ARTHROGENIC ALPHAVIRUS VACCINE

Abstract

The invention relates to a vaccine comprising live attenuated recombinant alphavirus comprising mutated capsid protein. The invention also relates to a method of preventing a subject from contracting an alphaviral infection that would otherwise produce clinical signs of disease. In an embodiment the mutated capsid protein is Chikungunya virus (CHIKV) capsid protein having a mutated nucleolar localisation signal/sequence (NoLS), preferably the mutant NLS 101/95.

Claims

1-50. (canceled)

51. A vaccine selected from the group consisting of: (a) an isolated, purified, synthetic or recombinant alphaviral mutated capsid protein; (b) an isolated, purified, synthetic or recombinant alphaviral nascent structural polyprotein comprising a mutated capsid protein; (c) a recombinant alphaviral genome encoding a mutated capsid protein; (d) a recombinant alphavirus comprising mutated capsid protein; (e) a live attenuated recombinant alphavirus comprising mutated capsid protein; (f) a chimeric alphavirus comprising mutated capsid protein; and (g) a live attenuated chimeric alphavirus comprising mutated capsid protein.

52. The vaccine of claim 51, wherein, the mutated capsid protein is derived from an alphavirus selected from the group consisting of Chikungunya virus (CHIKV), Ross River virus (RRV), Barmah Forest virus (BFV), O'nyong-nyong virus (ONNV), Mayaro virus (MAYV), Sindbis virus group (causing Pogosta disease, Ockelbo disease and Karelian fever), and Semliki Forest virus (SFV).

53. The vaccine of claim 51, wherein the alphaviral genome or alphavirus of (c) to (g) is selected from the group consisting of Chikungunya virus (CHIKV), Ross River virus (RRV), Barmah Forest virus (BFV), O'nyong-nyong virus (ONNV), Mayaro virus (MAYV), Sindbis virus group (causing Pogosta disease, Ockelbo disease and Karelian fever), and Semliki Forest virus (SFV).

54. The vaccine of claim 51, wherein the mutated capsid protein is incapable or substantially incapable of nucleolar localisation.

55. The vaccine of claim 51, wherein the mutated capsid protein enables assemblage of a nucleocapsid core of a recombinant alphavirus but substantially reduces the ability of the recombinant alphavirus to replicate.

56. The vaccine of claim 51, wherein the mutated capsid protein causes a defect in infectious virus particle formation, causing reduction and delay of release of viral progeny.

57. The vaccine of claim 51, wherein the mutated capsid protein has at least a mutated nucleolar localisation region (NoLS) compared with wildtype capsid protein.

58. The vaccine of claim 51, wherein charged amino acids required for nucleolar transportation of a capsid protein are mutated compared with wildtype capsid protein.

59. The vaccine of claim 51, wherein the mutated capsid protein is a nucleolar localisation region (NoLS) mutant of wild-type CHIKV capsid protein, and wherein the NoLS region of wild-type CHIKV capsid protein is shown in SEQ. ID NO. 3.

60. The vaccine of claim 51, wherein the mutated capsid protein comprises one or more amino acid substitutions, additions and/or deletions present between residues 58 and 110 of wild type CHIKV capsid protein, wherein the corresponding region of wild-type CHIKV capsid protein is shown in SEQ. ID NO. 3.

61. The vaccine of claim 51, wherein the mutated capsid protein comprises one or more of amino acid positions 62, 63, 65, 66, 68, 69, 84, 85, 95, 96, 101 and 102 of wild-type CHIKV capsid protein shifted in position, replaced and/or deleted, and wherein the corresponding region of wild-type CHIKV capsid protein is shown in SEQ. ID NO. 3.

62. The vaccine of claim 51, wherein the mutated capsid protein comprises at least amino acid positions 62, 63, 65, 66, 68, 69, 84, 85, 95, 96, 101 and 102 of wild-type CHIKV capsid protein shifted in position, replaced and/or deleted, and wherein the corresponding region of wild-type CHIKV capsid protein is shown in SEQ. ID NO. 3.

63. The vaccine of claim 51, wherein the mutated capsid protein comprises a mutated nucleolar localisation region (NoLS) sequence selected from the group consisting of SEQ. ID NO. 4, SEQ. ID NO. 5, SEQ. ID NO. 6 and SEQ. ID NO. 7.

64. The vaccine of claim 63, wherein the mutated capsid protein comprises the NoLS sequence of SEQ. ID NO. 4.

65. The vaccine of claim 63, wherein the mutated capsid protein comprises the NoLS sequence of SEQ. ID NO. 5.

66. The vaccine of claim 63, wherein the mutated capsid protein comprises the NoLS sequence of SEQ. ID NO. 6.

67. The vaccine of claim 51, wherein the mutated capsid protein comprises the NoLS sequence of SEQ. ID NO. 7.

68. The vaccine of claim 51, wherein the mutated capsid protein is a nucleolar localisation region (NoLS) mutant of wild-type Semliki Forest Virus capsid protein.

69. The vaccine of claim 68, wherein the mutated capsid protein comprises one or more amino acid substitutions, additions and/or deletions between residues 66 to 83 and/or 92 to 105 of wild type Semliki Forest Virus capsid protein, with the sequence of wild type Semliki Forest Virus capsid protein being shown in SEQ. ID NO. 2.

70. A vaccine or sub-unit vaccine selected from the group consisting of recombinant CHKV or SFV alphavirus comprising mutated capsid protein, recombinant CHKV or SFV alphaviral nascent structural polyprotein comprising a mutated capsid protein, and recombinant CHKV or SFV alphaviral genome encoding a mutated capsid protein, wherein the mutated capsid protein is a nucleolar localisation region (NoLS) mutant of wild-type CHIKV capsid protein incapable or substantially incapable of nucleolar localisation.

Description

BRIEF DESCRIPTION OF FIGURES

[0161] FIG. 1 (A) Partial sequence alignment of wild-type CHIKV capsid protein (WTalso known as Capsid-WT) [SEQ. ID NO. 3] and mutated CHIKV capsid proteins (Capsid-NLS 63/66/69 [SEQ. ID NO. 4], Capsid-NLS 85/95/101 [SEQ. ID NO. 5], Capsid-101 [SEQ. ID NO. 6] and Capsid-101/95 [SEQ. ID NO. 7]also known as Capsid-NoLS). (B) EGFP-tagged capsid protein subcellular localisation. Vero cells separately transfected with pEGFP, pCapsid-WTEGFP, pCapsid-NLS 63/66/69-EGFP, pCapsid-NLS 85/95/101-EGFP, pCapsid-101-EGFP and pCapsid-NoLSEGFP. Vero cells in the top panel show EGFP (enhanced green fluorescent protein) fluorescence. Vero cells in the middle panel show indirect immunofluorescence using nucleolin-specific antibody. Vero cells in the bottom panel show a merge of the top and middle panels.

