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

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

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

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

PK analysis of favipiravir in male Hartley guinea pigs dosed by oral instillation or intraperitoneal (i.p.) injection.

<p>Favipiravir (100 mg/kg) was administered orally in carrot baby food vehicle or by i.p. injection in 2.9% sodium bicarbonate. Longitudinal plasma favipiravir levels are shown from 3 animals per treatment group at 15 and 30 minutes, and 1, 2, and 4 h after treatment. Data points represent the mean and standard error of the mean.</p

Survival outcome following oral treatment of JUNV-infected guinea pigs with favipiravir.

<p>Guinea pigs (n = 10/group) were challenged i.p. with 1300 PFU of JUNV. They were dosed by instillation of favipiravir, ribavirin, or carrot baby food vehicle (placebo) into the back of the oral cavity. Treatments (Tx) with the indicated concentrations of drugs were initiated 24 h post-infection (p.i.) and administered twice daily for 14 days (capped hashed line). A) survival, B) mean body weight (relative to initial starting weight), and C) temperature were monitored for 40 days. ***<i>P</i><0.001 compared to placebo-treated animals by the log-rank test.</p

Effect of i.p. favipiravir treatment on day 14 viral loads in JUNV-challenged guinea pigs.

<p>Animals were infected and treated as described in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002614#pntd-0002614-g003" target="_blank">Figure 3</a>. Three pre-designated animals in each treatment group were sacrificed on day 14 post-infection for analysis of A) serum, B) brain, C) heart, D) kidney, E) liver, F) lung and G) spleen virus titers. Two serum samples and 1 liver sample collected from 2 moribund animals from the placebo group euthanized on days 12 and 14 were also included in the analysis. Unique symbols in each treatment group represent values for the same animal across all parameters and hashed lines indicate the assay limits of detection in tissue samples. **<i>P</i><0.01, ***<i>P</i><0.001 compared to placebo.</p