Patent Description:
The Rift Valley fever virus (RVFV) is a mosquito-borne bunyavirus that belongs to the Phenuiviridae family of the genus Phlebovirus. RVFV causes Rift Valley Fever (RVF) in ruminants, which after epizootic outbreaks, is transmitted to humans mainly through mosquito bites.

Rift Valley Fever is currently present in the African continent, in the southern Arabian Peninsula and in the Indian Ocean islands. In addition, RVF has the potential to extend to other geographical areas, in particular in relation to climate change and globalization. Currently, there is no treatment or vaccine against Rift Valley Fever on the market.

RVFV is an RNA virus. The structure of the RVFV virion consists of a lipid envelope with two membrane glycoproteins (Gn and Gc) arranged in an icosahedral lattice that protects an internal nucleocapsid composed of the viral nucleoprotein (N) and an RNA-dependent polymerase (RdRp) linked to the viral RNA.

The RVFV genome is made up of three segments of single-stranded RNA of different size (large (L), medium (M), small (S)) with negative polarity (L and M) or bipolarity (S). The L segment encodes the RNA-dependent RNA polymerase (RdRp). The M segment encodes for two <NUM> kDa and <NUM>-<NUM> kDa non-structural proteins (NSm) and for Gn and Gc membrane glycoproteins. The S segment encodes a <NUM> kDa non-structural protein (NSs), considered the primary virulence factor of the virus, as well as the N protein.

Live attenuated vaccines induce long-lasting and highly protective immunity after single-dose administration in both animals and humans. They are called live vaccines because they contain the microorganism causing the infection in a live or viable form, but with a very reduced (attenuated) capacity for infection, reproduction or, in general, virulence. This makes live attenuated vaccines an excellent basis for the development of successful immunization programs in the affected countries or to implement preventive control measures in countries with a higher risk of introduction or expansion of the disease.

Mutagenic treatments have frequently been used as viral attenuation methods. One example of this is the MP-<NUM> live attenuated FVR vaccine (Ikegami et al. , <NUM>), obtained by mutagenic treatment with <NUM>-fluorouracil. Currently, the MP-<NUM> vaccine is in phase II trials as a vaccine candidate for humans.

Nucleoside analogues with antiviral activity against RVFV have been described, such as ribavirin (<NUM>-β-D-ribofuranosyl-<NUM>-H-<NUM>,<NUM>,<NUM>-triazole-<NUM>-carboxyamide), favipiravir (<NUM>-fluoro-<NUM>-hydroxy-<NUM>-pyrazinecarboxyamide) and BCX4430 [(<NUM>,<NUM>,4R,5R)-<NUM>-(<NUM>-amino-<NUM>-pyrrolo [<NUM>,<NUM>-d]pyrimidin-<NUM>-yl)-<NUM>-(hydroxymethyl)pyrrolidine-<NUM>,<NUM>-diol] (galidesivir). Favipiravir is a pyrazine derivative (<NUM>-fluoro-<NUM>-hydroxy-<NUM>-pyrazinecarboxyamide) that acts as a pyrimidine analogue and has potent antiviral activity against different RNA viruses.

Favipiravir has mutagenic activity on several RNA viruses, including hepatitis C virus, foot-and-mouth disease virus, West Nile fever virus, norovirus, influenza virus and RVFV. It has been described in the prior art that the mechanism of action of favipiravir against RVFV is due to the accumulation of mutations in the viral genome leading to a progressive decrease of viable viral progeny (Borrego et al.

The development of live attenuated RVF vaccines is an active field of research and there is a need to develop new live attenuated RVF vaccines, which can serve to develop safe and effective RVF control strategies, for animal and/or human use.

For the purposes of the present invention, the amino acids are mentioned by their full name or are represented using the three-letter symbols of the IUPAC nomenclature, also used in the ST. <NUM> standard for the presentation of lists of nucleotide and amino acid sequences in patent applications of the World Intellectual Property Organization (WIPO). Thus, for example, the amino acid alanine is represented by the symbol "Ala", the amino acid aspartic acid is represented by "Asp", etc..

For the purposes of the present invention, nucleotide sequences of RNA molecules are described by the corresponding nucleotide sequence of DNA molecules. It is known in the art how to determine RNA sequences from the corresponding DNA sequences, which is performed by replacing the thymine nucleotides with uracil nucleotides.

For the purposes of the present invention, "multiplicity of infection (MOI)" refers to the ratio of agents (e.g., viruses) to infection targets (e.g., cells). By way of example, when referring to a group of cells inoculated with virus particles, the multiplicity of infection or MOI is the ratio between the number of virus particles and the number of target cells present.