[0162] FIG. 2 (A) Fluorescence loss in photobleaching (FLIP) analysis performed on live Vero cells transfected with either pCapsid-WTEGFP (labelled WT) or capsid protein mutants pCapsid-101-EGFP (labelled 101) and pCapsid-NoLSEGFP (labelled 101/95). (B) Fluorescence loss in the cytoplasm was assessed over a 280 sec period during continual photobleaching of a section of the nucleus. Fluorescence recovery curves were constructed; data were normalized following the bleaching period so that the initial pre-bleaching was set as 1 and the fully bleached fluorescence intensity was set as 0.

[0163] FIG. 3 Mutation of capsid protein NoLS does not affect capsid protein autoprotease activity. Vero cells were transfected with pEGFP, pCapsid-EGFP, pCapsid-101-EGFP or pCapsid-NoLS-EGFP or plasmids expressing EGFP-tagged WT Capsid-W, Capsid-101-W and Capsid-NoLS-W. EGFP-tagged WT Capsid-W, Capsid-101-W and Capsid-NoLS-W contain the C-terminal tryptophan (W261) required for capsid protein autoproteolytic cleavage. Cells were lysed at 24 h post transfection and cell lysates were analysed for cleavage of EGFP from capsid protein by Western blot and EGFP-specific antibody.

[0164] FIGS. 4 (A) and (B) Subcellular localisation of capsid protein in CHIKV-WT or CHIKV-NoLS infected mammalian and mosquito cells. Vero (A) or C6/36 (B) cells were infected with CHIKV-WT or CHIKV-NoLS at an MOI 1 pfu/cell. Cells were fixed and permeabilised at 24 h post infection and indirect immunofluorescence performed using capsid protein-specific antibodies. Images are representative of at least 6 fields of view. The white bar represents 15 m.

[0165] FIGS. 4 (C), (D) and (E) CHIKV containing the NoLS mutation in capsid protein shows attenuation in vitro. Multistep growth kinetics in BHK-21 (C) and C6/36 (D) cells were obtained by infecting cells with CHIKV-WT or CHIKV-NoLS at an MOI of 0.1 pfu/cell. Supernatants were collected at indicated time points and infectious virus quantified by plaque assay. **, P<0.01; ***, P<0.001 using two-way ANOVA with Bonferroni post-tests. Each symbol represents the meanstandard error for 3 independent experiments. (E) Plaque size (mm) in infected BHK-21 cells. ***, P<0.001 using Student's unpaired t-tests. Each symbol represents the diameter of a single plaque.

[0166] FIG. 5. Viraemia post infection. (A) Graphs showing CHIKV-WT and CHIKV-NoLS viral titers in ankle joint of CHIKV-infected mice at day 3 post-infection, titrated by plaque assay. (B) Graphs showing mRNA expression of inflammatory mediators of CHIKV disease in the ankle joint as analysed by qRT-PCR at day 3 post-infection, the inflammatory mediators being monocyte chemoattractant protein-1 (MCP-1), interferon gamma (IFN) and tumor necrosis factor alpha (TNF). (C) Graph showing CHIKV-WT and CHIKV-NoLS viral titers in serum and ankle joint of CHIKV-infected mice post-infection, titrated by plaque assay.

[0167] FIG. 6 (A) Photographs of CHIKV-WT and CHIKV-NoLS infected C57BL/6 mice at day 3 post-infection. (B) Graph showing CHIKV-induced footpad swelling (widthbreadth), monitored daily.

[0168] FIG. 7 (A) Photographs of challenged/infected CHIKV-NoLS immunised C57BL/6 mice at day 6 post-challenge/infection. The photograph on the left shows a mock challenge, the middle photograph shows CHIKV-NoLS immunised mice challenged with CHIKV-WT (labelled WT Challenge), and the photograph on the right shows CHIKV-NoLS immunised mice challenged with CHIKV-NoLS (labelled NoLS Challenge). (B) Graph showing CHIKV titer of CHIKV-NoLS immunised mice challenged with CHIKV-WT (labelled WT Challenge), CHIKV-NoLS (labelled NoLS Challenge) or mock challenge. (C) Graph showing CHIKV-induced footpad swelling (widthbreadth) of challenged/infected CHIKV-NoLS immunised mice, monitored dailymock challenge (labelled Mock-CHIKV Ch), CHIKV-WT challenge (labelled CHIKV-WT-CHIKV Ch), and CHIKV-NoLS challenge (labelled CHIKV-NoLS-CHIKV Ch).

[0169] FIG. 8. Neutralising capacity of pooled mouse sera collected at day 7, 15 and 30 post-infection with mock challenge (labelled Mock), CHIKV-WT challenge (labelled WT), and CHIKV-NoLS challenge (labelled NoLS). (A) Pooled mouse sera (n>7) was serially diluted and mixed with CHIKV-ZsGreen (MOI 1) for 2 hours before infection of Vero cells for 1 hour and incubation at 37 C. for six hours. Infectivity was measured as % ZsGreen+ve live cells by flow cytometry. (B) Graph showing % ZsGreen+ve live cells at days 7, 15 and 30.

[0170] FIG. 9. Graph showing the ability of CHIKV-NoLS immunised mice (day 30 post immunisation) to protect against the development of viraemia following Ross River virus (RRV) challengemock challenge (labelled Mock Challenge ), CHIKV-WT challenge (labelled WT Challenge), and CHIKV-NoLS challenge (labelled NoLS Challenge). 2way ANOVA with Bonferoni post test; ***P<0.001; *P<0.05.

[0171] FIG. 10. Western blot of capsid protein EGFP-tagged constructs to confirm expression levels in Vero cells. The blot compares the expression levels of pEGFP (labelled GFP), pCapsid-WTEGFP (labelled C prot wt), pCapsid-101-EGFP (labelled 101) and pCapsid-NoLSEGFP (labelled 101/95) using EGFP and Actin-specific antibodies. Actin was used as a load control.

[0172] FIG. 11. Protein sequence alignment of CHIKV wild-type capsid protein [SEQ. ID NO. 1] and SFV wild-type capsid protein [SEQ. ID NO. 2], with amino acids found important for nucleolar transportation shown in underline.

[0173] FIG. 12. CHIKV RNA synthesis is not affected by mutation of the capsid protein NoLS. BHK-21 and C6/36 cells were infected with CHIKV-WT or CHIKV-NoLS at an MOI of 0.1 pfu/cell. The viral genome copy number in the culture supernatants and viral RNA copy number in infected cells were determined by quantitative RT-PCR at the indicated times post infection. (A) BHK-21 cell-associated virus, (B) C6/36 cell-associated virus, (C) BHK-21 culture supernatant, (D) C6/36 culture supernatant. ***, P<0.001 using two-way ANOVA with Bonferroni post-tests. Each bar represents the meanstandard error for 3 independent experiments.

[0174] FIG. 13 Graph of virus titer versus hours post infection showing multistep growth kinetics of CHIKV-WT and CHIKV-NoLS in Vero cells to assess phenotypic stability. **, P<0.01 and ***, P<0.001 using two-way ANOVA with Bonferroni post-tests.

[0175] FIG. 14. CHIKV-NoLS or CHIKV-WT plaque size (mm) plotted against viral passage number in infected Vero cells. Each symbol represents the diameter of a single plaque with the meanstandard deviation.

