HAL is a multi-disciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. A SAP30 complex inhibits IFN-beta expression in Rift Valley fever virus infected cells. Nicolas Le May, Zeyni Mansuroglu, Psylvia Léger, Thibaut Josse, Guillaume Blot, Agnès Billecocq, Ramon Flick, Yves Jacob, Eliette Bonnefoy, Michèle Bouloy

Innovative vaccine platforms are needed to develop effective countermeasures against emerging and re-emerging diseases. These platforms should direct antigen internalization by antigen presenting cells and promote immunogenic responses. This work describes an innovative systems approach combining two novel platforms, αGalactose (αGal)-modification of antigens and amphiphilic polyanhydride nanoparticles as vaccine delivery vehicles, to rationally design vaccine formulations. Regimens comprising soluble αGal-modified antigen and nanoparticle-encapsulated unmodified antigen induced a high titer, high avidity antibody response with broader epitope recognition of antigenic peptides than other regimen. Proliferation of antigen-specific CD4(+) T cells was also enhanced compared to a traditional adjuvant. Combining the technology platforms and augmenting immune response studies with peptide arrays and informatics analysis provides a new paradigm for rational, systems-based design of next generation vaccine platforms against emerging and re-emerging pathogens.

Rift Valley fever virus (RVFV) is an arbovirus that causes significant morbidity and mortality in both humans and livestock. With increased world travel and the threat of bioterrorism, there is a real risk of RVFV spreading to naïve geographical areas (Trans. R. Soc. Trop. Med. Hyg., 73, 1979, 618; MMWR Morb. Mortal. Wkly Rep., 49, 2000, 905). The introduction of RVFV would cause critical public health, agricultural and economic damage. Despite the clear need for an efficacious vaccine, there are no United States (US) Food and Drug Administration or US Department of Agriculture approved vaccines against RVFV. To address this need, a virus-like particle (VLP)-based vaccine candidate was developed. First, a non-replicating chimeric RVF VLP vaccine candidate was generated that protected mice and rats against a lethal RVFV challenge. This was followed by the development and optimization of conditions for production of RVF VLPs in insect and mammalian cells. Immunological studies demonstrated that VLP-based vaccine candidates elicit both humoral and cellular immune responses. Subsequent challenge studies using a lethal wild-type RVFV strain under high-containment conditions showed that RVF VLP vaccine candidates can completely protect mice and rats.

The aim of this study was to evaluate the contribution of positively charged amino acid residues for the Uukuniemi virus (UUKV) N protein functionality. Based on phlebovirus nucleocapsid (N) protein alignments and 3D-structure predictions of UUKV N protein, 14 positively charged residues were chosen as targets for the mutagenesis. The impact of mutations to the N protein functionality was analyzed using minigenome-, virus-like particle-, and mammalian two-hybrid-assays. Seven of the mutations affected the functional competence in all three assays, while others had milder impact or no impact at all. In the 3D-model of UUKV N protein, five of the affected residues, R61, R64, R73, R98 and R115, were located either within or in close proximity to the central cavity that could potentially bind RNA.

Rift Valley fever virus is recognized as an important bioterror and agroterror threat to Western countries including the United States. Once introduced, the virus would be readily spread by native mosquito populations and potentially become endemic. While infection often results in severe morbidity and mortality in both humans and livestock, there are currently no FDA or USDA-licensed vaccines. The development of effective countermeasures and implementing surveillance and diagnostic capabilities are critical. Ultimately, the presence of RVFV would lead to severe long-term negative impacts for healthcare, agricultural and travel economic sectors.

Rift Valley fever virus (RVFV) is an arthropod-borne pathogen that often results in severe morbidity and mortality in both humans and livestock. As its geographic range continues to expand, it presents a real threat to naïve populations around the world by accidental introduction (e.g., the result of increased travel) or intentional release (e.g., a bioterror event). While there is a clear need for a safe and efficacious vaccine against this emerging and re-emerging pathogen, no FDA-approved vaccine is currently available. This need was addressed by the establishment of novel mammalian and insect suspension cell line systems for the efficient production of RVF virus-like particle (VLP)-based vaccine candidates. A direct comparison of the production of RVF VLPs in these systems was performed. Optimization and characterization resulted in a production platform suitable for scale-up. Furthermore, RVF VLP-based vaccines were tested in a lethal challenge model and showed full protection, demonstrating that RVF VLPs present promising RVFV vaccine candidates.