For the purposes of the present invention, the term "ELISA" refers to enzyme-linked immunoabsorbent assay, which is an immunoassay technique in which an immobilized antigen is detected by an antibody bound to an enzyme (peroxidase, alkaline phosphatase, etc.,) capable of generating a detectable product from a substrate by a colour change or some other type of change caused by the enzymatic action on said substrate. In said technique, there may be a primary antibody that recognizes the antigen and that in turn is recognized by a secondary antibody bound to said enzyme. The antigen can be detected indirectly in the sample by colour changes measured by spectrophotometry.

For the purposes of the present invention, "challenge", in the context of immunology, refers to the deliberate exposure of an animal to an infectious agent, e.g., a virus, to study the response of the animal to exposure to said infectious agent. The dose used in the challenge is called the "challenge dose".

For the purposes of the present invention, RACE (Rapid amplification of cDNA ends) refers to a technique of rapid amplification of complementary DNA (cDNA) ends. RACE is a technique used in molecular biology to obtain the full-length sequence of an RNA molecule, in which a cDNA copy of the RNA sequence of interest is produced by reverse transcription, followed by polymerase chain reaction (PCR) amplification of the cDNA copies.

For the purposes of the present invention, "ANOVA" or "analysis of variance" is a set of statistical models used to analyze differences between means within a statistical sample.

ANOVA is based on the law of total variance, where the variance observed in a particular variable is divided into components attributable to different sources of variation. ANOVA provides a statistical test to determine if two or more population means are the same.

The present invention provides an attenuated variant of Rift Valley Fever Virus (RVFV), wherein:.

In one embodiment of the variant of the RVFV of the invention, further:.

In one embodiment of the attenuated variant Rift Valley fever virus (RVFV):.

wherein said amino acids correspond to amino acid substitutions of the sequence of wild strain <NUM>/<NUM> of the RVF virus.

In one embodiment of the RVFV variant of the invention, the amino acid sequence encoded by the L segment of the RNA of said variant is SEQ ID NO: <NUM>; the amino acid sequence encoded by the M segment of the RNA of said variant is SEQ ID NO: <NUM>; the NSs protein consists of the sequence SEQ ID NO: <NUM>; and the N protein consists of the sequence SEQ ID NO: <NUM>.

In the present disclosure, the amino acid sequence encoded by the L segment of the RNA of wild strain <NUM>/<NUM> of the RVF virus is SEQ ID NO: <NUM>; the amino acid sequence encoded by the M segment of the RNA of wild strain <NUM>/<NUM> of the RVF virus is SEQ ID NO: <NUM>; the NSs protein of the wild type <NUM>/<NUM> strain of the RVF virus consists of the sequence SEQ ID NO: <NUM>; and the N protein of wild-type strain <NUM>/<NUM> of the RVF virus consists of the sequence SEQ ID NO: <NUM>.

In the present disclosure, the amino acid sequence encoded by the L segment of the RNA of wild strain ZH548 of the RVF virus is SEQ ID NO: <NUM>; the amino acid sequence encoded by the M segment of the RNA of the ZH548 wild strain of the RVF virus is SEQ ID NO: <NUM>; the NSs protein of the wild type ZH548 strain of the RVF virus consists of the sequence SEQ ID NO: <NUM>; and the N protein of the wild type ZH548 strain of the RVF virus consists of the sequence SEQ ID NO: <NUM>.

In one embodiment of the RVFV variant of the invention, comprising an RNA encoding said variant, the L segment of said RNA consists of the sequence SEQ ID NO: <NUM>; and the M segment of said RNA consists of the sequence SEQ ID NO: <NUM>; and the S segment of said RNA consists of the sequence SEQ ID NO: <NUM>.

In the present disclosure, the wild strain <NUM>/<NUM> of the RVF virus comprises an RNA encoding said wild strain, wherein the L segment of said RNA consists of the sequence SEQ ID NO: <NUM>; and the M segment of said RNA consists of the sequence SEQ ID NO: <NUM>; and the S segment of said RNA consists of the sequence SEQ ID NO: <NUM>.

In the present disclosure, wild type strain ZH548 of the RVF virus comprises an RNA encoding said wild type strain, wherein the L segment of said RNA consists of the sequence SEQ ID NO: <NUM>; and the M segment of said RNA consists of the sequence SEQ ID NO: <NUM>; and the S segment of said RNA consists of the sequence SEQ ID NO: <NUM>.

The present invention also provides a pharmaceutical or veterinary composition comprising the RVFV variant of the invention, together with at least one pharmaceutically acceptable excipient or for veterinary use.