[0176] FIG. 15. Graph of average CHIKV-NoLS titres before and after 0.22 m filtration. A T-175 flask of P0 CHIKV-NoLS was thawed and the cell suspension was spun at 2000 rpm for 5 min to pellet cell debris. The supernatant (unfiltered) was collected and 10 ml supernatant was filtered through either a 0.22 m pore size hydrophilic Polyethersulfone (PES) membrane or 0.22 m pore size hydrophilic PVDF membrane.

[0177] FIG. 16. (A) Graph showing the effect of temperature on the titre of CHIKV-NoLS during long term storage. (B) Graph showing the effect of temperature on the titre of CHIKV-WT during long term storage. CHIKV-NoLS and CHIKV-WT were diluted to 510.sup.5 pfu/ml and vials stored at 21 C., 4 C., 20 C. and 80 C. At the indicated time points infectious CHIKV-NoLS (A) and CHIKV-WT (B) were quantified by plaque assay.

MATERIALS AND METHODS

[0178] Site-directed Mutagenesis of Capsid Protein

[0179] Capsid protein cDNA was amplified from the ICRES vector (University of Tartu) and cloned into commercially available EGFP (enhanced GFP) plasmid. Site-directed mutants of CHIKV capsid protein were generated using a QuikChange II site-directed mutagenesis kit (Agilent), as per the manufacturer's instructions.

[0180] Western Blot Analysis

[0181] Cells were transfected with pEGFP, pCapsid-WTEGFP (wild type capsid protein), or the capsid protein mutants pCapsid-NLS 63/66/69-EGFP, pCapsid-NLS 85/95/101-EGFP, pCapsid-101-EGFP and pCapsid-NoLSEGFP using Lipofectamine 2000 transfection reagent (Thermo Fisher Scientific) as per the manufacturer's instructions. After 24 h, the cell lysates were analyzed by Western blot analysis using EGFP and Actin-specific antibodies. Actin served as a loading control.

[0182] Confocal Imaging

[0183] Cells were grown on polylysine-treated coverslips. Cells were fixed in 4% paraformaldehyde and permeabilized in 1% Triton X-100. The cells were then blocked in 1% bovine serum albumin (BSA) made in PBS and incubated at 37 C. for 1 h. Primary antibody, nucleolin, was diluted 1:100 in 1% BSA and incubated with the cells for 1 h at 37 C. Texas Red anti-mouse (Vector Laboratories) was diluted 1:500 in 1% BSA and incubated with the cells for 1 h at 37 C. Coverslips were mounted in Vectorshield mounting medium (Vector Laboratories), and staining was visualized on an Olympus FV1000 confocal microscope.

[0184] Live Cell Imaging

[0185] Cells were plated on glass-based 33-mm culture dishes and imaged at 24 h post-transfection using an Upright LSM 510 META Axioplan 2 confocal microscope (Zeiss). Cells were maintained at 37 C. and, during imaging, the cell culture medium was exchanged for CO.sub.2-independent medium (Thermo Fisher). Fluorescence loss was measured using the ROI mean module of the LSM 510 software.

[0186] Flow Cytometry for Cytotoxicity

[0187] Transfected cells were collected and stained with 1 g/mL propidium iodide. Cells were fixed in 4% paraformaldehyde and analysed using the CyAn ADP flow cytometer (Beckman Coutler) with Kaluza software.

[0188] Multi Step Growth Kinetics

[0189] Cells were infected at a multiplicity of infection (MOI) of 0.1 pfu/cell. Following adsorption of virus for 1 h at 37 C., cell monolayers were washed and fresh growth medium was added. Supernatants were collected at indicated time points and infectious virus quantified by plaque assay.

[0190] Ankle Cytokines

[0191] RNA was extracted from mouse tissues using TRIzol (Invitrogen, Melbourne, Victoria, Australia) according to the manufacturer's instructions. 1 g of RNA was reverse transcribed using random primers and reverse transcriptase (Sigma Aldrich, Sydney, Australia) according to the manufacturer's instructions. Quantitative PCR was performed with 50 ng of template cDNA, QuantiTect Primer Assay kits (Qiagen, Hilden, Germany) and SYBR Green Real-time PCR reagent in a CFX96 Touch Real-Time PCR System. Data were normalized to the housekeeping gene HPRT1 and the fold change in messenger RNA (mRNA) expression relative to mock-infected PBS treated samples for each gene was calculated using the Ct method. Briefly, Ct=Ct (RRV-infected)Ct (Mock-infected) where Ct=Ct (gene of interest)Ct (housekeeping geneHPRT). The fold change for each gene was calculated as 2-Ct.

[0192] In Vivo Infections and Disease Monitoring

[0193] 21-day-old C57BL/6 mice were subcutaneously infected with 10.sup.4 pfu CHIKV-WT or CHIKV-NoLS in the ventral/lateral side of the foot and monitored daily for signs of CHIKV-induced footpad swelling (widthbreadth). Animal experiments were approved by the Animal Ethics Committee of Griffith University (GLY/05/15/AEC). All procedures involving animals conformed to the National Health and Medical Research Council Australian code of practice for the care and use of animals for scientific purposes 8th edition 2013.

[0194] Viral Titer Assay

[0195] Ankle joint and serum were collected and assayed for viral titer using plaque assay. Tissue samples were homogenized in 1 mL of PBS and 10-fold serial dilutions of homogenate and sera were added in triplicate to Vero cells. Virus was allowed to incubate for 1 h at 37 C. in a 5% CO.sub.2 incubator before virus was removed and the cells overlaid with OPTI-MEM (Invitrogen, Melbourne, Victoria, Australia) containing 3% FCS and 1% agarose (Sigma Aldrich, Sydney, Australia) and incubated for 48 h in a 5% CO.sub.2 incubator. Cells were fixed in 1% formalin and virus plaques were made visible by staining with 0.1% crystal violet.

[0196] In vivo Immunisation and Challenge

[0197] 21-day-old C57BL/6 mice were subcutaneously immunised with one dose of 10.sup.4 pfu CHIKV-WT or CHIKV-NoLS in the ventral/lateral side of the foot. At 30 days post immunisation, mice were either challenged with 10.sup.4 pfu CHIKV-WT in the ventral/lateral side of the foot and monitored daily for signs of CHIKV-induced footpad swelling or 10.sup.4 pfu Ross River virus (RRV) subcutaneously in the thorax and viraemia measured. Animal experiments were approved by the Animal Ethics Committee of Griffith University (GLY/05/15/AEC). All procedures involving animals conformed to the National Health and Medical Research Council Australian code of practice for the care and use of animals for scientific purposes 8th edition 2013.

[0198] In Vitro Neutralisation

[0199] Pooled mouse sera (n>7) was serially diluted and mixed with CHIKV-ZsGreen (MOI 1) for 2 h before infection of Vero cells for 1 h and incubation at 37 C. for 6 h. Infectivity was measured as % ZsGreen+ve live cells by flow cytometry.

[0200] Oligonucleotides, Plasmids, and Antibodies.

