Rift Valley Fever Virus Gn Research Articles

A Lipid Nanoparticle-Formulated Self-Amplifying RNA Rift Valley Fever Vaccine Induces a Robust Humoral Immune Response in Mice.

Rift Valley fever (RVF) is a mosquito-borne viral zoonosis that causes high fetal and neonatal mortality rates in ruminants and sometimes severe to fatal complications like encephalitis and hemorrhagic fever in humans. There is no licensed RVF vaccine for human use while approved livestock vaccines have suboptimal safety or efficacy. We designed self-amplifying RNA (saRNA) RVF vaccines and assessed their humoral immunogenicity in mice. Plasmid DNA encoding the Rift Valley fever virus (RVFV) medium (M) segment consensus sequence (WT consensus) and its derivatives mutated to enhance cell membrane expression of the viral surface glycoproteins n (Gn) and c (Gc) were assessed for in vitro expression. The WT consensus and best-expressing derivative (furin-T2A) were cloned into a Venezuelan equine encephalitis virus (VEEV) plasmid DNA replicon and in vitro transcribed into saRNA. The saRNA was formulated in lipid nanoparticles and its humoral immunogenicity in BALB/c mice was assessed. High quantities of dose-dependent RVFV Gn IgG antibodies were detected in the serum of all mice immunized with either WT consensus or furin-T2A saRNA RVF vaccines. Significant RVFV pseudovirus-neutralizing activity was induced in mice immunized with 1 µg or 10 µg of the WT consensus saRNA vaccine. The WT consensus saRNA RVF vaccine warrants further development.

Multifunctional human monoclonal antibody combination mediates protection against Rift Valley fever virus at low doses

The zoonotic Rift Valley fever virus (RVFV) can cause severe disease in humans and has pandemic potential, yet no approved vaccine or therapy exists. Here we describe a dual-mechanism human monoclonal antibody (mAb) combination against RVFV that is effective at minimal doses in a lethal mouse model of infection. We structurally analyze and characterize the binding mode of a prototypical potent Gn domain-A-binding antibody that blocks attachment and of an antibody that inhibits infection by abrogating the fusion process as previously determined. Surprisingly, the Gn domain-A antibody does not directly block RVFV Gn interaction with the host receptor low density lipoprotein receptor-related protein 1 (LRP1) as determined by a competitive assay. This study identifies a rationally designed combination of human mAbs deserving of future investigation for use in humans against RVFV infection. Using a two-pronged mechanistic approach, we demonstrate the potent efficacy of a rationally designed combination mAb therapeutic.

Bacterium-Like Particles Displaying the Rift Valley Fever Virus Gn Head Protein Induces Efficacious Immune Responses in Immunized Mice.

Rift Valley fever virus (RVFV), a mosquito-borne zoonotic phlebovirus, causes serious disease in humans and ruminants. According to the World Health Organization, Rift Valley fever is classified as a priority disease, and as such, vaccine development is of high priority due to the lack of licensed vaccines. In this study, a bacterium-like particle vaccine (BLP), RVFV-BLPs, is constructed. A novel display system is described, which is based on non-living and non-genetically modified Gram-positive bacterial cells, designated as Gram-positive enhancer matrix (GEM). The RVFV Gn head protein was displayed on the surface of GEM by co-expression with the peptidoglycan-binding domain (protein anchor) at the C-terminus. We determined that the RVFV Gn head-PA fusion protein was successfully displayed on the GEM. Mice immunized with RVFV-BLPs produced humoral and cellular immunity. Interestingly, comparing the production of RVFV Gn head-specific IgG and its subtype by vaccinating with different antigen doses of the RVFV-BLPs determined that the RVFV-BLPs (50 μg) group showed a greater effect than the other two groups. More importantly, antibodies produced by mice immunized with RVFV-BLPs (50 μg) exhibited potent neutralizing activity against RVFV pseudovirus. RVFV-BLPs (50 μg) also could induce IFN-γ and IL-4 in immunized mice; these mice generated memory cells among the proliferating T cell population after immunization with RVFV-BLPs with effector memory T cells as the major population, which means that RVFV-BLPs is an effective vaccine to establish a long-lived population of memory T cells. The findings suggest that the novel RVFV-BLPs subunit vaccine has the potential to be considered a safe and effective candidate vaccine against RVFV infection.