The present invention also provides the RVFV variant of the invention for use as a medicament.

In one embodiment, the present invention provides the use of the RVFV variant of the invention for the manufacture of a medicament.

In another embodiment, the present invention provides a therapeutic method for preventing Rift Valley fever comprising administering to an animal an effective amount of the attenuated variant of the RVFV of the invention. Preferably, said animal is a human or a ruminant.

For the purposes of the present invention, "effective amount" refers to the amount of the RVFV variant of the invention that provides an objectively identifiable improvement in the state of the animal, recognized by a qualified observer, and wherein said animal is treated with a pharmaceutical composition comprising said amount of the RVFV variant.

Additionally, the present invention provides the RVFV variant of the invention for use in the prevention of Rift Valley fever.

In one embodiment, the present invention provides the use of the RVFV variant of the invention for the manufacture of a medicament for the prevention of Rift Valley fever. Preferably, said medicament is a vaccine.

In one embodiment, the RVFV variant of the invention is for use in animals.

In a preferred embodiment of the variant of the RVFV for use of the invention, said animals are ruminants. Preferably, said ruminants are selected from: cows, sheep, goats, camels and buffaloes.

The RVFV variant of the invention is for use in domestic ruminants and wild ruminants. In the use in wild ruminants, the RVFV variant of the invention is useful for preventing RVF in wild animals in reserves, zoos, etc. In fact, it is suspected that the African buffalo may be a carrier of the virus.

Thus, in one embodiment of the variant of the RVFV for use of the invention, such ruminants are domestic or wild ruminants.

In a preferred embodiment, said domestic ruminants are selected from: cows, sheep, goats and camels.

In another preferred embodiment, said wild ruminants are buffalo.

In another preferred embodiment, the RVFV variant of the invention is for use in humans.

The present invention also provides a Rift Valley fever vaccine comprising the RVFV variant of the invention.

In a preferred embodiment, the vaccine of the invention comprises at least one pharmaceutically acceptable excipient or for veterinary use.

For the purposes of the present invention, inert ingredients such as, but not limited to, buffers, co-solvents, surfactants, oils, humectants, emollients, preservatives, stabilizers, antioxidants, dyes, air protectors and/or moisture and binders are pharmaceutically acceptable excipients or for veterinary use. An example of a buffer is phosphate buffered saline (PBS).

In one embodiment, said pharmaceutical or veterinary composition comprises Dulbecco's Modified Eagle Medium (DMEM).

The pharmaceutical, veterinary composition or vaccine of the invention can be formulated with pharmaceutically acceptable excipients or for veterinary use, as well as with any other type of pharmaceutically acceptable carriers or diluents or for veterinary use, according to conventional techniques in pharmaceutical or veterinary practice.

The pharmaceutical, veterinary composition or the vaccine of the invention can be administrated in single or multiple doses.

The pharmaceutical, veterinary composition or the vaccine of the invention can be administered by any route of administration for which said composition will be formulated in the pharmaceutical form suitable for the chosen route of administration. Examples of routes of administration include, but are not limited to, subcutaneous, intravenous and intramuscular.

The present specification also provides primers for amplification of genomic regions of the RVF virus RNA comprising nucleic acids at least <NUM>% identical to any of the sequences selected from: SEQ ID NO: <NUM>, SEQ ID NO: <NUM>, SEQ ID NO: <NUM>, SEQ ID NO: <NUM>, SEQ ID NO: <NUM>, SEQ ID NO: <NUM>, SEQ ID NO: <NUM>, SEQ ID NO: <NUM>, SEQ ID NO: <NUM>, SEQ ID NO: <NUM>, SEQ ID NO: <NUM>, SEQ ID NO: <NUM>, SEQ ID NO: <NUM>, SEQ ID NO: <NUM>, SEQ ID NO: <NUM>, SEQ ID NO: <NUM>, SEQ ID NO: <NUM>, SEQ ID NO: <NUM>, SEQ ID NO: <NUM>, SEQ ID NO: <NUM>, SEQ ID NO: <NUM>, SEQ ID NO: <NUM>, SEQ ID NO: <NUM>, SEQ ID NO: <NUM>, SEQ ID NO: <NUM>, SEQ ID NO: <NUM>, SEQ ID NO: <NUM>, SEQ ID NO: <NUM>, SEQ ID NO: <NUM>, SEQ ID NO: <NUM>, SEQ ID NO: <NUM> and SEQ ID NO: <NUM>.

In a preferred disclosure, said sequence identity is selected from: <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>% and <NUM>%.