[0201] To generate pCapsid-EGFP, cDNA corresponding to CHIKV capsid protein was amplified by PCR using primers CHIKCprotF (5 GCGGCGCAAGCTTATGGAGTTCATCCCAACCC 3-SEQ. ID NO. 8) and CHIKCprotR (5 CGCGGATCCGACTCTTCGGCCCCCTCG 3-SEQ. ID NO. 9) and cloned into pEGFP-N1 (Takara Bio USA, Inc.). To generate pCapsidW-EGFP containing tryptophan residue required for capsid protein autoproteolytic cleavage at the C-terminal of capsid protein, primers CHIKCprotF and CHIKCprotWR (5 CGCGGATCCGACCACTCTTCGGCC 3-SEQ. ID NO. 10) were used and the obtained fragment was cloned into pEGFP-N1. pSP6-CHIKV-ZsGreen, a plasmid containing cDNA of CHIKV variant expressing the ZsGreen marker protein, was constructed using a full-length infectious cDNA clone of the La Reunion CHIKV isolate LR2006-OPY1 as described previously (Pohjala L, Utt A, Varjak M, Lulla A, Merits A, Ahola T, Tammela P. 2011. Inhibitors of Alphavirus Entry and Replication Identified with a Stable Chikungunya Replicon Cell Line and Virus-Based Assays. Plos One 6). Oligonucleotides used in site-directed mutagenesis are listed in Table 1. Mutants were generated using a QuikChange II site-directed mutagenesis kit (Agilent Technologies USA, Inc.). Antibodies to nucleolin (Santa Cruz Biotech USA, Inc.), EGFP (BD Biosciences, USA), and actin (Santa Cruz Biotech USA, Inc.) were purchased from the respective suppliers. Monoclonal capsid protein antibody was made in house and characterised as described previously (Goh L Y H, Hobson-Peters J, Prow N A, Gardner J, Bielefeldt-Ohmann H, Suhrbier A, Hall R A. 2015. Monoclonal antibodies specific for the capsid protein of chikungunya virus suitable for multiple, applications. Journal of General Virology 96:507-512; Goh LYH, Hobson-Peters J, Prow N A, Baker K, Piyasena T B H, Taylor C T, Rana A, Hastie M L, Gorman J J, Hall R A. 2015. The Chikungunya Virus Capsid Protein Contains Linear B Cell Epitopes in the N- and C-Terminal Regions that are Dependent on an Intact C-Terminus for Antibody Recognition. Viruses-Basel 7:2943-2964. A cocktail of anti-capsid monoclonal antibodies (1.7B2 and 4.1H11) was used for immunofluorescence.

TABLE-US-00001 TABLE1 Oligonucleotidesusedinsite-directedmutagenesis Primername Sequence SEQ.IDNO. K84/85Asense 5 caaaacaacacaaatcaagcggcgcagccacctaaaaagaaac 11 K84/85Aantisense 3 gttttgttgtgtttagttcgccgcgtcggtggatttttctttg 12 K95/96Asense 5 gaaaccggctcaagcggcaaagaagccgggc 13 K95/96Aantisense 3 ctttggccgagttcgccgtttcttcggcccg 14 R101/102Asense 5 gaagccgggcgccgcagagaggatgtgcatgaaaatcg 15 R101/102Aantisense 3 cttcggcccgcggcgtctctcctacacgtacttttagc 16 R62/63Asense 5 gcggtaccccaacagaagccagccgcgaatcggaagaataag 17 R62/63Aantisense 3 cgccatggggttgtcttcggtcggcgcttagccttcttattc 18 K68/69Asense 5 gaatcggaagaatgcggcgcaaaagcaaaaacaacaggcgcc 19 K68/69Aantisense 3 cttagccttcttacgccgcgttttcgtttttgttgtccgcgg 20 RK65/66Asense 5 cagccgcgaatgcggcgaatgcggcgcaaaag 21 RK65/66Aantisense 3 gtcggcgcttacgccgcttacgccgcgttttc 22

[0202] Cell Culture, Transfection and Virus Propagation.

[0203] Vero and BHK-21 cells were cultured in Opti-MEM, Gibco (Thermo Fisher Scientific, Australia), supplemented with 3% fetal calf serum (FCS). C6/36 cells were cultured in Leibovitz's L-15 medium, Gibco (Thermo Fisher Scientific, Australia), supplemented with 10% tryptose phosphate broth and 10% FCS. Plasmid transfections were carried out with Lipofectamine 2000 (Thermo Fisher Scientific, Australia) according to the manufacturer's instructions.

[0204] Mice

[0205] C57BL/6 WT mice were obtained from the Animal Resources Centre (Perth, Australia) and bred in-house. All animal experiments were performed in accordance with the guidelines set out by the Griffith University Animal Ethics Committee. Twenty one-day-old C57BL/6 male and female mice, in equal distribution, were inoculated in the ventral/lateral side of the foot with 10.sup.4 plaque-forming units (pfu) CHIKV-WT or CHIKV-NoLS diluted in PBS to a volume of 20 l. Mock-infected mice were inoculated with PBS alone. Mice were weighed and scored for disease signs every 24 h and sacrificed by CO.sub.2 asphyxiation at experimental end points. CHIKV-induced footpad swelling was assessed by measuring the height and width of the perimetatarsal area of the hind foot, using Kincrome digital vernier callipers. At 30 days post infection mice were challenged in the ventral/lateral side of the foot with 10.sup.4 pfu CHIKV-WT, weighed and scored for disease signs every 24 h and viraemia measured at day 1, 2 and 3 post challenge or 10.sup.4 pfu Ross River virus (RRV) subcutaneously in the thorax and viraemia measured at day 1 and 2 post challenge.

[0206] Neutralisation Assay

[0207] The neutralising capacity of antibody from CHIKV-WT or CHIKV-NoLS infected mice at day 30 post infection was analysed by immunofluorescence-based cell infection assays using Vero cells and CHIKV-ZsGreen. Infectious virus, taken at an amount sufficient for multiplicity of infection (MOI) 0.4, was mixed with diluted (10.sup.1, 10.sup.2 and 10.sup.3), heat-inactivated (56 C. for 30 mins) pooled mouse sera, followed by incubation for 2 h at 37 C. Virus-antibody mixtures were added to Vero cells and incubated at 37 C. for 1 h. The virus inoculum was removed, cells washed with PBS, and Opti-MEM containing 3% FCS added, followed by incubation for 6 h at 37 C. Cells were gently resuspended, stained with LIVE/DEAD Near Infrared cell stain (Thermo Fisher Scientific, Australia) and fixed in 4% paraformaldehyde. Infectivity was measured as % ZsGreen+ve live cells using BD LSR II Fortessa Cell Analyser and quantified with FlowJo software (Treestar USA Inc.).

