Rift valley fever vaccine

Abstract

Certain embodiments are directed to an improved RVF vaccine for human use, and method for producing the same.

Claims

1. A composition comprising a Rift Valley Fever Virus (RVFV) comprising: (i) an L segment, (ii) an M segment having a nucleic acid sequence of SEQ ID NO:4, and (iii) an S segment.

2. The composition of claim 1, wherein the L segment has at least 90% identity to the nucleic acid sequence of SEQ ID NO:1.

3. The composition of claim 1, wherein the S segment has at least 90% identity to the nucleic acid sequence of SEQ ID NO:3.

4. The composition of claim 1, wherein the L segment has 100% identity to the nucleic acid sequence of SEQ ID NO:1.

5. The composition of claim 1, wherein the S segment has 100% identity to the nucleic acid sequence of SEQ ID NO:3.

6. An immunogenic composition comprising the composition of claim 1 and a pharmaceutically acceptable carrier.

7. A host cell expressing the L segment, M segment, and S segment of claim 1.

8. A method of producing an immune response in a mammal comprising administering the RVFV virus of claim 1 to a mammal.

9. The method of claim 8, wherein the mammal is a human.

10. The method of claim 8, wherein the vector is administered by injection, inhalation, or instillation.

11. The method of claim 10, wherein the vector is administered by intramuscular injection.

12. The method of claim 8, wherein the administering confers immunity to Rift Valley Fever caused by Phenuiviridae Phlebovirus.

13. A method of producing an immune response in a mammal comprising administering the immunogenic composition of claim 6 to a mammal.

14. A method of producing the RVFV of claim 1 comprising: (a) culturing a non-rodent cell comprising: (i) cDNA of full-length RVFV L segment, (ii) cDNA of full-length RVFV M segment or RVFV M segment lacking 78 kD/NSm ORF, (iii) cDNA of full-length RVFV S segment, (iv) cDNA of RVFV N open reading frame (ORF), (v) cDNA of RVFV M ORF, and (vi) cDNA of RVFV L ORF, (b) isolating RVFV produced by culturing the non-rodent cell.

15. The method of claim 14, wherein cDNAs (i), (ii), and (iii) are, independently, immediately downstream of human or rhesus monkey RNA polymerase I promoter and upstream of mouse RNA polymerase I terminator.

16. The method of claim 14 wherein expression of ORFs (iv), (v) and (vi) are independently controlled by a chicken β-actin promoter.

Description

DESCRIPTION OF THE DRAWINGS

(1) The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of the specification embodiments presented herein.

(2) FIG. 1. Replication kinetics of RVax-1 in culture cells.

(3) FIG. 2. Illustration of vector maps for some examples of the described nucleotides.

(4) FIGS. 3A-C. The reverse genetics system for Rift Valley fever virus using the precursor rRNA promoter of Macaca mulatta. (FIG. 3A) Schematic representation of the repetitive units of ribosomal DNA (rDNA). The rDNA operon consists of the 5′ external transcribed spacer (ETS), 18S rRNA, internal transcribed spacer (ITS) 1, 5.8S rRNA, ITS2, 28S rRNA, and 3′ETS, and is flanked by the intergenic spacers (IGS). (FIG. 3B) The sequence alignment of precursor rRNA promoter sequences (−234 to −1) of Homo sapience (chromosome 21, GenBank #NC_000021.9, position 8388797 . . . 8389034), Macaca mulatta (chromosome 20, GenBank #NC_041773.1, position 29808263 . . . 29808496), and Vero cells (GenBank #DI217998.1). The precursor rRNA promoter encodes two functional elements (i) upstream control element (UCE: −156 to −107 relative to the transcription start site+1) and (ii) core control element (CCE: −45 to +18). The CCE determines the species-specific strength of promoter activity. (FIG. 3C) Schematic representation of plasmids encoding full-length antiviral-sense L-, M-, or S-segments of RVFV flanked by the precursor rRNA promoter (−234 to −1) of Macaca mulatta and the murine RNA polymerase I terminator.