Rift Valley fever virus Gn V5-epitope tagged virus enables identification of UBR4 as a Gn interacting protein that facilitates Rift Valley fever virus production

Rift Valley fever virus (RVFV) is an arbovirus that was first reported in the Rift Valley of Kenya which causes significant disease in humans and livestock. RVFV is a tri-segmented, negative-sense RNA virus consisting of a L, M, and S segments with the M segment encoding the glycoproteins Gn and Gc. Host factors that interact with Gn are largely unknown. To this end, two viruses containing an epitope tag (V5) on the Gn protein in position 105 or 229 (V5Gn105 and V5Gn229) were generated using the RVFV MP-12 vaccine strain as a backbone. The V5-tag insertion minimally impacted Gn functionality as measured by replication kinetics, Gn localization, and antibody neutralization assays. A proteomics-based approach was used to identify novel Gn-binding host proteins, including the E3 ubiquitin-protein ligase, UBR4. Depletion of UBR4 resulted in a significant decrease in RVFV titers and a reduction in viral RNA production.

Lrp1 is a host entry factor for Rift Valley fever virus

Rift Valley fever virus (RVFV) is a zoonotic pathogen with pandemic potential. RVFV entry is mediated by the viral glycoprotein (Gn), but host entry factors remain poorly defined. Our genome-wide CRISPR screen identified low-density lipoprotein receptor-related protein 1 (mouse Lrp1/human LRP1), heat shock protein (Grp94), and receptor-associated protein (RAP) as critical host factors for RVFV infection. RVFV Gn directly binds to specific Lrp1 clusters and is glycosylation independent. Exogenous addition of murine RAP domain 3 (mRAPD3) and anti-Lrp1 antibodies neutralizes RVFV infection in taxonomically diverse cell lines. Mice treated with mRAPD3 and infected with pathogenic RVFV are protected from disease and death. A mutant mRAPD3 that binds Lrp1 weakly failed to protect from RVFV infection. Together, these data support Lrp1 as a host entry factor for RVFV infection and define a new target to limit RVFV infections.

Preliminary Evaluation of a Recombinant Rift Valley Fever Virus Glycoprotein Subunit Vaccine Providing Full Protection against Heterologous Virulent Challenge in Cattle.

Rift Valley fever virus (RVFV) is a mosquito-borne zoonotic pathogen that causes periodic outbreaks of abortion in ruminant species and hemorrhagic disease in humans in sub-Saharan Africa. These outbreaks have a significant impact on veterinary and public health. Its introduction to the Arabian Peninsula in 2003 raised concerns of further spread of this transboundary pathogen to non-endemic areas. These concerns are supported by the presence of competent vectors in many non-endemic countries. There is no licensed RVF vaccine available for humans and only a conditionally licensed veterinary vaccine available in the United States. Currently employed modified live attenuated virus vaccines in endemic countries lack the ability for differentiating infected from vaccinated animals (DIVA). Previously, the efficacy of a recombinant subunit vaccine based on the RVFV Gn and Gc glycoproteins, derived from the 1977 human RVFV isolate ZH548, was demonstrated in sheep. In the current study, cattle were vaccinated subcutaneously with the Gn only, or Gn and Gc combined, with either one or two doses of the vaccine and then subjected to heterologous virus challenge with the virulent Kenya-128B-15 RVFV strain, isolated from Aedes mosquitoes in 2006. The elicited immune responses by some vaccine formulations (one or two vaccinations) conferred complete protection from RVF within 35 days after the first vaccination. Vaccines given 35 days prior to RVFV challenge prevented viremia, fever and RVFV-associated histopathological lesions. This study indicates that a recombinant RVFV glycoprotein-based subunit vaccine platform is able to prevent and control RVFV infections in target animals.

Characterization of Two Neutralizing Antibodies against Rift Valley Fever Virus Gn Protein.