[0208] Immunofluorescence Microscopy and FLIP (Fluorescence Loss in Photobleaching)

[0209] Cells grown on polylysine-treated coverslips were fixed in 4% paraformaldehyde and permeabilised in 1% Triton X-100. Cells were then blocked in 1% bovine serum albumin (BSA) made in PBS and incubated at 37 C. for 1 h. Primary antibodies were diluted 1:100 in 1% BSA and incubated with the cells for 1 h at 37 C. Alexa Fluor 647 conjugated secondary antibody, Invitrogen (Thermo Fisher Scientific, Australia), was diluted 1:500 in 1% BSA and incubated with the cells for 1 h at 37 C. Coverslips were mounted in Vectorshield mounting medium (Vector Laboratories, USA) and staining was visualised on an Olympus FluoView FV1000 confocal microscope. For FLIP analysis, Vero cells were plated on glass-based 33-mm culture dishes and imaged at 24 h post transfection using an LSM 510 META confocal microscope (Zeiss, Oberkochen, Germany). Cells were maintained at 37 C. and, during imaging, the cell culture medium was exchanged for CO.sub.2-independent medium, Invitrogen (Thermo Fisher Scientific, Australia). Fluorescence loss at the region of interest (ROI) was normalised using the relative fluorescence intensity from the LSM 510 software; initial fluorescence intensity was set as 1.

[0210] In Vitro Viral Replication Kinetics

[0211] BHK-21 and C6/36 cells were infected with CHIKV-WT or CHIKV-NoLS at MOI 0.1, allowed to incubate for 1 h at 37 C. in a 5% CO.sub.2 incubator before virus was removed and the cells washed with PBS and overlaid with Opti-MEM containing 3% FCS. At various times post infection supernatant aliquots were harvested and vial titre measured by plaque assay as outlined below. To determine the virus RNA genome copy number in culture supernatants and virus positive strand RNA copy number in infected cells supernatant was collected and monolayers washed three times in PBS. RNA extraction was performed using TRIzol, Invitrogen (Thermo Fisher Scientific, Australia), according to the manufacturer's instructions. Extracted RNA was reverse transcribed using random nonamer primers and M-MLV reverse transcriptase (Sigma-Aldrich USA, Inc.) according to the manufacturer's instructions. Standard curve was generated using serial dilutions of a full-length infectious cDNA clone of the La Reunion CHIKV isolate LR2006-OPY1. Quantification of viral load was performed using SYBR Green Real-time PCR reagent in 12.5 L reaction volume to detect E1 region. Primers CHIKV E1F (5 CCCGGTAAGAGCGGTGAA 3-SEQ. ID NO. 23) and CHIKV E1R (5 CTTCCGGTATGTCGATG3-SEQ. ID NO. 24) were used to detect CHIKV genomic, antigenomic and subgenomic RNAs. All reactions were performed using a CFX96 Touch Real-Time PCR System. Standard curve was plotted and copy numbers of amplified products were interpolated from standard curve using Graphpad Prism software to determine viral RNA copy number.

[0212] Viral Titre Assay

[0213] Mice were sacrificed at days 1, 2, 3, and 4 post infection with the ankle joint and serum collected and assayed for viral titre using plaque assay. Tissue samples were homogenised in 1 ml of PBS and 10-fold serial dilutions of homogenate and sera were added in triplicate to Vero cells. Virus was allowed to incubate for 1 h at 37 C. in a 5% CO.sub.2 incubator before virus was removed and the cells overlaid with Opti-MEM containing 3% FCS and 1 agarose (Sigma-Aldrich USA, Inc.) and incubated for 48 h in a 5% CO.sub.2 incubator. Cells were fixed in 1% formalin and virus plaques were made visible by staining with 0.1% crystal violet. Results were expressed as pfu/ml or pfu per gram of tissue (pfu/g).

[0214] Quantitative RT-PCR

[0215] RNA was extracted from tissues using TRIzol, Invitrogen (Thermo Fisher Scientific, Australia), according to the manufacturer's instructions. 1 g of total RNA was reverse transcribed using random nonamer primers and M-MLV reverse transcriptase (Sigma-Aldrich USA, Inc.) according to the manufacturer's instructions. Quantitative PCR was performed with 50 ng of template cDNA, QuantiTect Primer Assay kits (Qiagen, Hilden, Germany) and SYBR Green Real-time PCR reagent in a CFX96 Touch Real-Time PCR System using a standard three-step melt program (95 C. for 15 s, 55 C. for 30 s and 72 C. for 30 s). Data were normalised to HPRT1 and the fold change in mRNA expression relative to mock-infected PBS treated samples for each gene was calculated using the C.sub.T method. Briefly, C.sub.T=C.sub.T (Virus infected)C.sub.T (Mock infected) where C.sub.T=C.sub.T (gene of interest)C.sub.T (housekeeping gene). The fold change for each gene is calculated as 2.sup.CT.

[0216] Statistical Analysis

[0217] Two-way ANOVA with Bonferroni post-tests was used to examine in vitro viral growth kinetic data and viraemia. Student's unpaired t-tests were used to analyse quantitative RT-PCR and ankle titers at day 3 post infection. One-way ANOVA with Bonferroni post-tests was used to examine neutralisation assay. A P-value<0.05 was considered to be significant.

[0218] Results and Discussion

Example 1Identification of the CHIKV Capsid Protein Nucleolar Localisation Sequence

[0219] In order to identify the minimal CHIKV capsid protein nucleolar localisation sequence (NoLS), site-directed mutagenesis was performed on recombinant EGFP-tagged CHIKV capsid protein at a region in the N-terminus rich in basic amino acids. Amino acids of the protein were replaced with alanine. Ten amino acids, constituting the minimal NoLS of the CHIKV capsid protein, were identified.

[0220] Four mutant capsid proteins were generated and their sequence differences are shown in the sequence alignment of FIG. 1(A) together with the sequence of wild-type CHIKV Capsid protein (Capsid-WT, labelled as WT). The mutated CHIKV capsid proteins were Capsid-NLS 63/66/69 (labelled NLS 63/66/69), Capsid-NLS 85/95/101 (labelled NLS 85/95/10), Capsid-101 (labelled 101), and Capsid-101/95 (labelled 101/95) which will also be referred to herein as Capsid-NoLS.

[0221] The 10 amino acid changes in Capsid-101/95 are likely to be the minimal residues required for nucleolar localisation of the capsid protein.

Example 2Subcellular Localisation of Mutant CHIKV Capsid Proteins

[0222] The subcellular localisation of the mutant capsid proteins in Vero cells was investigated using confocal microscopy. As seen in FIG. 1(B), Capsid-WT (labelled as WT) localised to the nucleolus. Capsid-101 (labelled as 101) showed a reduced ability to localise to the nucleolus, but was still observed in the nucleolus. Capsid-101/95 (labelled as 101/95) comprised the minimal nucleolar localisation sequence and showed a complete absence from the nucleolus.

[0223] Indirect immunofluorescence, using capsid-specific antibodies, was further used to analyse the subcellular localisation of CHIKV capsid protein in CHIKV-WT and CHIKV-NoLS infected Vero cells and mosquito (Aedes albopictus) derived C6/36 cells. Results show that in CHIKV-WT infected Vero cells capsid protein accumulates in subnuclear bodies reminiscent of the nucleolus at 24 h post infection (FIG. 4A). In CHIKV-NoLS infected Vero cells these punctae are absent. Thus, in the context of the virus the NoLS mutation causes similar disruption of capsid protein subnuclear localisation in infected Vero cells. The NoLS mutation is therefore stable in the virus, resulting in a phenotypic disruption of capsid protein subnuclear localisation.