In a more preferred disclosure, said primers of the invention consist of nucleic acids consisting of the sequences selected from: SEQ ID NO: <NUM>, SEQ ID NO: <NUM>, SEQ ID NO: <NUM>, SEQ ID NO: <NUM>, SEQ ID NO: <NUM>, SEQ ID NO: <NUM>, SEQ ID NO: <NUM>, SEQ ID NO: <NUM>, SEQ ID NO: <NUM>, SEQ ID NO: <NUM>, SEQ ID NO: <NUM>, SEQ ID NO: <NUM>, SEQ ID NO: <NUM>, SEQ ID NO: <NUM>, SEQ ID NO: <NUM>, SEQ ID NO: <NUM>, SEQ ID NO: <NUM>, SEQ ID NO: <NUM>, SEQ ID NO: <NUM>, SEQ ID NO: <NUM>, SEQ ID NO: <NUM>, SEQ ID NO: <NUM>, SEQ ID NO: <NUM>, SEQ ID NO: <NUM>, SEQ ID NO: <NUM>, SEQ ID NO: <NUM>, SEQ ID NO: <NUM>, SEQ ID NO: <NUM>, SEQ ID NO: <NUM>, SEQ ID NO: <NUM>, SEQ ID NO: <NUM> and SEQ ID NO: <NUM>.

In one disclosure of such primers of the invention, said RNA is from the RVFV variant of the invention or from the wild strain <NUM>/<NUM> of the RVFV.

Vero cells (ATCC No. Catalogue CCL-<NUM>) were grown in Dulbecco's Modified Eagle Medium (DMEM) supplemented with <NUM>% to <NUM>% fetal calf serum (FCS) and L-glutamine (<NUM>), penicillin (<NUM> U/ml) and streptomycin (<NUM>/ml) in a humid atmosphere of <NUM>% CO<NUM> at <NUM>. Insect cells of Aedes albopictus C6/<NUM> (ATCC No. Catalogue CRL1660) were grown in Eagle minimal essential medium (EMEM) supplemented with <NUM>% fetal calf serum (FCS), L-glutamine (<NUM>), gentamicin (<NUM>µg/ml) and vitamin MEM solution (Sigma) in a humid atmosphere of <NUM>% CO<NUM> at <NUM>.

The starting parental virus originated in a sheep experimentally infected with wild strain <NUM>/<NUM> isolate of the VRVR <NUM>/<NUM> virus (parental virus) (Borrego et al. , <NUM>); (Busquets et al. The virus was re-isolated from infected sheep plasma and cultured in a C6/<NUM> mosquito cell line (ATCC CRL-<NUM>). Assays to quantify plate-forming units (pfu) were performed in semi-solid medium including <NUM>% carboxymethylcellulose (CMC; Sigma). pfu units are used in virology to describe the number of virus particles capable of forming plates per unit volume. Viral particles that are defective or that fail to infect their target cell will not form a plaque, and are not counted.

RNA was extracted from the supernatants of the infected cells using the Speedtools RNA virus extraction kit (Biotools B&M Labs, S. , Madrid, Spain) according to the manufacturer's instructions. Reverse transcription polymerase chain reaction (RT-PCR) was performed using SuperScript IV reverse transcriptase (Invitrogen) and Phusion high-fidelity DNA polymerase (Finnzymes), as directed by the manufacturers, using primers designed to amplify the L segments (Table 1A), M segments (Table 1B), and S segments (Table 1C) of the viral genome. Table 1D shows the primers used for the amplification of the genomic ends by the RACE technique. The overlapping PCR amplicons were purified and subjected to automatic Sanger sequencing. The Laser Gene software was used for the analysis of the results.

Groups of transgenic 129Sv/Ev IFNAR-/- mice of <NUM>-<NUM> months of age or wild 129Sv/Ev mice of <NUM> months of age (B&K Universal) were inoculated intraperitoneally with different doses of the viruses, as indicated in the corresponding experiments. After viral inoculation, animals were monitored daily for weight and development of clinical signs, including signs on the coat, hunched posture, reduced activity, and conjunctivitis. At the indicated times, the animals were bled through the maxillary vein. Serums were inactivated by heat at <NUM> for <NUM> minutes and maintained at -<NUM> until use. All mice were housed in a BSL-<NUM> containment area with food and water supplied ad libitum. All experimental procedures were managed in accordance with the guidelines of the EU Directive <NUM>/<NUM>/EU for animal experiments and protocols approved by the Biosafety and Ethics Committees for Animal Experiments of INIA (EAEC permit codes <NUM>/<NUM> and CBS <NUM>/<NUM>).