Rift Valley fever virus (RVFV) is an arthropod-borne pathogen that often results in severe morbidity and mortality in both humans and livestock. As its geographic range continues to spread, it presents a real threat to naïve populations around the world by accidental introduction (e.g., the result of increased world travel) or a bioterror event. The lack of prophylactic and therapeutic measures, the potential for human-to-human transmission, and the significant threat to livestock associated with RVFV make infection with these pathogens a serious public health concern. Rift Valley fever epizootics and epidemics might rapidly overwhelm the capacities of the public health and veterinary medical communities to provide rapid diagnostic testing, distribution of countermeasures and adequate medical care.

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Background: F1-V from Yersinia pestis has been shown to be a protective antigen against disease. We modified the F1-V antigen with α-galactose residues and immunized α galactosyl transferase (α-GT) KO mice in order to mimic the same defect in humans that results in circulating antibodies against α-gal epitopes enhancing immune responsiveness. Aim: To demonstrate that α-gal modified F1-V induces a more robust immune response than unmodified F1-V. Methods: α-GT KO mice were injected subcutaneously with different doses of α-gal-F1-V or F1-V. All mice were boosted with F1-V at 36 dpi and necropsied on day 40 in order to evaluate in vitro recall response of lymphocytes and to measure anti-F1-V antibody titers. Results: Mean IgG titer values were 50,000 and 10,000 for mice immunized with the α-gal-F1-V or F1-V, respectively. The relative avidity of the antibody response was also higher for the α-gal-F1-V immunized mice. Moreover, lymphocytes from α-gal-F1-V immunized mice demonstrated enhanced proliferation in response to F1-V compared to cells from F1-V immunized mice. Conclusions: α-gal modified F1-V increases both the avidity and amount of anti-F1-V produced following vaccination. As a functional assay, α-gal-F1-V antisera efficiently opsonized Y. pestis, facilitated phagocytosis by human monocytes and prevented Y. pestis-induced monocyte cytotoxicity.

Virus-like particles (VLPs) present viral antigens in a native conformation and are effectively recognized by the immune system and therefore are considered as suitable and safe vaccine candidates against many viral diseases. Here we demonstrate that chimeric VLPs containing Rift Valley fever virus (RVFV) glycoproteins GN and GC, nucleoprotein N and the gag protein of Moloney murine leukemia virus represent an effective vaccine candidate against Rift Valley fever, a deadly disease in humans and livestock. Long-lasting humoral and cellular immune responses are demonstrated in a mouse model by the analysis of neutralizing antibody titers and cytokine secretion profiles. Vaccine efficacy studies were performed in mouse and rat lethal challenge models resulting in high protection rates. Taken together, these results demonstrate that replication-incompetent chimeric RVF VLPs are an efficient RVFV vaccine candidate.

Uukuniemi virus (UUKV) belongs to the Phlebovirus genus in the family Bunyaviridae. As a non-pathogenic virus for humans UUKV has served as a safe model bunyavirus in a number of studies addressing fundamental questions such as organization and regulation of viral genes, genome replication, structure and assembly. The present study is focused on the oligomerization of the UUKV nucleocapsid (N) protein, which plays an important role in several steps of virus replication. The aim was to locate the domains involved in the N protein oligomerization and study the process in detail.