[0224] Interestingly, in CHIKV-WT infected C6/36 cells capsid protein did not accumulate in subnuclear bodies and was found predominantly in the cytoplasm at 24 h post infection (FIG. 12B). In CHIKV-NoLS infected C6/36 cells capsid protein was also found to predominate in the cytoplasm, similar to the localisation observed in CHIKV-WT infected C6/36 cells. Subnuclear localisation of capsid protein is therefore not a characteristic of CHIKV infection in insect cells and mutation of the NoLS has no effect on the subcellular localisation of capsid protein in infected C6/36 cells.

Example 3Trafficking Ability of Mutant CHIKV Capsid Protein

[0225] Fluorescence loss in photobleaching (FLIP) analysis was performed on live Vero cells transfected with either EGFP-tagged wild-type capsid protein or the capsid protein mutants. FLIP analysis was used to investigate the mobility of Capsid-101/95 (labelled 101/95) compared to Capsid-WT (labelled wt) and mutant Capsid-101 (labelled 101). Fluorescence loss in the cytoplasm was assessed over a 280 sec period during continual photobleaching of a section of the nucleus. This allowed analysis of the nuclear trafficking rates of the mutants. Fluorescence recovery curves were constructed; data were normalized following the bleaching period so that the initial prebleaching was set as 1 and the fully bleached fluorescence intensity was set as 0.

[0226] As seen in FIGS. 2(A) and (B), Capsid-WT showed almost total fluorescence loss after 280 sec, indicating mobility of the protein and trafficking into the nucleus from the cytoplasm. However, mutants Capsid-101 and Capsid-101/95 showed a distinct inability to traffic to the nucleus with fluorescence intensity remaining relatively high. At later times there also appeared to be a cumulative effect of the additional mutations, with Capsid-101/95 more immobile than Capsid-101. These results suggest that mutation of the nucleolar localisation sequence has a major effect on the trafficking ability of capsid protein and perhaps consequently on its subcellular localization.

Example 4Mutation of the NoLS did not Affect Capsid Protein Autoproteolytic Cleavage

[0227] Mutation of the NoLS and its effect on capsid protein autoprotease activity was analysed. Constructs expressing EGFP-tagged WT Capsid-W, Capsid-101-W and Capsid-NoLS-W, containing the C-terminal tryptophan (W261) required for capsid protein autoproteolytic cleavage, were generated. FIG. 3 shows that all constructs lacking W261 residue from the conserved cleavage site were unable to cleave EGFP from capsid protein. However, capsid protease efficiently cleaved EGFP from capsid protein in all constructs that contained W261, including the NoLS mutants of capsid protein (FIG. 3). Thus, mutation of the NoLS has no effect on the autocatalytic protease activity of CHIKV capsid protein.

Example 5CHIKV Containing the NOLs Mutation in Capsid Protein Shows Attenuation In Vitro

[0228] To assess the importance of capsid protein nucleolar localisation on CHIKV replication, the effect of the NoLS mutation in the context of a full-length CHIKV infectious clone-derived virus was examined. CHIKV containing the NoLS mutation in capsid protein (CHIKV-NoLS) was rescued and propagated in Vero cells. Plaque purification of virus and Sanger sequencing of the entire CHIKV genome confirmed the NoLS mutation was maintained in passaged virus in the absence of additional mutations. Indirect immunofluorescence, using capsid-specific antibodies, was used to analyse the subcellular localisation of CHIKV capsid protein in CHIKV-WT and CHIKV-NoLS infected Vero cells and mosquito (Aedes albopictus) derived C6/36 cells.

[0229] Results show that in CHIKV-WT infected Vero cells capsid protein accumulates in subnuclear bodies reminiscent of the nucleolus at 24 h post infection (FIG. 4A). In CHIKV-NoLS infected Vero cells these punctae are absent. Thus, in the context of the virus the NoLS mutation causes similar disruption of capsid protein subnuclear localisation in infected Vero cells. The NoLS mutation is therefore stable in the virus, resulting in a phenotypic disruption of capsid protein subnuclear localisation. Interestingly, in CHIKV-WT infected C6/36 cells capsid protein did not accumulate in subnuclear bodies and was found predominantly in the cytoplasm at 24 h post infection (FIG. 4B). In CHIKV-NoLS infected C6/36 cells capsid protein was also found to predominate in the cytoplasm, similar to the localisation observed in CHIKV-WT infected C6/36 cells. Subnuclear localisation of capsid protein is therefore not a characteristic of CHIKV infection in insect cells and mutation of the NoLS has no effect on the subcellular localisation of capsid protein in infected C6/36 cells.

[0230] To examine the replication kinetics of CHIKV-WT and CHIKV-NoLS in mammalian (BHK-21) and mosquito (C6/36) cells, cells were infected at a multiplicity of infection (MOI) of 0.1 pfu/cell and multistep growth kinetics analysed. CHIKV-NoLS grew to significantly lower titers than CHIKV-WT in both BHK-21 cells (FIG. 4C) and C6/36 cells (FIG. 4D). Furthermore, CHIKV-NoLS had a small plaque phenotype in BHK-21 cells (FIG. 4E), indicating a reduced ability of the virus to spread from the initial site of infection and thus attenuation.

Example 6CHIKV Mutant Capsid ProteinEffect on Disease and Viraemia

[0231] Viral titers in serum and ankle joint, and expression of inflammatory mediators of CHIKV disease, were investigated in mutant Capsid-101/95 infected mice. The results are shown in FIGS. 5(A) to (C) and FIGS. 6(A) and (B). Viral titers in serum and ankle joint, and expression of inflammatory mediators of CHIKV disease in Capsid-101/95 infected mice were significantly reduced compared to Capsid-WT infected mice.

[0232] At day 3 post-infection the expression of inflammatory mediators of CHIKV disease in the ankle joint was analysed by qRT-PCR. Monocyte chemoattractant protein-1 (MCP-1), interferon gamma (IFN) and tumor necrosis factor alpha (TNF) were dramatically under expressed in the ankle tissue of Capsid-101/95 (labelled CHIKV-NoLS) infected mice compared to Capsid-WT (labelled CHIKV-WT) infected mice (see FIG. 5(B)). These soluble immune mediators are key markers of disease progression and severity in CHIKV infected mice. Low expression of these mediators is likely linked to the lack of disease signs in Capsid-101/95 (CHIKV-NoLS) infected mice.

[0233] The amount of infectious virus in the blood and joint tissue is intimately linked to disease severity in CHIKV-infected mice. At day 3 post-infection the amount of live virus in the serum and ankle tissue of Capsid-101/95 (CHIKV-NoLS) infected mice was dramatically reduced compared to Capsid-WT (labelled CHIKV-WT) infected mice (see FIG. 5(A)). Reduced Capsid-101/95 (CHIKV-NoLS) titers are also likely linked to the reduced disease severity in Capsid-101/95 (CHIKV-NoLS) infected mice.