Two ewes were inoculated with a dose of <NUM><NUM> pfu of FMH-P8 and compared to three additional ewes inoculated with virus <NUM>/<NUM> (control group). One sheep from each group was slaughtered on day <NUM> post infection to analyze the degree of liver injury caused by the infection. Rectal temperature was taken daily after the challenge and blood and serum samples were taken daily for at least <NUM> consecutive days. The blood and serum samples obtained were used to perform a quantification of liver transaminase levels, as well as in vitro tests for neutralizing antibodies.

Neutralization tests were performed on <NUM>-well culture plates following the test prescribed by the OIE (Chapter <NUM>. <NUM> OIE Terrestrial Manual <NUM>). Briefly, sera were diluted in base <NUM> from an initial <NUM>/<NUM> dilution in DMEM medium containing <NUM>% fetal bovine serum, mixed with an equal volume of infectious virus containing <NUM> TCID<NUM> (<NUM>% infectious tissue culture dose) and incubated <NUM> minutes at <NUM>. A suspension of Vero cells was then added and the plates were incubated for <NUM> days. The monolayers were controlled for the development of cytopathic effect, fixed and stained. Each sample was tested in <NUM> wells. The titer is expressed as the last dilution of serum that gives a reduction of the cytopathic effect in <NUM>% of the wells.

For the detection of antibodies against the nucleoprotein (N protein), an ELISA assay was performed. The ELISA plates were adsorbed with <NUM> ng/well of recombinant N-protein produced in E. coli and purified, diluted in carbonate buffer (pH <NUM>). After blocking with <NUM>% skimmed milk-PBS-<NUM>% Tween <NUM>, the sera were analyzed in duplicate in serial dilutions in base <NUM> starting at <NUM>/<NUM>. The bound antibodies were detected with goat antibodies conjugated to horseradish peroxidase (HRP), mouse-HRP anti-IgG(H+L) (BioRad) and the bound conjugate was detected using <NUM>,<NUM>',<NUM>,<NUM>' - tetramethylbenzidine (TMB, Invitrogen/Life Technologies) for <NUM> minutes, followed by a volume of stop solution (3N H<NUM>SO<NUM>). The optical densities were measured at <NUM> (OD<NUM>).

Data analysis was performed with GraphPad prism version <NUM> software.

The parental virus isolate RVFV <NUM>/<NUM> was subjected to serial passages in Vero cells in the presence of <NUM> favipiravir. Viral titration of the culture supernatants indicated that the production of viral progeny progressively decreased, being undetectable in steps <NUM>, <NUM> and <NUM>. However, in steps <NUM> and <NUM> infectivity was recovered with normal viral titers, indicating the generation of a virus resistant to favipiravir, which was called FMH-P8 (from "Favipiravir-Mutagenized Hyperattenuated Passage <NUM>"). The viral production of FMH-P8 virus was analyzed in the presence of different concentrations of favipiravir, obtaining a <NUM>% reduction in viral production at a concentration of <NUM> of favipiravir. These results indicated that FMH-P8 virus was more resistant to favipiravir compared to parental virus.

Overlapping RT-PCR reactions were performed from RNA extracted from infection supernatants of attenuated RVF virus of the invention, FMH-P8, obtained in the previous example. Amplicons were produced in these reactions, covering all <NUM> segments of the viral genome. We proceeded to sequence these amplicons by automatic sequencing (Sanger sequencing). The deduced amino acid sequences were aligned and compared to those of the parental virus RVFV <NUM>/<NUM>. The description of the sequences of the attenuated RVF virus of the invention FMH-P8 and the parental virus RVFV <NUM>/<NUM> are shown in Table <NUM>.

Comparing the sequences of the attenuated RVF virus of the invention FMH-P8 with that of the parental virus RVFV <NUM>/<NUM>, a total of <NUM> nucleotide changes have been found that result in <NUM> amino acid changes. In particular, in the L segment, which encodes the viral polymerase, the target of favipiravir, <NUM> nucleotide changes were identified, with <NUM> amino acid changes. The distribution of all changes found in the <NUM> genomic segments is shown in Table <NUM>.

Nucleotide changes found in segment S led to only <NUM> amino acid substitutions, both in the NSs protein: Val52Ile and Pro82Leu. Pro82 belongs to the second Pro-X-X-Pro motif involved in the nuclear localization of the NSs protein and the activation of Interferon-β (IFN-β). The N nucleoprotein was the only FMH-P8 virus protein that showed an amino acid sequence identical to that of the parental virus, with only two (silent) nucleotide substitutions.