The Rift Valley fever virus (RVFV) is an arthropod-borne virus that can not only cause severe disease in domestic animals but also in humans. However, the licensed vaccines or available therapeutics for humans do not exist. Here, we report two Gn-specific neutralizing antibodies (NAbs), isolated from a rhesus monkey immunized with recombinant human adenoviruses type 4 expressing Rift Valley fever virus Gn and Gc protein (rHAdV4-GnGcopt). The two NAbs were both able to protect host cells from RVFV infection. The interactions between NAbs and Gn were then characterized to demonstrate that these two NAbs might preclude RVFV glycoprotein rearrangement, hindering the exposure of fusion loops in Gc to endosomal membranes after the virus invades the host cell. The target region for the two NAbs is located in the Gn domain III, implying that Gn is a desired target for developing vaccines and neutralizing antibodies against RVFV.

A DNA Vaccine Encoding the Gn Ectodomain of Rift Valley Fever Virus Protects Mice via a Humoral Response Decreased by DEC205 Targeting.

The Rift Valley fever virus (RVFV) is responsible for a serious mosquito-borne viral disease in humans and ruminants. The development of a new and safer vaccine is urgently needed due to the risk of introduction of this arbovirus into RVFV-free continents. We recently showed that a DNA vaccine encoding eGn, the ectodomain of the RVFV Gn glycoprotein, conferred a substantial protection in the sheep natural host and that the anti-eGn IgG levels correlated to protection. Addressing eGn to DEC205 reduced the protective efficacy while decreasing the antibody and increasing the IFNγ T cell responses in sheep. In order to get further insight into the involved mechanisms, we evaluated our eGn-encoding DNA vaccine strategy in the reference mouse species. A DNA vaccine encoding eGn induced full clinical protection in mice and the passive transfer of immune serum was protective. This further supports that antibodies, although non-neutralizing in vitro, are instrumental in the protection against RVFV. Addressing eGn to DEC205 was also detrimental to protection in mice, and in this species, both the antibody and the IFNγ T cell responses were strongly decreased. Conversely when using a plasmid encoding a different antigen, i.e., mCherry, DEC205 targeting promoted the antibody response. Altogether our results show that the outcome of targeting antigens to DEC205 depends on the species and on the fused antigen and is not favorable in the case of eGn. In addition, we bring evidences that eGn in itself is a pertinent antigen to be included in a DNA vaccine and that next developments should aim at promoting the anti-eGn antibody response.

In Situ Hybridization for Detection of Rift Valley Fever Virus

Rift Valley fever virus (RVFV) is a mosquito‐borne zoonotic arbovirus with a tripartite genome consisting of a small, a medium (M), and a large (L) segment. This RNA virus causes frequent epidemics in Africa and the Arabian Peninsula. Outbreaks of Rift Valley fever typically result in abortion storms, the deaths of young ruminants and less often the deaths of adult ruminants. As a zoonotic virus, RVFV can also cause symptoms ranging from a mild flu‐like illness to encephalitis and death in humans. As such, RVFV‐infected tissues must be handled with caution. Formalin‐fixed, paraffin‐embedded (FFPE) tissues offer a safer format for researchers and diagnosticians testing for the presence of this virus. Currently, immunohistochemistry (IHC) serves as a confirmatory diagnostic test in South Africa alongside RT‐qPCR on fresh frozen tissue. However, PCR does not enable the ability to visualize the virus within a histological context and IHC has limitations in its ability to detect viral antigen in suboptimally preserved samples. These limitations create a need for more powerful confirmatory diagnostic tools. Here we demonstrate the use of RNAscope® in situ hybridization (ISH) technology for the detection of RVFV viral RNA (vRNA) in FFPE tissues.Samples tested included a broad set of previously published, confirmed positive field samples from sheep naturally infected during the 2010 South African outbreak. Prior, a DNA probe was designed to target the nucleotides between 20‐997 of the RVFV L segment (NC_014397.1) and the assay was validated on experimentally infected tissues. Here we tested this pan‐RVFV ISH assay on naturally infected liver, spleen, kidney, lung, thyroid, brain, skeletal and cardiac muscle, lymph node, salivary gland, adrenal gland, gastrointestinal tract, and male and female reproductive organs. We also compared ISH results to other tests, a previously established triplex FFPE RT‐qPCR targeting all three gene segments as well as IHC targeting the RVFV Gn glycoprotein. Finally, we were able to dual label samples with pan‐RVFV ISH and IHC for analysis of both RVFV RNA and protein distribution as well as RVFV RNA and cell type markers, providing finer grained detail regarding cellular tropism.Pan‐RVFV ISH appropriately labeled vRNA and this signal correlated with visible lesions in the various organs tested. Additionally, vRNA was detected in macrophages in the lymph nodes, a result previously seen by IHC. The positive ISH results mirrored those obtained by FFPE RT‐qPCR at the tissue block level. In comparison to IHC, ISH subjectively appears to produce a stronger and more reliable signal. This was observed in multiple organs where histopathology was present but RVFV antigen IHC was negative. We believe that this highly sensitive and specific ISH assay has potential as a RVFV confirmatory diagnostic test and research applications.Support or Funding InformationThis work was funded by the Department of Diagnostic Medicine, College of Veterinary Medicine, Kansas State University; the Department of Paraclinical Sciences, University of Pretoria, USDA Agricultural Research Services, and APHIS.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Chimaeric Rift Valley Fever Virus-Like Particle Vaccine Candidate Production in Nicotiana benthamiana.