[0234] As seen in FIGS. 6(A) and (B), Capsid-101/95 (CHIKV-NoLS) infected mice showed no signs of acute CHIKV disease. Capsid-101/95 (CHIKV-NoLS) infected mice developed no footpad swelling. The results suggest that Capsid-101/95 (CHIKV-NoLS) is highly attenuated in mice. With low reactogenicity, Capsid-101/95 (CHIKV-NoLS) is a suitable candidate for a live attenuated vaccine.

Example 7Mice Immunised with CHIKV Capsid Protein Show Protective Immunity

[0235] As seen in FIGS. 7(A) to (C), Capsid-101/95 (CHIKV-NoLS) immunised mice were protected from CHIKV disease when challenged with Capsid-WT (labelled CHIKV-WT). Mice immunised with Capsid-101/95 (CHIKV-NoLS) showed no signs of footpad swelling upon challenge with Capsid-WT at day 30 post immunisation (see FIG. 7(C)) and developed no detectable viraemia from days 1-3 post challenge (see FIG. 7(B)). Immunisation with CHIKV-NoLS protected mice from CHIKV challenge for up to 30 days, indicating Capsid-101/95 (CHIKV-NoLS) is immunogenic after one dose and immunity is long lived.

Example 8Sera from CHIKV Capsid Protein Infected Mice Neutralise Infectious CHIKV

[0236] As seen in FIGS. 8(A) and (B), sera from Capsid-101/95 (CHIKV-NoLS) infected mice preincubated with CHIKV was able to neutralise infectious CHIKV in vitro. Antibodies induced by Capsid-101/95 (CHIKV-NoLS) infection efficiently neutralised CHIKV in vitro.

Example 9CHIKV Capsid Protein Immunisation Reduces Peak Viraemia in Ross River Virus Challenged Mice

[0237] As seen in FIG. 9, Capsid-101/95 (CHIKV-NoLS) immunisation reduced peak viraemia in Ross River virus (RRV) challenged mice. Capsid-101/95 (CHIKV-NoLS) immunised mice showed significantly reduced peak (day 2) and early viraemia, day 1 and 2 post challenge, upon challenge with related alphavirus RRV. By reducing viraemia, an indicator of disease outcome, Capsid-101/95 (CHIKV-NoLS) has the potential to offer cross protection against the disease caused by other arthritogenic alphaviruses such as RRV, BFV, SFV, MAYV and/or ONNV.

Example 10CHIKV Capsid Protein Immunisation Reduces Peak Viraemia in Ross River Virus Challenged Mice

[0238] To determine whether the mutant CHIKV capsid proteins Capsid-101 (labelled 101) and Capsid-101/95 (labelled 101/95) expressed at levels similar to the wild-type Capsid-WT (labelled C prot wt), each mutant was transfected into Vero cells, and cell lysates were assayed by Western blot analysis using an GFP-specific antibody and, loading control, Actin antibody. The results showed that both Capsid-101 (labelled 101) and Capsid-101/95 (labelled 101/95) expressed at levels similar to that for the wild-type capsid protein Capsid-WT.

Example 11SFV Capsid Protein NoLS

[0239] The SFV capsid protein is the only other alphavirus capsid protein currently known to localise to the nucleolus. [Favre D1, Studer E, Michel M R. Arch Virol. 1994;137(1-2):149-55. Two nucleolar targeting signals present in the N-terminal part of Semliki Forest virus capsid protein]. Two nucleolar targeting signals have previously been identified in the N-terminal part of the SFV capsid protein.

[0240] FIG. 11 shows a protein sequence alignment of CHIKV wild-type capsid protein and SFV wild-type capsid protein, with CHKV amino acids found important for nucleolar transportation shown in underline. Based on the CHIKV and SFV sequence similarities, a mutated SFV capsid protein could be developed which would not localise within the nucleolusthat is, SFV mutant capsid proteins similar to the mutant CHIKV capsid proteins Capsid-101 and Capsid-101/95 could be developed. Mutation of the NoLS of SFV the capsid protein would attenuate its replication and subsequently act as a SFV vaccine and offer cross protection to other alphaviruses.

Example 12Cross-Reactivity of Alphavirus Antibodies and Chimeric Alphavirus

[0241] A growing body of evidence indicates cross-reactivity of alphavirus antibodies with broadly neutralising effects both in vitro and in vivo. A live attenuated vaccine comprising mutated CHIKV capsid protein is likely to offer cross protection against other arthritogenic alphaviruses, such as RRV, BFV, SFV, ONNV and MAYV, which share a greater degree of structural and genetic homology to CHIKV than other types of alphaviruses. It is less likely to be effective against encephalitic alphaviruses, such as Eastern equine encephalitis virus (EEEV) and Venezuelan equine encephalitis virus (VEEV), which are more distantly related. For this reason, a chimeric alphavirus may be constructed (such as taught by Roy C J, Adams A P, Wang E, Leal G, Seymour R L, Sivasubramani S K, Mega W, Frolov I, Didier P J, Weaver S C. Vaccine. 2013. A chimeric Sindbis-based vaccine protects cynomolgus macaques against a lethal aerosol challenge of eastern equine encephalitis virus.), containing all or part of the structural polyprotein or non-structural polyprotein of an encephalitic alphavirus and, for example, the NoLS mutant capsid protein of CHIKV. On this point, it must be remembered that the entire structural polyprotein of alphaviruses can be swapped from one alpahvirus to another forming chimeric viruses. A chimeric alphavirus containing the CHIKV capsid with the NoLS mutation may offer much better vaccine protection, greater immunogenicity and/or neutralisation to the desired alphavirus, for not only arthritogenic alphaviruses but also encephalitic alphaviruses (subject to a similar level of attenuation).

Example 13Identification of Antibody Subtypes

[0242] The humoral and cellular responses in subjects following vaccination with a mutant CHIKV capsid protein (or other alphaviral capsid protein) can be measured. A global view of antibody responses (i.e. identify different antibody subtypes) can be obtained. It is likely that the live attenuated recombinant vaccine will induce a strong antibody response with some antibody subtypes dominating the response. Identifying these antibody subtypes associated with strong neutralization may have diagnostic value. For instance, the absence of CHIKV-specific antibody subtype in an unvaccinated subject infected with CHIKV may serve as a specific marker of subjects with increased risk of developing severe disease that can progress to chronic disease. Also, any antibody subtype identified as neutralizing can be administered to subjects with high viremia. The vaccine may also influence cellular response and T cell responses which can also be measured.