In the sequence corresponding to the M segment of the FMH-P8 virus, a total of <NUM> amino acid substitutions were identified, three in the NSm protein (Arg26Lys, His108Tyr, Glu118Lys), eight in the Gn protein (Arg210Lys-mix-, Asp333Asn, Ala427Thr, Ala432Val, Glu487Gly, His540Lys, Ala582Thr, Val587lle) and four in the Gc protein (Ala950Val, Val1090lle, Ala1116Val and Arg1182Lys). The Arg1182Gly change in Gc has been identified as an attenuation marker for MP-<NUM> virus (Ikegami et al.

Seven amino acid substitutions were identified in the FMH-P8 virus L protein, distributed throughout the sequence. Two changes were located in the N-terminal region of the L protein (Met100Thr and His375Tyr); two in the C-terminal region (Leu1629Phe and Glu2071Lys), the three remaining substitutions (Gly924Ser, Ile1050Val and Ala1303Thr) in the central region of the protein. Positions <NUM> and <NUM> are located within the RdRp core, where the conserved catalytic motifs A to H of the polymerase reside.

Since viral RNA polymerase is known to be a target of favipiravir, the conservation level of mutated residues of FMH-P8 RVF virus located within the catalytic nucleus of RdRp has been evaluated. The L-protein sequences corresponding to <NUM> different strains of RVFV have been compared and several virus species belonging to the genus phlebovirus have also been included (Table <NUM>). Residues Gly924 and Ala1303 were found to be extremely conserved in all viruses included in the alignment. Position <NUM> showed only conservative changes, mainly showing isoleucine (such as parental virus <NUM>/<NUM>) or valine (such as attenuated FMH-P8 RVFV), while the other substituted positions were conserved among the RVFV strains but varied in other viruses of the phlebovirus genus.

The kinetics and total yield of FMH-P8 attenuated RVF virus were analyzed. For comparison purposes, parental virus RVFV <NUM>/<NUM> was also tested before and after spread over <NUM> passes, in the absence of favipiravir. As mosquitoes play an important role in the natural transmission cycle of RVFV, infections were also carried out in the C6/<NUM> cell line derived from Aedes albopictus (ATCC CRL1660).

Infections performed on Vero cells showed similar growth curves for all three viruses (<FIG>). Titration of supernatants collected at different times post infection in several independent experiments showed only small differences between the three viruses. The growth pattern of parental virus RVFV <NUM>/<NUM> after <NUM> passes showed no differences with parental virus RVFV <NUM>/<NUM> prior to such passes. FMH-P8 RVFV grew a little faster and with higher viral production yields <NUM>-<NUM> days post infection, although the differences were not statistically significant (multiple t-test).

Both viral growth and final yield in C6/<NUM> mosquito cells were clearly affected by FMH-P8 RVF virus (<FIG>). Infected C6/<NUM> mosquito cells remain viable for longer in cell culture than Vero cells and the analysis was extended up to <NUM> days. In the insect cells, the growth of the FMH-P8 RVF virus was significantly delayed, with viral titers of <NUM><NUM> pfu/ml to <NUM>-<NUM> days post infection, at least <NUM> log units lower than those produced by the control viruses. The total virus yields at the last points analyzed (<NUM>-<NUM> days post infection), although they reached a titer of <NUM><NUM> pfu/mL, were below those reached by the parental virus RVFV <NUM>/<NUM> (> <NUM><NUM> pfu/mL). No significant changes were found for the parental virus RVFV <NUM>/<NUM> after <NUM> passes with respect to the parental virus RVFV <NUM>/<NUM> before said passes.

The phenotype of the Vero cell plate in the presence of the FMH-P8 RVF virus differed substantially from the parental virus, producing plates smaller than those produced by the RVFV <NUM>/<NUM> parental virus before or after <NUM> passes (<FIG>).

To check in vivo the attenuation of the FMH-P8 RVF virus, an infection experiment was performed using the A129 mouse strain (IFNAR-/-). A129 mice cannot cope with acute viral infection and are highly susceptible to RVFV infection and offer a highly sensitive assessment of FMH-P8 RVFV attenuation.