(5) FIGS. 4A-C. Analysis of the RVFV S-segment RNA replication in transfected Vero cells. The RNA replication of the S-GFP genome, in which the NSs gene of the S-segment was replaced with the GFP gene, was evaluated in transfected Vero cells. Although the S-GFP genome encodes the GFP gene, the GFP protein can be expressed only when GFP mRNA is transcribed from the S-GFP, via the support of viral N and L proteins. To evaluate the background transfection efficiency, Vero cells were separately transfected with the EGFP-N1 plasmid, which can constitutively express GFP proteins. The GFP expression from the EGFP-N1 plasmid occurred in up to 20% of cells based on the image analysis (FIG. 4A, left panel), whereas the GFP expression from the S-GFP replication was much less frequent (FIG. 4A, middle panel). Resuspended transfected Vero cells at 72 hours post transfection were subsequently evaluated for the numbers of GFP- and DAPI-positive cells by an automated cell counter. Vero cells expressing EGFP-N1, S-GFP (with N, L, and GnGc proteins), or S-GFP (without N, L, and GnGc proteins) showed GFP signals in 18.3%, 0.47%, or 0% population, respectively (FIG. 4B). Northern blot analysis of total RNA was performed to evaluate the L, M, and S-segment RNA replication in transfected Vero cells (FIG. 4C). Vero cells were mock-transfected or transfected with pProK-sPI-vS(+), pProK-sPI-vM(+), pProK-sPI-vL(+), pCAGGSK-vN, pCAGGSK-vL, or pCAGGSK-vG. Without protein expression plasmids, only the positive-sense S-segment RNA could be visualized, while positive-sense M- or L-segment were too faint to be visualized. Co-expression of N and L proteins with or without GnGc proteins led to accumulations of negative-sense S and M segments, whereas the negative-sense L segment was still not detectable in the analysis.

(6) FIG. 5. Rescue of rMP-12 and RVax-1 infectious clones from Vero cells. The recovery of infectious clones of parental recombinant RVFV MP-12 strain (rMP-12) and RVax-1 was performed using Vero cells. Six different wells (#1 to #6) were separately transfected with pProK-sPI-vS(+), pProK-sPI-vM(+), pProK-sPI-vL(+), pCAGGSK-vN, pCAGGSK-vL, and pCAGGSK-vG. Virus titers in culture supernatants and the appearance of cytopathic effect (CPE) in monolayers were analyzed for 16 days post transfection (dpt). Since the 6-well plate became 100% confluent by 72 hpt, cells were re-spread from a well in 6-well plate into a 10 cm dish at 72 hpt. By 7 dpt, all plates showed 10 to 15 small plaque-like foci on monolayers. All foci disappeared gradually in following days in the wells #1 and #3 for rMP-12 or #2, #3, #4, #5, and #6 for RVax-1. The remaining wells #2, #4, #5, and #6 for rMP-12 and #1 for RVax-1 showed a few foci increasing in size, which eventually led to widespread CPE such as rounding or detachment of cells in the monolayers.