Rift Valley fever virus (RVFV) is an emerging mosquito-borne virus and hemorrhagic fever agent, which causes abortion storms in farmed small ruminants and potentially causes miscarriages in humans. Although live-attenuated vaccines are available for animals, they can only be used in endemic areas and there are currently no commercially available vaccines for humans. Here the authors describe the production of chimaeric RVFV virus-like particles transiently expressed in Nicotiana benthamiana by Agrobacterium tumefaciens-mediated gene transfer. The glycoprotein (Gn) gene is modified by removing its ectodomain (Gne) and fusing it to the transmembrane domain and cytosolic tail-encoding region of avian influenza H5N1 hemagglutinin. This is expressed transiently in N. benthamiana with purified protein yields calculated to be ≈57 mg kg-1 fresh weight. Transmission electron microscopy shows putative chimaeric RVFV Gne-HA particles of 49-60 nm which are immunogenic, eliciting Gn-specific antibody responses in vaccinated mice without the use of adjuvant. To our knowledge, this is the first demonstration of the synthesis of Gne-HA chimaeric RVFV VLPs and the first demonstration of a detectable yield of RVFV Gn in plants.

A Protective Monoclonal Antibody Targets a Site of Vulnerability on the Surface of Rift Valley Fever Virus

SummaryThe Gn subcomponent of the Gn-Gc assembly that envelopes the human and animal pathogen, Rift Valley fever virus (RVFV), is a primary target of the neutralizing antibody response. To better understand the molecular basis for immune recognition, we raised a class of neutralizing monoclonal antibodies (nAbs) against RVFV Gn, which exhibited protective efficacy in a mouse infection model. Structural characterization revealed that these nAbs were directed to the membrane-distal domain of RVFV Gn and likely prevented virus entry into a host cell by blocking fusogenic rearrangements of the Gn-Gc lattice. Genome sequence analysis confirmed that this region of the RVFV Gn-Gc assembly was under selective pressure and constituted a site of vulnerability on the virion surface. These data provide a blueprint for the rational design of immunotherapeutics and vaccines capable of preventing RVFV infection and a model for understanding Ab-mediated neutralization of bunyaviruses more generally.

A Rift Valley fever virus Gn ectodomain-based DNA vaccine induces a partial protection not improved by APC targeting

Rift Valley fever virus, a phlebovirus endemic in Africa, causes serious diseases in ruminants and humans. Due to the high probability of new outbreaks and spread to other continents where competent vectors are present, vaccine development is an urgent priority as no licensed vaccines are available outside areas of endemicity. In this study, we evaluated in sheep the protective immunity induced by DNA vaccines encoding the extracellular portion of the Gn antigen which was either or not targeted to antigen-presenting cells. The DNA encoding untargeted antigen was the most potent at inducing IgG responses, although not neutralizing, and conferred a significant clinical and virological protection upon infectious challenge, superior to DNA vaccines encoding the targeted antigen. A statistical analysis of the challenge parameters supported that the anti-eGn IgG, rather than the T-cell response, was instrumental in protection. Altogether, this work shows that a DNA vaccine encoding the extracellular portion of the Gn antigen confers substantial—although incomplete—protective immunity in sheep, a natural host with high preclinical relevance, and provides some insights into key immune correlates useful for further vaccine improvements against the Rift Valley fever virus.

