The dominant-negative inhibition of double-stranded RNA-dependent protein kinase PKR increases the efficacy of Rift Valley fever virus MP-12 vaccine

Abstract

Rift Valley fever virus (RVFV), belonging to the genus Phlebovirus, family Bunyaviridae, is endemic to sub-Saharan Africa and causes a high rate of abortion in ruminants and hemorrhagic fever, encephalitis, or blindness in humans. MP-12 is the only RVFV strain excluded from the select-agent rule and handled at a biosafety level 2 (BSL2) laboratory. MP-12 encodes a functional major virulence factor, the NSs protein, which contributes to its residual virulence in pregnant ewes. We found that 100% of mice subcutaneously vaccinated with recombinant MP-12 (rMP12)-murine PKRN167 (mPKRN167), which encodes a dominant-negative form of mouse double-stranded RNA (dsRNA)-dependent protein kinase (PKR) in place of NSs, were protected from wild-type (wt) RVFV challenge, while 72% of mice vaccinated with MP-12 were protected after challenge. rMP12-mPKRN167 induced alpha interferon (IFN-α) in sera, accumulated RVFV antigens in dendritic cells at the local draining lymph nodes, and developed high levels of neutralizing antibodies, while parental MP-12 induced neither IFN-α nor viral-antigen accumulation at the draining lymph node yet induced a high level of neutralizing antibodies. The present study suggests that the expression of a dominant-negative PKR increases the immunogenicity and efficacy of live-attenuated RVFV vaccine, which will lead to rational design of safe and highly immunogenic RVFV vaccines for livestock and humans.

Fig 1

Schematics of S segments of MP-12, rMP12-C13type, and rMP12-mPKRN167. rMP12-C13type (C13type) lacks 69% of the NSs ORF, as described previously (56). rMP12-mPKRN167 (N167) encodes a dominant-negative form of mouse PKR in place of NSs. The expected phenotype corresponding to each S segment is also presented. +++ and − represent the presence and absence of suppression, respectively.

Fig 2

Dominant-negative effect of rMP12-mPKRN167. Wild-type MEFs were mock infected or infected with MP-12, rMP12-C13type, or rMP12-mPKRN167 at an MOI of 3. Cells were collected at 12 h p.i. (A) or 16 h p.i. (B), and the abundance of eIF2α, phosphorylated eIF2α (eIF2α-P) (Ser 51), NSs, N, Flag-PKR N167, and β-actin were analyzed by Western blotting. The mean ratios and standard deviations of the density of phosphorylated eIF2α and eIF2α were determined from 3 independent experiments. The asterisks represent statistical significance (Student's unpaired t test; P < 0.01). (C and D) MEFs were mock infected or infected with MP-12, rMP12-C13type, or rMP12-mPKRN167 at an MOI of 3. Cell lysates were collected at 1 h p.i. (C) and 16 h p.i. (D), and the abundance of N proteins was measured by antigen capture ELISA for RVFV N proteins (15). The asterisks represent statistical significance (Mann-Whitney U test; *, P < 0.05; **, P < 0.01 versus rMP12-C13type at each dilution). (E) As a control to monitor initial N protein derived from inocula, heat-inactivated MP-12 or rMP12-mPKRN167 was also tested.

Fig 3

Immunogenicity of rMP12-mPKRN167. Five-week-old CD1 mice were mock vaccinated with PBS (n = 6) or vaccinated subcutaneously with 1 × 10⁵ PFU of MP-12, rMP12-C13type (C13type), or rMP12-mPKRN167 (N167) (n = 9). Sera were collected at 1, 2, 3, 30, 90, and 180 days p.i. (A) Abundance of IFN-α in mouse serum samples (mock, n = 6; other groups, n = 9) at 1 day p.i. Serum IFN-α was measured with the VeriKine Mouse Interferon Alpha ELISA Kit (PBL International). A Mann-Whitney U test was performed for statistical analyses (*, P < 0.05; **, P < 0.01). (B) Viral titers of mouse serum samples (n = 10 per group). The viral titers in serum samples at days 1, 2, and 3 were determined by plaque assay in Vero E6 cells. *, P < 0.05 by the Kruskal-Wallis test. (C) Neutralizing antibody titers of serum samples: MP-12 (n = 9), C13type (n = 10), or N167 (n = 10). A PRNT₈₀ was performed to determine the neutralizing antibody titer. A Mann-Whitney U test was performed for statistical analyses between PRNT₈₀ titers at 180 days p.i. (*, P < 0.05; **, P < 0.01). The error bars indicate standard deviations.

Fig 4

Protection efficacy of rMP12-mPKRN167 in mice. Five-week-old CD1 mice were mock vaccinated (n = 10) or vaccinated subcutaneously with 1 × 10⁵ PFU of MP-12 (n = 10), rMP12-C13type (C13type) (n = 10), or rMP12-mPKRN167 (N167) (n = 9). Sera were collected at 42 days p.i., and mice were challenged with 1 × 10³ PFU of wt RVFV strain ZH501 (i.p.) at 44 days p.i. The mice were observed for 21 days postchallenge. (A) Kaplan-Meier survival curves of vaccinated mice after wt RVFV challenge. (B) Daily weight changes of mock-vaccinated mice or surviving vaccinated mice after challenge. The error bars indicate standard deviations.

Fig 5

Viral N protein accumulation in the draining lymph nodes of mice after immunization. (A) Relative abundance of RVFV N in viral stocks prepared from Vero E6 cells. Representative data from three independent experiments are shown. (B) Five-week-old outbred CD1 mice were mock vaccinated with PBS or vaccinated in the footpad with 1 × 10⁵ PFU (30 μl) of MP-12, rMP12-C13type (C13type), or rMP12-mPKRN167 (N167) or heat-inactivated nonreplicable (NR) MP-12 at 56°C for 30 min. Popliteal (P) and inguinal (I) lymph nodes were collected at 1 day p.i., and the lysates were used for antigen capture ELISA. The cutoff of 0.104 is shown as a dotted line. The asterisk represents statistical significance (Mann-Whitney U-test; P < 0.05). (C) Antigen capture ELISA was performed by using popliteal and inguinal lymph nodes of mice vaccinated with MP-12, which were collected at 2 and 3 days p.i. The cutoff value of 0.145 is shown as a dashed line. The error bars indicate standard deviations.

Fig 6

Localization of RVFV N antigens at the draining lymph nodes. Popliteal lymph nodes of mice that were mock vaccinated (B) or vaccinated with rMP12-mPKRN167 virus (A and C to E) or rMP12-C13type (F) were stained with a mixture of control antibodies (normal rabbit serum and biotin-conjugated anti-rat IgG2a) (A), a mixture of anti-RVFV N antibody and biotin-conjugated anti-CD45R/B220 antibody (B to E), or anti-RVFV N antibody only. Nuclei were counterstained with DAPI (4′,6-diamidino-2-phenylindole). The scale is indicated in each panel.

Fig 7

Measurement of cytokines and chemokines in the sera of vaccinated mice. Five-week-old CD1 mice were mock vaccinated with PBS (n = 3) or vaccinated in the footpad with 1 × 10⁵ PFU (30 μl) of MP-12, rMP12-C13type (C13type), or rMP12-mPKRN167 (N167) (n = 4). Sera were collected at 2 days p.i., and the abundance of cytokines and chemokines was measured by using the Bio-Plex Pro Mouse Cytokine 23-Plex Assay (Bio-Rad). The fold increases relative to the highest values of mock samples are shown. An unpaired Student's t test (versus mock vaccinated) was performed for statistical comparison (*, P < 0.05; **, P < 0.01). The error bars indicate standard deviations.