(7) FIGS. 6A-E. Generation and characterization of RVax-1. (FIG. 6A) The genome structure of RVax-1. The RVax-1 encodes a truncation in the M-segment (Δ21-384), which abolishes the expression of the NSm and 78 kD proteins. The RVax-1 encodes 73, 167, or 326 silent mutations in the S-, M-, or L-segment. (FIG. 6B) The replication kinetics of rMP-12 (parental recombinant MP-12 strain) and RVax-1 are shown as plaque-forming unit (PFU)/ml. Vero cells (ATCC CCL-81) were infected with rMP-12 or RVax-1 at MOI 0.01. Both stock viruses were rescued from Vero cells and amplified once in Vero cells. After 1 hr incubation at 37° C. with 5% CO.sub.2, cells were washed three times with media. Cells were then incubated at 35° C. with 5% CO.sub.2, up to 96 hpi. Virus titers were measured via the plaque assay using Vero cells. (FIG. 6C) Plaques of rMP-12 or RVax-1, which were stained with crystal violet. (FIG. 6D) Western blot analysis of Vero cells infected with rMP-12 or RVax-1 at MOI 5 confirmed a lack of NSm/78 kD protein expression in cells infected with RVax-1. GAPDH was used as a loading control. (FIG. 6E) Cell viability of Vero cells mock-infected or infected with rMP-12 or RVax-1 (MOI 5) was analyzed by MTT assay. Due to a lack of NSm proteins, RVax-1 could induce cell death earlier than parental rMP-12.

(8) FIGS. 7A-C. Genetic stability of RVax-1 in serial passages in Vero cells. The parental rMP-12 or RVax-1 were serially passaged in Vero cells at 35° C. with 5% CO.sub.2, to characterize the genetic stability of silent mutations. (FIG. 7A) The MOI at each passage in Vero cells is shown. The range of MOI was 0.004 to 0.037. (FIG. 7B) Northern blot analysis of viral RNA in infected Vero cells at passages 2, 5, and 10. Membranes were reacted with a mixture of RNA probes detecting negative-sense (upper panel) or positive-sense (bottom panel) L-, M-, and S-segment RNA. (FIG. 7C) The RNA-seq analysis of total RNA from Vero cells infected with rMP-12 or RVax-1 was performed with the support of the UTMB NGS core (Director: Steven G. Widen, PhD). None of silent mutations unique to the RVax-1 (n=566) were not changed during 10 passages in Vero cells. The threshold of the variant detection was 1%. The genetic instability of NSs gene ORF was, however, found in both rMP-12 and RVax-1 during their serial passages in Vero cells (data not shown).

(9) FIGS. 8A-F. Immunogenicity and protective efficacy of RVax-1 in mice. Inbred C57BL/6 mice (6-week-old, M/F=1:1, n=10 per group) were intramuscularly (i.m.) mock-vaccinated with PBS or i.m. vaccinated with 1×10.sup.5 PFU dose of rMP-12 (control) or RVax-1. Sera were collected at 3, 21, 70, and 98 days post vaccination (dpv). All mice were then intraperitoneally (i.p.) challenged with 1×10.sup.3 PFU dose of pathogenic recombinant RVFV strain ZH501 (rZH501). Mice were monitored for health status and body weight for 21 days post challenge (dpc). (FIG. 8A) Schematic representation of mouse challenge experiment. (FIG. 8B) The levels of viremia at 3 dpv were measured in sera collected from mice vaccinated with rMP-12 or RVax-1. (FIG. 8C) Body weight changes of mock-vaccinated or vaccinated mice are shown for 121 days period. Body weight was normalized to that at Day 0. The means±standard errors of body weight changes per group are shown. (FIG. 8D) The percentage of mouse survival after the challenge with RVFV rZH501 strain. (FIG. 8E) Neutralizing antibody titers of mice vaccinated with rMP-12 or RVax-1 were measured by the Plaque Reduction Neutralizing Antibody Test (PRNT) 80 using MP-12 strain. (FIG. 8F) Anti-RVFV N IgG titers were measured by IgG-ELISA coated with recombinant RVFV N antigen purified with His-tag.

DESCRIPTION

(10) The following discussion is directed to various embodiments of the invention. The term “invention” is not intended to refer to any particular embodiment or otherwise limit the scope of the disclosure. Although one or more of these embodiments may be preferred, the embodiments disclosed should not be interpreted, or otherwise used, as limiting the scope of the disclosure, including the claims. In addition, one skilled in the art will understand that the following description has broad application, and the discussion of any embodiment is meant only to be exemplary of that embodiment, and not intended to intimate that the scope of the disclosure, including the claims, is limited to that embodiment.

