Characterization of Rift Valley Fever Virus MP-12 Strain Encoding NSs of Punta Toro Virus or Sandfly Fever Sicilian Virus

<div><p>Rift Valley fever virus (RVFV; genus <i>Phlebovirus</i>, family <i>Bunyaviridae</i>) is a mosquito-borne zoonotic pathogen which can cause hemorrhagic fever, neurological disorders or blindness in humans, and a high rate of abortion in ruminants. MP-12 strain, a live-attenuated candidate vaccine, is attenuated in the M- and L-segments, but the S-segment retains the virulent phenotype. MP-12 was manufactured as an Investigational New Drug vaccine by using MRC-5 cells and encodes a functional NSs gene, the major virulence factor of RVFV which 1) induces a shutoff of the host transcription, 2) inhibits interferon (IFN)-β promoter activation, and 3) promotes the degradation of dsRNA-dependent protein kinase (PKR). MP-12 lacks a marker for differentiation of infected from vaccinated animals (DIVA). Although MP-12 lacking NSs works for DIVA, it does not replicate efficiently in type-I IFN-competent MRC-5 cells, while the use of type-I IFN-incompetent cells may negatively affect its genetic stability. To generate modified MP-12 vaccine candidates encoding a DIVA marker, while still replicating efficiently in MRC-5 cells, we generated recombinant MP-12 encoding Punta Toro virus Adames strain NSs (rMP12-PTNSs) or Sandfly fever Sicilian virus NSs (rMP12-SFSNSs) in place of MP-12 NSs. We have demonstrated that those recombinant MP-12 viruses inhibit IFN-β mRNA synthesis, yet do not promote the degradation of PKR. The rMP12-PTNSs, but not rMP12-SFSNSs, replicated more efficiently than recombinant MP-12 lacking NSs in MRC-5 cells. Mice vaccinated with rMP12-PTNSs or rMP12-SFSNSs induced neutralizing antibodies at a level equivalent to those vaccinated with MP-12, and were efficiently protected from wild-type RVFV challenge. The rMP12-PTNSs and rMP12-SFSNSs did not induce antibodies cross-reactive to anti-RVFV NSs antibody and are therefore applicable to DIVA. Thus, rMP12-PTNSs is highly efficacious, replicates efficiently in MRC-5 cells, and encodes a DIVA marker, all of which are important for vaccine development for Rift Valley fever.</p> </div

rMP12-PTNSs and rMP12-SFSNSs inhibit IFN-β mRNA synthesis.

<p>MEF cells were mock-infected or infected with MP-12, rMP12-C13type, rMP12-PTNSs or rMP12-SFSNSs at a m.o.i of 3. Total RNA was harvested at 7 hpi. Northern blotting was performed with strand-specific RNA probes to detect mouse IFN-β or ISG56 mRNA, or RVFV anti-sense S-segment/N mRNA, respectively. The 18S rRNA was shown as loading control. Representative data from three independent experiments are shown.</p

Replication of rMP12-PTNSs and rMP12-SFSNSs in cell culture.

<p>(A) VeroE6 cells, (B) MEF cells or (C) MRC-5 cells were mock-infected or infected with MP-12, rMP12-C13type, rMP12-PTNSs, or rMP12-SFSNSs at a m.o.i of 0.01. Culture supernatants were collected at 72 hpi (A and B), or indicated time points (C) and virus titer was determined by plaque assay with VeroE6 cells. Means+standard deviations of three independent experiments are shown in the graph. Asterisk represents statistical significance (Unpaired t-test, **p<0.01, vs. MP-12).</p

Generation of rMP12-PTNSs and rMP12-SFSNSs.

<p>(A) Schematics of MP-12 S-segments encoding mutation or foreign gene in place of MP-12 NSs. The rMP12-C13type (C13type) lacks 69% of the NSs ORF as described previously <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002181#pntd.0002181-Muller2" target="_blank">[44]</a>. The rMP12-PTNSs, and rMP12-SFSNSs encode NSs of Punta Toro virus Adames strain and Sandfly fever Sicilian virus, respectively. The expected phenotype corresponding to each S-segment is also presented. (B) Plaque phenotypes of MP-12, rMP12-PTNSs and rMP12-SFSNSs at 4 dpi. Plaque assay was performed with VeroE6 cells overlaid with 0.6% noble agar and stained with Neutral red.</p

Efficacy and immunogenicity of rMP12-PTNSs or rMP12-SFSNSs in mice.