Example 14Attenuation of CHIKV NoLs in Mammalian and Insect Cells Based on Synthesis of Viral RNA and Virus Gene Copy Number

[0243] To further investigate the attenuation of CHIKV-NoLS in mammalian and insect cells, complementary RT-qPCR analysis of virus genome copy number in the culture supernatants and virus RNA copy number in infected cells was performed. Results suggest that synthesis of viral RNA remains unperturbed by the NoLS mutation in both BHK-21 (FIG. 12A) and C6/36 (FIG. 12B) cells. Furthermore, by 12 h post infection the copy number of CHIKV-NoLS RNAs in infected BHK-21 (FIG. 12A) and C6/36 (FIG. 12B) cells significantly exceeded these in CHIKV-WT infected cells. The difference is potentially due to increased survival of CHIKV-NoLS infected cells allowing prolonged or more efficient synthesis of viral RNA and/or due to reduced competition between viral replicase and capsid protein for binding viral genomic RNAs. However, the genome copy numbers of CHIKV-NoLS in culture supernatants did not show any increase up to 12 h post infection in both BHK-21 (FIG. 12C) and C6/36 (FIG. 12D) cells indicating that no or very little virus was released. This result correlates with the delayed release and reduced titers of infectious CHIKV-NoLS recovered from culture supernatants (FIGS. 4C and 4D). In contrast, for CHIKV-WT infected cells virus titres started to increase at 8 h post infection. Results suggest that, although synthesis of viral RNA in infected cells was not reduced, mutation of the capsid protein NoLS causes a defect in infectious virus particle formation causing reduction and delay of release of viral progeny. Together, these data suggests that subnuclear localisation of CHIKV capsid protein in not a hallmark of infection across different host cells and that the attenuation of CHIKV-NoLS in both mammalian and insect cells is likely the result of a defect in infectious virus particle formation due to the NoLS mutation.

Example 15Multi Step Growth Kinetics of P5 CHIKV-NoLS

[0244] The CHIKV-NoLS vaccine candidate was passaged in Vero cells to assess phenotypic stability. T-75 flasks were grown to 90-95% confluency and infected at a multiplicity of infection (MOI) of 0.1 PFU/cell with CHIKV-NoLS or CHIKV-WT. Following 24 h of incubation at 37 C., culture media was used to infect another flask of Vero cells at MOI of 0.1. After 5 serial passages the growth kinetics of P5 CHIKV-NoLS was compared to CHIKV-WT and CHIKV-NoLS at passage 0 (P0).

[0245] C636 insect cells were infected at a multiplicity of infection (MOI) of 0.1 pfu/cell. Following adsorption of virus for 1 h at 37 C., cell monolayers were washed and fresh growth medium was added. Supernatants were collected at indicated time points and infectious virus quantified by plaque assay.

[0246] As seen in FIG. 13, P5 CHIKV-NoLS shows similar multi step replication kinetics to P0 CHIKV-NoLS. P0 CHIKV-NoLS and P5 CHIKV-NoLS show significantly reduced infectious titers at 24 and 48 hours post infection. This indicates that after 5 passages in Vero cells, replication of CHIKV-NoLS in insect cells remains significantly impaired compared to CHIKV-WT. The attenuated replication phenotype of CHIKV-NoLS remains stable after 5 passages in Vero cells.

Example 16CHIKV NoLs Stability Following Cell Culture PassagePlaque Size

[0247] The CHIKV-NoLS vaccine candidate was passaged in Vero cells to assess phenotypic stability. T-75 flasks were grown to 90-95% confluency and infected at a multiplicity of infection (MOI) of 0.1 PFU/cell with CHIKV-NoLS or CHIKV-WT. Following 24 h of incubation at 37 C., culture media was used to infect another flask of Vero cells at MOI of 0.1. After 10 serial passages Vero plaque sizes were measured and compared to assess stability. Results are shown in FIG. 14.

[0248] CHIKV-NoLS has a small plaque phenotype in mammalian cells, indicating a reduced ability of the virus to spread from the initial site of infection and thus attenuation. To assess its phenotypic stability, CHIKV-NoLS was passaged 10 times in Vero cells at 37 C. using a multiplicity of infection of 0.1 PFU/cell.

[0249] The average plaque size of CHIKV-NoLS remained notably smaller than CHIKV-WT after 10 passages. CHIKV-NoLS also exhibited more homogeneous plaque morphology than CHIKV-WT. Results show that, after 10 passages, CHIKV-NoLS does not revert to a CHIKV-WT plaque phenotype and suggest that the CHIKV-NoLS vaccine candidate remains attenuated in its ability to spread from the initial site of infection.

Example 17Effect of Filtration on CHIKV NoLs

[0250] This example shows that a viable live attenuated vaccine candidate can be produced in large quantities, with little loss of vaccine yield following filtration.

[0251] To examine the loss of CHIKV-NoLS yield during virus propagation filtration, the titer of CHIKV-NoLS before and after 0.22 m filtration was measured. The results are shown in FIG. 15.

[0252] Average CHIKV-NoLS titers before and after filtration: Unfiltered1.5710.sup.6 PFU/ml; PVDF (hydrophilic PVDF membrane 0.22 m pore size)1.0910.sup.6 PFU/ml; and PES (hydrophilic Polyethersulfone membrane 0.22 m pore size)1.1910.sup.6 PFU/ml.

Example 18Effect of Temperature on CHIKV NoLs During Long-Term Storage

[0253] This example shows that a viable live attenuated vaccine candidate can be stored long-term at either 20 C. or 80 C.

[0254] Although able to be stored stably at 80 C., storage of CHIKV-NoLS at 20 C. or 4 C. would reduce the cost of storing CHIKV-NoLS. Storage at 20 C., the temperature of a regular freezer, would also increase the accessibility of CHIKV-NoLS to wider populations.

[0255] The effect of temperature (21 C., 4 C., 20 C. and 80 C.) on long-term CHIKV-NoLS storage is shown in FIG. 16(A) and the effect of temperature (21 C., 4 C., 20 C. and 80 C.) on long-term CHIKV-WT storage is shown in FIG. 16(B).

[0256] No infectious CHIKV-NoLS or CHIKV-WT was detected after 28 days when stored at 21 C., room temperature. After 56 days at 4 C. the titer of CHIKV-NoLS fell to 110 pfu/ml and CHIKV-WT to 450 pfu/ml. The titer of CHIKV-NoLS and CHIKV-WT remained stable after 56 days when stored at either 20 C. or 80 C.

[0257] The above Examples demonstrate the following:

[0258] Mutating the NoLs of CHIKV capsid protein attenuates CHIKV replication in vitro.

[0259] Capsid-101/95 (CHIKV-NoLS) infected mice showed no disease signs, reduced viraemia and reduced expression of inflammatory mediators, making Capsid-101/95 (CHIKV-NoLS) an ideal live attenuated vaccine candidate.

[0260] Capsid-101/95 (CHIKV-NoLS) immunised mice are protected from disease when challenged with CHIKV wild-type capsid protein.

[0261] Capsid-101/95 (CHIKV-NoLS) immunised mice develop CHIKV specific neutralising antibodies.

[0262] Capsid-101/95 (CHIKV-NoLS) is likely to offer alphaviral cross protection, including viraemia upon Ross River virus challenge.

[0263] A viable live attenuated vaccine candidate can be produced in large quantities, with little loss of vaccine yield following filtration.

[0264] A viable live attenuated vaccine candidate can be stored long-term at either 20 C. or 80 C.

[0265] In compliance with the statute, the invention has been described in language more or less specific to structural or methodical features. It is to be understood that the invention is not limited to specific features shown or described since the means herein described comprises preferred forms of putting the invention into effect. The invention is, therefore, claimed in any of its forms or modifications within the proper scope of the appended claims (if any) appropriately interpreted by those skilled in the art.