An equine herpesvirus type 1 (EHV-1) vector expressing Rift Valley fever virus (RVFV) Gn and Gc induces neutralizing antibodies in sheep

Rift Valley fever virus (RVFV) is an arthropod-borne bunyavirus that can cause serious and fatal disease in humans and animals. RVFV is a negative-sense RNA virus of the Phlebovirus genus in the Bunyaviridae family. The main envelope RVFV glycoproteins, Gn and Gc, are encoded on the M segment of RVFV and known inducers of protective immunity. In an attempt to develop a safe and efficacious RVF vaccine, we constructed and tested a vectored equine herpesvirus type 1 (EHV-1) vaccine that expresses RVFV Gn and Gc. The Gn and Gc genes were custom-synthesized after codon optimization and inserted into EHV-1 strain RacH genome. The rH_Gn-Gc recombinant virus grew in cultured cells with kinetics that were comparable to those of the parental virus and stably expressed Gn and Gc. Upon immunization of sheep, the natural host, neutralizing antibodies against RVFV were elicited by rH_Gn-Gc and protective titers reached to 1:320 at day 49 post immunization but not by parental EHV-1, indicating that EHV-1 is a promising vector alternative in the development of a safe marker RVFV vaccine.

Evaluation of Fluorescence Microsphere Immunoassay for Antibody Detection to Rift Valley Fever Nucleocapsid Protein and Glycoproteins

Rift Valley Fever Virus (RVFV) is a zoonotic disease that infects ruminants including cattle, sheep, goats, camels and buffalo. There is a need to create a safe, rapid, and accurate assay that can simultaneously detect antibodies directed against multiple RVFV antigens. We describe the improvement and validation of a fluorescence microsphere immunoassay (FMIA) for the detection of IgG antibodies against the RVFV glycoproteins and the immunodominant nucleocapsid (N) protein. The purpose of this assay is to simultaneously screen and confirm sera from animals for positive or negative disease status. Well‐characterized vaccinated and experimentally infected sheep and calf sera were used for the evaluation of the assay. Recombinant viral proteins were produced then coupled to polystyrene magnetic beads for analysis using the Luminex MAGPIX system with xMAP technology. An unrelated viral antigen coupled control bead set was added to the reaction in order to account for non‐specific binding of antibodies to the antigen of the antigen‐bead complex. Median Fluorescence Intensity (MFI) results were converted to Sample/Positive ratios in order to standardize test results. Our results revealed highest MFI values for the detection of IgG antibodies against the RVFV N protein, indicating that this RVFV antigen could serve as a good candidate for a screening assay. The glycoprotein Gn target has been shown to differentiate vaccinated from non‐vaccinated animals in a candidate subunit vaccine formulation based on the RVFV Gn and Gc proteins. The FMIA demonstrated a high correlation (R2) of 0.91 when compared side by side with plaque reduction neutralization tests. The addition of further RVFV targets such as the non‐structural proteins NSs and NSm is currently under investigation. The results presented in this report demonstrate that FMIA provides a rapid and robust serological diagnostic tool for the detection of antibodies against RVFV. The targets developed in this assay will provide the basis for the development of a multiplex sero‐diagnostic assay system that can simultaneously screen for several ruminant diseases including transboundary animal diseases.Support or Funding InformationThis material is based upon work supported by the Kansas State NBAF Transition Funds, Kansas State University College of Veterinary Medicine, University of Pretoria Department of Veterinary Tropical Diseases, the USDA Agricultural Research project number 5430‐32000‐005‐00D and the U.S. Department of Homeland Security under Grant Award Number DHS‐2010‐ST‐061‐AG0001. The views and conclusions contained in this document are those of the authors and should not be interpreted as necessarily representing the official policies, either expressed or implied, of the U.S. Department of Homeland Security.