(11) I. Rift Valley Fever Virus (RVFV)

(12) Rift Valley Fever Virus (RVFV) is a virus belonging to the family Phenuiviridae and genus Phlebovirus. RVF virus has a single-stranded, negative-sense genome composed of three genome segments, S, M and L. The S segment is an ambisense genome segment, meaning it encodes proteins in both the positive-sense and negative-sense orientations. The RVFV genome encodes both structural and non-structural proteins. A “structural” protein is a protein found in the virus particle, whereas a “non-structural” protein is only expressed in a virus-infected cell. RVFV structural proteins include nucleoprotein (NP or N, used interchangeably), two glycoproteins (Gn and Gc) and the viral RNA-dependent RNA polymerase (L protein). Non-structural RVF virus proteins include NSs, NSm and the 78 kD protein. As used herein, a “full-length” RVFV genome segment is one containing no deletions. Full-length genome segments can contain mutations or substitutions, but retain the same length as the wild-type virus. A “complete deletion” of an ORF of a RVFV genome segment means either every nucleotide encoding the ORF is deleted from genome segment, or nucleotides encoding the ORF are deleted such that no proteins are translated from the ORF.

(13) Further provided is a collection of plasmids comprising (i) a plasmid encoding a full-length anti-genomic copy of the L segment of RVF virus; (ii) a plasmid encoding a full-length anti-genomic copy of the M segment of RVF virus, or an anti-genomic copy of the M segment of RVF virus comprising a deletion of the 78 kD/NSm ORF; and (iii) a plasmid encoding an anti-genomic copy of the S segment of RVF virus. In some embodiments, the plasmids further comprise at least one promoter (e.g., RNA polymerase I promoter or a chicken β actin promoter) and one terminator (e.g., RNA polymerase I terminator).

(14) II. Vaccines or Immunogenic Compositions

(15) Provided are immunogenic compositions comprising the recombinant RVFV described herein and a pharmaceutically acceptable carrier. Suitable pharmaceutical carriers are described herein and are well known in the art. The pharmaceutical carrier used depends on a variety of factors, including the route of administration. In one embodiment, the immunogenic composition further comprises an adjuvant. The adjuvant can be any substance that improves the immune response to the recombinant RVFV.

(16) Provided herein are recombinant RVFVs, wherein the genome of the recombinant RVF viruses comprise (i) a full-length L segment (e.g., SEQ ID NO:1); (ii) a full-length M segment (e.g., SEQ ID NO:2) or an M segment comprising a deletion of the NSm open reading frame (ORF) (e.g., SEQ ID NO:4); and (iii) an S segment (SEQ ID NO:3).

(17) Certain embodiments are directed to vaccines or immunogenic compositions comprising one or more of the RVFVs described herein. In one embodiment, the present invention features vaccines or immunogenic compositions comprising a RVFV as described herein.

(18) The RVFV can be administered in vivo, for example where the aim is to produce an immunogenic response in a subject. A “subject” in the context of the present invention may be any animal, such as livestock, or humans. In some embodiments it may be desired to express nucleotides of the invention in a producer cell line.

(19) For such in vivo applications the RVFV can be administered as a component of an immunogenic composition which may comprise the RVFV in admixture with a pharmaceutically acceptable carrier. The immunogenic compositions of the invention are useful to stimulate an immune response against RVFV and may be used as one or more components of a prophylactic or therapeutic vaccine against RVFV for the prevention, amelioration or treatment of Rift Valley Fever.

(20) The compositions of the invention may be injectable suspensions, solutions, sprays, lyophilized powders, syrups, elixirs and the like. Any suitable form of composition may be used. To prepare such a composition, a RVFV having the desired degree of purity is mixed with one or more pharmaceutically acceptable carriers and/or excipients. The carriers and excipients must be “acceptable” in the sense of being compatible with the other ingredients of the composition. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to, water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, or combinations thereof, buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptide; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG). The immunogenic compositions may contain additional substances, such as wetting or emulsifying agents, buffering agents, or adjuvants to enhance the effectiveness of the vaccines (Remington's Pharmaceutical Sciences, 18th edition, Mack Publishing Company, (ed.) 1980).