Different doses of virus were inoculated intraperitoneally to groups of <NUM>-<NUM> mice and were monitored daily for <NUM> weeks to check the development of signs of disease and survival (<FIG>). After <NUM> passes the parental virus RVFV <NUM>/<NUM> caused <NUM>% mortality <NUM> days post inoculation with <NUM><NUM> pfu, while mice inoculated with the same dose of the parental virus RVFV <NUM>/<NUM> before such passes showed a survival rate of <NUM>% (<NUM>/<NUM>). Although these data suggest increased virulence of the parental virus RVFV <NUM>/<NUM> after <NUM> passes, the results were not statistically significant. Higher doses of the parental virus RVFV <NUM>/<NUM> caused death of inoculated animals in the first <NUM> days post infection: <NUM>% in those inoculated with <NUM><NUM> pfu; <NUM>% in those inoculated with <NUM><NUM> pfu, with no survivors at day <NUM>.

In contrast, animals inoculated with the FMH-P8 RVF virus showed survival rates above <NUM>% even with a high challenge dose (<NUM><NUM> pfu), with a significant number of survivors at the end of the experiment: <NUM>/<NUM> (<NUM>%) in those who received <NUM><NUM> pfu and <NUM>/<NUM> (<NUM>%) in those inoculated with <NUM><NUM> pfu. No signs of disease were seen in any of these animals, except for slight weight loss on days <NUM>-<NUM> post infection (<FIG>).

Serum samples collected on day <NUM> (end of experiment) were analyzed by ELISA for the presence of N-nucleoprotein (anti-N) antibodies in survivors, indicative of viral replication (<FIG>). In some animals within the groups that received the lowest viral dose, <NUM><NUM> pfu, anti-N antibodies were undetectable, probably reflecting low or zero levels of viral replication (<NUM>/<NUM> in mice inoculated with RVFV <NUM>/<NUM> virus; <NUM>/<NUM> in mice inoculated with FMH-P8 virus). All animals inoculated with <NUM><NUM> and <NUM><NUM> pfu of FMH-P8 RVF virus, as well as three of the group inoculated with <NUM><NUM> pfu of FMH-P8 RVF virus developed specific anti-N antibodies. The titers of anti-N antibodies showed no significant differences (ordinary one-way ANOVA) within the groups inoculated with FMH-P8 RVF virus, regardless of the dose received.

An in vivo infectivity assay was performed in A129 mice with FMH-P8 RVF virus and with MP-<NUM> live attenuated vaccine (Ikegami et al. The MP-<NUM> vaccine administered in IFNAR-/- mice at the same dose (<NUM><NUM> cfu) causes the death of <NUM>% of the mice of the strain within <NUM> days (<FIG>). These results demonstrate the high vaccine potential of the FMH-P8 RVF virus.

FMH-P8 attenuated RVF virus was assayed in immunocompetent mice. To do this, wild 129Sv/Ev mice were inoculated intraperitoneally with <NUM><NUM> pfu of the FMH-P8 RVF virus, and <NUM> weeks later were challenged with a lethal dose (<NUM><NUM> pfu) of the parental RVF virus <NUM>/<NUM>. After inoculation with FMH-P8 RVF virus, mice showed no signs of disease, not even significant weight variations. In serum samples collected <NUM> days after inoculation (samples prior to lethal challenge with parental virus RVFV <NUM>/<NUM>), seven out of nine mice showed a strong neutralizing antibody response (<FIG>). N-nucleoprotein antibodies were detected in all these samples by indirect ELISA, including two samples that were negative in the neutralization assay, although their anti-N antibody titers were slightly lower (<FIG>). This indicates that the FMH-P8 RVF virus replicated in all inoculated mice at least to an extent sufficient to elicit an immune response. When mice were subjected to a lethal challenge with the virulent strain RVFV <NUM>/<NUM>, <NUM>% of the mice survived to the end of the experiment (<FIG>, <FIG>) without apparent clinical manifestation, including those in which no neutralizing antibody titers had been detected. In contrast, all mice in the control group became ill and died on day <NUM> (<FIG>, <FIG>).

Anti-N antibody titers increased following lethal challenge with parental virus RVFV <NUM>/<NUM> (<FIG>), indicating a booster effect of attenuated virus FMH-P8 RVFV in mice. Taken together, these results show that, despite its highly attenuated phenotype, the FMH-P8 RVF virus could be replicated in immunocompetent 129Sv/Ev mice at levels that allow for the induction of protective immune responses, even when no neutralizing antibodies are detected.

