Novel approaches to develop Rift Valley fever vaccines

Rift Valley fever (RVF) is endemic to sub-Saharan Africa, and has spread into Madagascar, Egypt, Saudi Arabia and Yemen. Rift Valley fever virus (RVFV) of the family Bunyaviridae, genus Phlebovirus causes hemorrhagic fever, neurological disorders or blindness in humans, and high rate abortion and fetal malformation in ruminants. RVFV is classified as a Category A Priority pathogen and overlap select agent by CDC/USDA due to its potential impact on public health and agriculture. There is a gap in the safety and immunogenicity in traditional RVF vaccines; the formalin-inactivated RVFV vaccine TSI-GSD-200 requires 3 doses for protection, and the live-attenuated Smithburn vaccine has a risk to cause abortion and fetal malformation in pregnant ruminants. In this review, problems of traditional vaccines and the safety and efficacy of recently reported novel RVF candidate vaccines including subunit vaccines, virus vector and replicons are discussed

Bicaudal D1-Dependent Trafficking of Human Cytomegalovirus Tegument Protein pp150 in Virus-Infected Cells ▿

Human cytomegalovirus (HCMV) virion assembly takes place in the nucleus and cytoplasm of infected cells. The HCMV virion tegument protein pp150 (ppUL32) is an essential protein of HCMV and has been suggested to play a role in the cytoplasmic phase of HCMV assembly. To further define its role in viral assembly and to identify host cell proteins that interact with pp150 during viral assembly, we utilized yeast two-hybrid analyses to detect an interaction between pp150 and Bicaudal D1 (BicD1), a protein thought to play a role in trafficking within the secretory pathway. BicD1 is known to interact with the dynein motor complex and the Rab6 GTPase. The interaction between pp150 and BicD1 was confirmed by coimmunoprecipitation and fluorescence resonance energy transfer. Depletion of BicD1 with short hairpin RNA (shRNA) caused decreased virus yield and a defect in trafficking of pp150 to the cytoplasmic viral assembly compartment (AC), without altering trafficking to the AC of another essential tegument protein, pp28, or the viral glycoprotein complex gM/gN. The C terminus of BicD1 has been previously shown to interact with the GTPase Rab6, suggesting a potential role for Rab6-mediated vesicular trafficking in HCMV assembly. Finally, overexpression of the N terminus of truncated BicD1 acts in a dominant-negative manner and leads to disruption of the AC and a decrease in the assembly of infectious virus. This phenotype was similar to that observed following overexpression of dynamitin (p50) and provided additional evidence that morphogenesis of the AC and virus assembly were dynein dependent

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

Identification of Host Factors for Rift Valley Fever Phlebovirus

Rift Valley fever phlebovirus (RVFV) is a zoonotic pathogen that causes Rift Valley fever (RVF) in livestock and humans. Currently, there is no licensed human vaccine or antiviral drug to control RVF. Although multiple species of animals and humans are vulnerable to RVFV infection, host factors affecting susceptibility are not well understood. To identify the host factors or genes essential for RVFV replication, we conducted CRISPR-Cas9 knockout screening in human A549 cells. We then validated the putative genes using siRNA-mediated knock-downs and CRISPR-Cas9-mediated knock-out studies. The role of a candidate gene in the virus replication cycle was assessed by measuring intracellular viral RNA accumulation, and the virus titers were analyzed using plaque assay or TCID50 assay. We identified approximately 900 genes with potential involvement in RVFV infection and replication. Further evaluation of the effect of six genes on viral replication using siRNA-mediated knock-downs revealed that silencing two genes (WDR7 and LRP1) significantly impaired RVFV replication. For further analysis, we focused on the WDR7 gene since the role of the LRP1 gene in RVFV replication was previously described in detail. WDR7 knockout A549 cell lines were generated and used to dissect the effect of WRD7 on a bunyavirus, RVFV, and an orthobunyavirus, La Crosse encephalitis virus (LACV). We observed significant effects of WDR7 knockout cells on both intracellular RVFV RNA levels and viral titers. At the intracellular RNA level, WRD7 affected RVFV replication at a later phase of its replication cycle (24 h) when compared with the LACV replication, which was affected in an earlier replication phase (12 h). In summary, we identified WDR7 as an essential host factor for the replication of two different viruses, RVFV and LACV, both of which belong to the Bunyavirales order. Future studies will investigate the mechanistic role through which WDR7 facilitates phlebovirus replication

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