(21) Adjuvants may also be included. Adjuvants include, but are not limited to, mineral salts (e.g., AlK(SO.sub.4).sub.2, AlNa(SO.sub.4).sub.2, AlNH(SO.sub.4).sub.2, silica, alum, Al(OH).sub.3, Ca.sub.3(PO.sub.4).sub.2, kaolin, or carbon), polynucleotides with or without immune stimulating complexes (ISCOMs) (e.g., CpG oligonucleotides, poly IC or poly AU acids, polyarginine with or without CpG (also known in the art as IC31), JuvaVax™, certain natural substances (e.g., wax D from Mycobacterium tuberculosis, substances found in Cornyebacterium parvum, Bordetella pertussis, or members of the genus Brucella), flagellin (Toll-like receptor 5 ligand, saponins such as QS21, QS17, and QS7, monophosphoryl lipid A, in particular, 3-de-O-acylated monophosphoryl lipid A (3D-MPL), imiquimod (also known in the art as IQM and commercially available as Aldara®; and the CCR5 inhibitor CMPD167.

(22) Suitable dosages of the immunogens in the immunogenic composition of the invention may be readily determined by those of skill in the art. For example, the dosage of the immunogens may vary depending on the route of administration and the size of the subject. Suitable doses may be determined by those of skill in the art, for example by measuring the immune response of a subject, such as a laboratory animal or a subject, using conventional immunological techniques, and adjusting the dosages as appropriate. Such techniques for measuring the immune response of the subject include but are not limited to, chromium release assays, tetramer binding assays, IFN-γ ELISPOT assays, IL-2 ELISPOT assays, intracellular cytokine assays, and other immunological detection assays, e.g., as detailed in the text “Antibodies: A Laboratory Manual” by Ed Harlow and David Lane.

(23) When provided prophylactically, the immunogenic compositions of the invention are ideally administered to a subject in advance of infection, or evidence of infection, or in advance of any symptom due to infection, especially in high-risk subjects and/or during identified viral breakouts. The prophylactic administration of the immunogenic compositions may serve to provide protective immunity of a subject against RVFV infection or to prevent or attenuate the progression of RVFV in a subject already infected with RVFV. When provided therapeutically, the immunogenic compositions may serve to ameliorate and treat Rift Valley Fever symptoms and are advantageously used as soon after infection as possible, preferably before appearance of any symptoms of Rift Valley Fever but may also be used at (or after) the onset of the disease symptoms.

(24) The immunogenic compositions may be administered using any suitable delivery method including, but not limited to, intramuscular, intravenous, intradermal, mucosal, and topical delivery. Such techniques are well known to those of skill in the art. More specific examples of delivery methods are intramuscular injection, intradermal injection, and subcutaneous injection. However, delivery need not be limited to injection methods. Further, delivery of DNA to animal tissue has been achieved by cationic liposomes. Alternatively, delivery routes may be oral, intranasal or by any other suitable route. Delivery may also be accomplished via a mucosal surface such as the anal, vaginal or oral mucosa. Immunization schedules (or regimens) are well known for animals (including humans) and may be readily determined for the particular subject and immunogenic composition. Hence, the immunogens may be administered one or more times to the subject. Preferably, there is a set time interval between separate administrations of the immunogenic composition. While this interval varies for every subject, typically it ranges from 10 days to several weeks, and is often 2, 4, 6 or 8 weeks. For humans, the interval is typically from 2 to 6 weeks. The immunization regimes typically have from 1 to 6 administrations of the immunogenic composition, but may have as few as one, two, three, or four. The methods of inducing an immune response may also include administration of an adjuvant with the immunogens. In some instances, annual, biannual or other long interval (5-10 years) booster immunization may supplement the initial immunization protocol.