SummaryThe αGal HyperAcute® Technology exploits a robust zoonotic blockade to enhance potency of antiviral vaccines. Naturally acquired immunity against the common αGal epitope [galactose‐alpha(1,3)‐galactose‐beta(1,4)N‐acetylglucosamine‐R (Gal‐α(1,3)‐Gal‐β(1,4)‐GlcNAc‐R)] is facilitated by the loss of a key enzyme in the epitope’s biosynthetic pathway. As human cells are devoid of this epitope, chronic stimulus from gut flora leads to high levels of circulating anti‐αGal antibodies and the development of a robust immune pathway. As the αGal epitope is immediately recognized as foreign, the naturally acquired αGal immune pathway in humans serves as a strong barrier to zoonotic infection. The αGal HyperAcute® Technology takes advantage of this natural process to facilitate the rapid presentation of modified antigens to antigen‐presenting cells, leading to a strong immune response. The evolutionary immunity to αGal ensures that the presence of αGal epitopes on antigens will lead to a robust immune response involving cross‐activation of TH1 immunity, characterized by cytokine secretion and increased phagocytic activity, and TH2 immunity characterized by high antibody titres. αGal epitopes can be applied to antiviral vaccines by biological, enzymatic or chemical means. Several detection methods that directly and indirectly verify αGal addition are discussed. Enhanced immunogenicity (humoral and cellular) of αGal‐modified vaccines is shown for several antiviral vaccine candidates. αGal modification of antiviral vaccine components leads to enhanced immunogenicity. The existing body of literature describing the utility of αGal epitopes as a safe and robust immunostimulatory and ‐modulatory agent in humans supports the basis for applying the αGal HyperAcute® Technology to the improvement of antiviral vaccines, both new and currently approved.

The advent of reverse genetics technology has revolutionized the study of RNA viruses, making it possible to manipulate their genomes and evaluate the effects of these changes on their biology and pathogenesis. The fundamental insights gleaned from reverse genetics-based studies over the last several years provide a new momentum for the development of designed therapies for the control and prevention of these viral pathogens. This review summarizes the successes and stumbling blocks in the development of reverse genetics technologies for Rift Valley fever virus and their application to the further dissection of its pathogenesis and the design of new therapeutics and safe and effective vaccines.

ABSTRACT To explore mechanisms of entry for Ebola virus (EBOV) glycoprotein (GP) pseudotyped virions, we used comparative gene analysis to identify genes whose expression correlated with viral transduction. Candidate genes were identified by using EBOV GP pseudotyped virions to transduce human tumor cell lines that had previously been characterized by cDNA microarray. Transduction profiles for each of these cell lines were generated, and a significant positive correlation was observed between RhoC expression and permissivity for EBOV vector transduction. This correlation was not specific for EBOV vector alone as RhoC also correlated highly with transduction of vesicular stomatitis virus GP (VSVG) pseudotyped vector. Levels of RhoC protein in EBOV and VSV permissive and nonpermissive cells were consistent with the cDNA gene array findings. Additionally, vector transduction was elevated in cells that expressed high levels of endogenous RhoC but not RhoA. RhoB and RhoC overexpression significantly increased EBOV GP and VSVG pseudotyped vector transduction but had minimal effect on human immunodeficiency virus (HIV) GP pseudotyped HIV or adeno-associated virus 2 vector entry, indicating that not all virus uptake was enhanced by expression of these molecules. RhoB and RhoC overexpression also significantly enhanced VSV infection. Similarly, overexpression of RhoC led to a significant increase in fusion of EBOV virus-like particles. Finally, ectopic expression of RhoC resulted in increased nonspecific endocytosis of fluorescent dextran and in formation of increased actin stress fibers compared to RhoA-transfected cells, suggesting that RhoC is enhancing macropinocytosis. In total, our studies implicate RhoB and RhoC in enhanced productive entry of some pseudovirions and suggest the involvement of actin-mediated macropinocytosis as a mechanism of uptake of EBOV GP and VSVG pseudotyped viral particles.

In this study we report the development and optimization of two minigenome rescue systems for Nipah virus, a member of the Paramyxoviridae family. One is mediated by the T7 RNA polymerase supplied either by a constitutively expressing cell line or by transfection of expression plasmids and is thus independent from infection with a helper virus. The other approach is based on RNA polymerase I-driven transcription, a unique approach for paramyxovirus reverse genetics technology. Minigenome rescue was evaluated by reporter gene activities of (i) the two different minigenome transcription systems, (ii) genomic versus antigenomic-oriented minigenomes, (iii) different ratios of the viral protein expression plasmids, and (iv) time course experiments. The high efficiency and reliability of the established systems allowed for downscaling to 96-well plates. This served as a basis for the development of a high-throughput screening system for potential antivirals that target replication and transcription of Nipah virus without the need of high bio-containment. Using this system we were able to identify two compounds that reduced minigenome activity.