mPKRN167 virus was rapidly cleared once type I IFNs were induced in response to abundant viral replication in primary infected cells.
In summary, we demonstrated that MP-12 encoding dominant-negative PKR is highly efficacious and applicable to DIVA. It protects all mice from wt RVFV challenge by a single subcutaneous inoculation. The viral antigens were detected in DCs in draining lymph nodes, and vaccinated mice elicited long-term neutralizing antibody response. In contrast, the parental MP-12 strain did not accumulate viral proteins in local draining lymph nodes and stimulated the host immune response in a manner distinct from that of strains lacking NSs. A novel approach using dominant-negative PKR will be useful to increase the efficacy and immunogenicity of vaccine candidates.