<p>Five-week-old CD1 mice were mock-vaccinated with PBS (n = 10) or vaccinated subcutaneously with 1×10⁵ pfu of MP-12 (n = 20), rMP12-NSsR173A (n = 10), rMP12-PTNSs (n = 9) or rMP12-SFSNSs (n = 10). Sera were collected at 42 days post vaccination, and mice were challenged with 1×10³ pfu of wt RVFV ZH501 strain (i.p) at 45 days post vaccination. Mice were observed for 21 days post-challenge. (A) Kaplan-Meier survival curves of vaccinated mice after wt RVFV challenge. (B) Neutralizing antibody titers of vaccinated mice (PRNT₈₀). Asterisk represents statistical significance (Mann-Whitney U-test, *p<0.05, **p<0.01 vs. rMP12-NSsR173A). (C) Anti-N IgG titer measured by IgG ELISA. Y-axis shows endpoint titers of sera. Asterisk represents statistical significance (Mann-Whitney U-test, *p<0.05, **p<0.01 vs. rMP12-NSsR173A). (D) Anti-NSs IgG level measured by IgG ELISA. Y-axis shows OD405 nm of sera at 1∶100 dilutions, and cut-off at 0.204 is shown as dotted line.</p

Host general transcriptional suppression by RVFV MP-12 mutants.

<p>(A) Flow cytometry analysis was performed in 293 cells. 293 cells were mock-infected or infected with MP-12, rMP12-C13type, rMP12-PTNSs or rMP12-SFSNSs at a m.o.i of 3 and treated with 0.5 mM EU at 8 hpi for 3 hours. Mock-infected cells were co-treated with ActD (5 µg/ml) at 8 hpi for 3 hours. Incorporated EU was stained with Alexa Fluor 647-azide, and RVFV antigens were stained with anti-RVFV antibodies and detected by Alexa Fluor 488 anti-mouse IgG. Subsequently, cells were analyzed by flow cytometry. Representative data from two independent experiments are shown. X-axis: signal intensity for RVFV antigen, Y-axis: signal intensity for EU. (B) Relative fluorescence intensity of EU-positive cells is shown as a histogram.</p

Antibody responses of mice vaccinated with MP-12 or the variants.

*<p>a-NSs IgG positive (cut-off = 0.204).</p><p>Pre: 42 days post vaccination, Post: 21 days post wt RVFV challenge.</p

Histopathological changes of spleens in mice infected with rSA51, wt Entebbe, or wt OS7 strains of Rift Valley fever phlebovirus (RVFV) at 2 days post infection (dpi).

<p>Mice intraperitoneally infected with 1x10³ PFU of rSA51 (A–C), wt Entebbe (D–F), or wt OS7 (G–I) were euthanized at 2 dpi. A mouse intraperitoneally mock-infected with PBS (J–L) was used as a control. Spleen sections were stained with hematoxylin-eosin staining (A, D, G, and J). Corresponding lesions were also incubated with anti-RVFV N rabbit polyclonal antibody to analyze immunohistochemistry (IHC; B, E, H, and K). Quantification of the area of positive signals normalized to the tissue area in each IHC image is also shown (C, F, I, and L). Arrowheads (A) indicate necrotic area in the marginal zone. Rp = red pulp; Fo = follicle; Mz = marginal zone. Bars represent 50 μm.</p

Histopathological changes in livers of mice infected with the rSA51, wt Entebbe, or wt OS7 strains of Rift Valley fever phlebovirus (RVFV) at 2 days post infection (dpi).

<p>Mice intraperitoneally infected with 1x10³ PFU of rSA51 (A–C), wt Entebbe clone 4 (D–F), or wt OS7 (G–I) were euthanized at 2 dpi. A mouse intraperitoneally mock-infected with PBS (J–L) was used as a control. Liver tissues, which were fixed with 10% neutral buffered formalin and embedded in paraffin blocks, were used for histopathological examinations. Thin sections were stained with hematoxylin-eosin staining (A, D, G, and J). Corresponding lesions were also incubated with anti-RVFV N rabbit polyclonal antibody to analyze immunohistochemistry (IHC; B, E, H, and K). Specific signals (red) derived from Vector Red Alkaline Phosphatase substrate were detected via the streptavidin-biotin method using the UltraVision Alk-Phos kit. Quantification of the area of positive signals normalized to the tissue area in each IHC image was performed via FIJI Image J software after color deconvolution (C, F, I, and L). Arrowheads (A, D, and G) indicate Councilman bodies, and inset images (A, D, and G) show hepatocytes with filamentous eosinophilic intranuclear inclusion bodies (arrows). C = central vein; P = portal vein. Bars represent 50 μm.</p

Virulence of wild-type (wt) Rift Valley fever phlebovirus (RVFV) strains in mice.

<p>Five-week-old outbred CD1 mice were intraperitoneally inoculated with 1x10³ PFU of nine different wt RVFV strains: (A) ZH501, Kenya 199800523, Kenya 90058, Saudi Arabia 200010911, Entebbe, or SA51; (B) OS1, OS7, or SA75. The Kaplan-Meier survival curves (n = 10 per group) of infected mice are shown. Asterisks represent the statistically significant differences based on log-rank testing (*p < 0.05, **p < 0.01, vs. ZH501).</p