|Period||01 / 2011 - 12 / 2012|
|Objective of the project
We recently established methods for the production of Rift Valley fever virus (RVFV) single-round infectious particles (SRIPs). In this project, we will develop an SRIP-based vaccine platform that allows extremely rapid development of (multivalent) vaccines against other pathogens
The bunyavirus family is the largest family of viruses. The success of these viruses is attributed to efficient transmission and broad host range. Efficient transmission is explained by their high infectivity in many mammalian species and remarkable virion stability outside the host. These properties are highly valued in the field of vector vaccine technology. However, because of their extremely high pathogenicity, use of bunyaviruses for such applications was never before considered. Now that we have developed methods for the efficient production of single-round infectious particles (SRIPs) of RVFV, the application of these viruses as vaccine vectors can finally be explored.
Like other bunyavirus family members, RVFV contains a three-segmented RNA genome, which is comprised of the large (L), medium (M) and small (S) segment. The L segment encodes the viral RNA polymerase, the M segment encodes the structural glycoproteins Gn and Gc. The S segment encodes the non-structural protein NSs and the nucleocapsid (N) protein.
We have developed methods to produce RVFV particles that lack the M genome segment. The L and S genome segment are packaged into virus-like particles by providing the RVFV glycoproteins (normally encoded by the M genome segment) either by a stable cell line, plasmid, or by a previously described recombinant paramyxovirus, NDV-GnGc, (Kortekaas et al., Vaccine 2010; 28:4394-4401). Although the particles are capable of infecting cells and expressing proteins of interest, they are not capable of autonomous spread and are therefore highly safe. The so-called single-round infectious particles (SRIPs) were produced to titers of up to 107 infectious particles/ml.
Task 1. RVFV contains a three-segmented genome. Pilot experiments have already demonstrated that foreign genes can be expressed from two of the three RVFV genome segments. However, it is known from literature that the viral promoters on each segment differ in strength. For this reason, reporter genes (eGFP and Renilla luciferase) will be expressed from each of the three genome segments and kinetics of expression levels will be determined. Alongside the aforementioned experiments, the packaging efficiency of each of the reporter genome segments will be determined. The combined results of these two tasks will determine which genome segment is optimal for gene delivery applications. (timeframe: 2011-2012)
Task 2. It will be highly valuable if more than a single gene can be expressed from the SRIP genome. In theory, this can be accomplished by various methods. First, different reporter genes will be inserted in the same genome segment (for example, green fluorescent and red fluorescent protein in the S segment) and it will be determined if both reporter genomes are maintained in the SRIP population. If this is the case, multiple genes can be delivered using the same genome segment (This work will also provide fundamental insights into RVFV biology, in this case revealing if cells can be superinfected with RVFV). Similar constructs can be developed using the M and L genome segments. The possibilities of combining S, M and L reporter genomes will also be studied.
It is plausible to assume that selective pressure is needed to maintain genome segments in the SRIP population. If this is indeed the case, at least two proteins should be produced from a single genome segment, of which one protein is essential for genome replication. We already successfully expressed eGFP from the S genome segment, which is ambisense by nature. The resulting genome segment produces both the RVFV N protein (which is essential for genome replication) and the eGFP protein. It would be highly interesting to evaluate if the L gene can be expressed from an S genome-like segment, providing an additional insertion site for a second foreign gene. (Time frame: 2012-2013
Task 3. Mice will be vaccinated with SRIPs containing the GFP reporter gene via different routes (nasal, oral, parenteral). RVFV-specific humoral and cellular immune responses will be studied in detail (this can be performed outside the BSL3 laboratory). For the humoral response, RVFV-specific IgG1 and IgG2a will be determined. CTL responses will be studied by monitoring Th1, Th2 and Th17 responses using cytokine assays. With this result we can investigate which type of immune responses are elicited by vaccination with SRIPs. To identify primary sites of SRIP replication and demonstrate safety (inability to spread in vivo) sacrificed mice will be monitored for GFP expression in all organs. (Time frame: 2011-2012)
Task 4. Expression kinetics of different versions (intracellular and secreted) of model antigens of influenza virus H1N1 will be determined. Immune responses (cellular (CTLs) and humoral) and protective efficacy elicited by SRIPs expressing these antigens in mice will be studied. The SRIPs will be produced, characterized and subsequently administered to mice via three different routes. Mice will be challenged with either RVFV or influenza virus. (Time frame 2011-2012)
Task 5. Study the feasibility of using SRIPs as a bivalent vaccine for the control of RVFV and PPRV in sheep. The genes encoding PPRV H and/or F will be introduced in the optimal SRIP expression site (see above). Expression kinetics and gene stability of the inserted genes will be analyzed. First, mice will be inoculated with the SRIPs via the optimal inoculation route determined in Task 3 and the humoral immune response will be analyzed. If successful, an experiment will be performed in sheep. One group of sheep will be challenged with RVFV and another group will be challenged with PPRV (Time frame: 2013-2014)
The bunyavirus family is the largest family of viruses and includes many highly pathogenic members (hantavirus, Crimean-congo hemorrhagic fever virus, etc). We are the first in the world to produce bunyavirus particles that can be handled outside biosafety containment. There is no doubt that there will be a huge interest in our technology when the first publications appear, and that this will result in new large collaborative projects.
- 1 Patent application on the methods to produce Bunyavirus SRIPs and their applications.
- It can be expected that this new technology will result in other patent applications during the course of the PhD project.
- 1 SRIP-based experimental RVFV vaccine for applications in humans and livestock.
- 1 SRIP-based bivalent vaccine for the control of PPR and RVFV.
- 1 cell line persistently infected with SRIPs expressing EGFP for sceening antiviral agents.
- 1 cell line persistently infected with SRIPs expressing Renilla luciferase for screening antiviral agents.
- 4 cell lines persistently infected with SRIPs expressing influenza virus HA and NA, PPRV H and F, for the production of recombinant proteins.
- Development of expertise on cellular and humoral immunity against bunyaviruses.
- 5 publications in peer-reviewed scientific journals of high impact.
- 2 contributions to conferences and scientific meetings.
- 1 PhD thesis
Although the project is in its first year it progresses already well
1 patent application on the methods to produce Bunyavirus SRIPs and their applications has recently been filed (september 20, 2010).
1 publication on the method to produce SRIPs and their potential use for vaccine- and diagnostic test development has been conditionally accepted (J. Virol. under revision).
a cell line persistently infected with SRIPs expressing EGFP has been established.
Publicaties bij dit project zijn beschikbaar via deze Link
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