A novel indirect ELISA based on glycoprotein Gn for the detection of IgG antibodies against Rift Valley fever virus in small ruminants

Rift Valley fever virus (RVFV) is an emerging zoonotic pathogen that causes high morbidity and mortality in humans and livestock. In this paper, we describe the cloning, expression and purification of RVFV glycoprotein Gn and its application as a diagnostic antigen in an indirect ELISA for the specific detection of RVF IgG antibodies in sheep and goats. The performance of this Gn based ELISA is validated using a panel of almost 2000 field samples from sheep and goats from Mozambique, Senegal, Uganda and Yemen. All serum samples were also tested by virus neutralization test (VNT), the gold standard method for RVFV serological testing. Compared to the VNT results the Gn based ELISA proved to have an excellent sensitivity (94.56%) and specificity (95.57%). Apart from establishing this new diagnostic assay, these results also demonstrate a close correlation between the presence of RVFV Gn and neutralizing antibodies.

Immunization with DNA Vaccine Expressing Gn Coupled to C3d Prevents Clinical Symptoms of Infection and Protects Mice against an Aerosol Rift Valley Fever Virus Infection.

Rift Valley fever virus (RVFV) is the causative agent of Rift Valley fever (RVF) and is an emerging infectious disease of zoonotic potential. However, aerosolization of RVFV has been proposed as a potential bioweapon and most vaccines have not been tested against aerosolized RVFV challenge. Previously, two vaccine platforms (DNA plasmids and alphavirus replicons) expressing a soluble form of the RVFV Gn glycoprotein alone or fused to three copies of complement protein, C3d, protected mice against an intraperitoneal (IP) RVFV infection. In this study, both vaccine candidates were used to determine the protective efficacy against an aerosolized RVFV challenge. Each vaccine was administered to mice alone or in a heterologous prime/replicon boost strategy and anti-RVFV immune responses were assessed. DNA plasmids expressing Gn-C3d and alphavirus replicons expressing Gn elicited high titer neutralizing antibodies that were similar to titers elicited by the live-attenuated MP12 virus. However, only Gn-C3d- DNA vaccine completely protected mice against virulent aerosolized RVFV challenge. Most mice receiving replicon based vaccines succumbed to RVFV infection. Surprisingly, even though live-attenuated MP12 vaccine protected mice against IP challenge, MP12 did not provide complete protection against aerosolized RVFV infection. Therefore, vaccine candidates that are effective against peripheral challenge should be tested against aerosolized challenge to determine the complete protection profile, since any bioterrorism attack using RVFV would most likely be in the form of an aerosol.

An Assembly Model of Rift Valley Fever Virus

Rift Valley fever virus (RVFV) is a bunyavirus endemic to Africa and the Arabian Peninsula that infects humans and livestock. The virus encodes two glycoproteins, Gn and Gc, which represent the major structural antigens and are responsible for host cell receptor binding and fusion. Both glycoproteins are organized on the virus surface as cylindrical hollow spikes that cluster into distinct capsomers with the overall assembly exhibiting an icosahedral symmetry. Currently, no experimental three-dimensional structure for any entire bunyavirus glycoprotein is available. Using fold recognition, we generated molecular models for both RVFV glycoproteins and found significant structural matches between the RVFV Gn protein and the influenza virus hemagglutinin protein and a separate match between RVFV Gc protein and Sindbis virus envelope protein E1. Using these models, the potential interaction and arrangement of both glycoproteins in the RVFV particle was analyzed, by modeling their placement within the cryo-electron microscopy density map of RVFV. We identified four possible arrangements of the glycoproteins in the virion envelope. Each assembly model proposes that the ectodomain of Gn forms the majority of the protruding capsomer and that Gc is involved in formation of the capsomer base. Furthermore, Gc is suggested to facilitate intercapsomer connections. The proposed arrangement of the two glycoproteins on the RVFV surface is similar to that described for the alphavirus E1-E2 proteins. Our models will provide guidance to better understand the assembly process of phleboviruses and such structural studies can also contribute to the design of targeted antivirals.