The attenuated FMH-P8 RVF virus was inoculated into ewes to assess its attenuation and immunogenicity in a natural host of the RVF virus. Animals received an elevated dose of <NUM><NUM> pfu of the attenuated FMH-P8 RVF virus and clinical signs were monitored daily and daily sampling was performed. Fever was not recorded in any of the animals on the days immediately following inoculation of the attenuated FMH-P8 RVF virus, and liver enzyme titration did not indicate alterations in sheep inoculated with FMH-P8 unlike control sheep that had been inoculated with parental virus <NUM>/<NUM> which showed a spike in fever from day <NUM> post infection. Even in the absence of clinical signs, seroconversion was observed, reaching a significant titer of neutralizing antibodies at day <NUM> post-inoculation. Although the neutralizing antibody titer obtained with RVFV is lower than that obtained with parental virus <NUM>/<NUM>, it is concluded that said neutralizing antibody titer is significant and sufficient to provide protection to sheep. The results of this example also demonstrate the safety provided by the attenuated FMH-P8 RVF virus and support the vaccine viability of the FMH-P8 RVF virus (<FIG>).

Wild mice <NUM> were inoculated with variants of the RVF virus and subsequently challenged with a lethal dose of the wild strain ZH548 of the RVF virus (a virulent strain).

Four groups of mice were inoculated with either the ZH548 strain of the RVF virus or different variants of the ZH548 strain, as indicated below.

The viraemia values and neutralizing antibody titers correspond to the group means (n=<NUM>, except G1 n=<NUM>).

viraemia: Cq value (quantification cycle) by RT-qPCR technique (reverse transcriptase quantitative polymerase chain reaction).

NEG: Values below the sensitivity level of the test (Cq = <NUM>).

Neutralizing antibody titer = PRNT80 (log10). In group C1, no survivors were recorded at that time post-infection. In groups G1 and B3 all animals were positive, while in group A2 there were <NUM> animals (<NUM>/<NUM>) with values below the limit of detection of the test (dilution <NUM>/<NUM>; log10 = <NUM>). * The indicated mean excludes these <NUM> negative values.

** After the challenge, in group B3, viraemia was detected at day <NUM> in a single animal (<NUM>/<NUM>), with a Cq = <NUM>.

The results of this example demonstrate a very clear virus attenuation effect of three amino acid substitutions in the ZH548 strain of the RVF virus: the Pro82Leu substitution in the protein encoded by the NSs gene of the S segment of the viral RNA and the Gly924Ser and Ala1303Thr substitutions in the RdRp protein of the L segment of the viral RNA.

The sequences of strain ZH548 are accessible through GenBank with the access codes DQ375403 (segment L), DQ380206 (segment M) and DQ380151 (segment S). The description of the sequences of strain ZH548 is shown in Table <NUM>.

A group of two sheep were inoculated with a lethal dose of the RVF virus strain ZH548.

A group of four sheep were inoculated with variant ZH548_L[Gly924Ser]_L[Ala1303Thr]_NSs[Pro82Leu] of the RVF virus (variant with Gly924Ser and Ala1303Thr substitutions in the protein encoded by the RdRp gene of viral RNA segment L and with Pro82Leu substitution in the protein encoded by the NSs gene of viral RNA segment S. Two sheep in this group were challenged with the ZH548 strain of the RVF virus three weeks later. The other two sheep in the group were sacrificed in a short time to compare the possible lesions with the control sheep given the lethal dose of the ZH548 strain of the RVF virus.

Ewes inoculated with the RVF virus strain ZH548_L[G924S/A1303T]_NSs[P82L] produced neutralizing antibodies and showed no lesions compared to those inoculated with the control virus. It was also not possible to detect infectious virus in the blood of immunized sheep compared to control.

Results of this example demonstrate the attenuation conferred by the three substitutions L[Gly924Ser], L[Ala1303Thr] and NSs[P82L]. They also confirm that said variant has the ability to induce an immune response capable of protecting sheep from a challenge with the wild strain ZH548.

The sequence listing free text is reproduced in Table <NUM>.

Claim 1:
Attenuated variant of the Rift Valley Fever virus (RVFV), wherein the substitutions in the RdRp protein encoded by the L segment of the RNA of said variant consists of:
- the amino acid at position <NUM> is serine (L[Gly924Ser]);
- the amino acid at position <NUM> is threonine (L[Ala1303Thr]);
wherein the sequence SEQ ID NO: <NUM> of wild strain <NUM>/<NUM> of the RVF virus or the sequence SEQ ID NO: <NUM> of the wild type ZH548 strain of the RVF virus, are the reference sequences for the numbering of the amino acids of said protein;
and the substitution in the NSs protein encoded by the S segment of the RNA of said variant consists of:
- the amino acid at position <NUM> is leucine (NSs[Pro82Leu]);
wherein the sequence SEQ ID NO: <NUM> of wild strain <NUM>/<NUM> of the RVF virus or the sequence SEQ ID NO: <NUM> of the wild type ZH548 strain of the RVF virus, are the reference sequences for the numbering of the amino acids of said protein.