(25) Methods may also include a variety of prime-boost regimens. In these methods, one or more priming immunizations are followed by one or more boosting immunizations. The actual immunogenic composition may be the same or different for each immunization and the type of immunogenic composition (e.g., containing protein or expression vector), the route, and formulation of the immunogens may also be varied. One useful prime-boost regimen provides for two priming immunizations, four weeks apart, followed by two boosting immunizations at 4 and 8 weeks after the last priming immunization.

(26) Certain embodiments of the invention provide methods of inducing an immune response against RVFV in a subject by administering an immunogenic composition described herein one or more times to a subject to induce a specific immune response in the subject. Such immunizations may be repeated multiple times at time intervals of at least 2, 4 or 6 weeks (or more) in accordance with a desired immunization regime.

(27) The immunogenic compositions of the invention may be administered alone, or may be co-administered, or sequentially administered, with other immunogens and/or immunogenic compositions, e.g., with “other” immunological, antigenic, vaccine, or therapeutic compositions thereby providing multivalent or “cocktail” or combination compositions of the invention and methods of employing them. Again, the ingredients and manner (sequential or co-administration) of administration, as well as dosages may be determined taking into consideration such factors as the age, sex, weight, species and condition of the particular subject, and the route of administration.

(28) It is to be understood and expected that variations in the principles of invention as described above may be made by one skilled in the art and it is intended that such modifications, changes, and substitutions are to be included within the scope of the present invention. The dose of the vaccine may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of antibody to elicit a desired response in the individual. Dosage regime may be adjusted to provide the optimum therapeutic response. The dose of the vaccine may also be varied to provide optimum preventative dose response depending upon the circumstances.

(29) Further provided is a method of immunizing a subject against RVF virus infection, comprising administering to the subject an immunogenic composition disclosed herein. In one embodiment, the subject is livestock. Livestock includes, but is not limited to sheep, goats, camels, and cattle. In another embodiment, the subject is a human. In one example, the immunogenic composition is administered in a single dose. In another embodiment, the immunogenic composition is administered in multiple doses, such as two, three or four doses. When administered in multiple doses, the time period between doses can vary. In some cases, the time period is days, weeks or months. The immunogenic composition can be administered using any suitable route of administration. Generally, the recombinant RVFV are administered parenterally, such as intramuscularly, intravenously or subcutaneously.

(30) III. Use of Recombinant RVF Viruses

(31) Recombinant RVFV generated using the reverse genetics system described herein can be used for both research and therapeutic purposes. Such recombinant RVFV can be used as vaccines to prevent infection of livestock and humans with wild-type RVF virus. The recombinant RVFV described herein can also be used as live-virus research tools, particularly those viruses that incorporate reporter genes, for instance a fluorescent protein such as GFP. For example, these viruses can be used for high-throughput screening of antiviral compounds in vitro.

(32) The enhanced safety, attenuation, and reduced possibility of virulent RVFV (via either RVF virus polymerase nucleotide substitution or gene segment reassortment with field-strains) while maintaining overall vaccine efficacy. A high level of protective immunity can be induced by a single dose of the rRVF viruses disclosed herein.

(33) IV. Kits

(34) Certain embodiments are directed to for example for preventing or treating an infection. For example, a kit may comprise one or more pharmaceutical compositions or vaccines as described above and optionally instructions for their use. In still other embodiments, the invention provides kits comprising one or more pharmaceutical compositions or vaccines and one or more devices for accomplishing administration of such compositions.

(35) Kit components may be packaged for either manual or partially or wholly automated practice of the methods described herein. In other embodiments involving kits, it is contemplated that a kit includes compositions described herein, and optionally instructions for their use. Such kits may have a variety of uses, including, for example, imaging, diagnosis, therapy, and other applications.