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.

1 . (a) An isolated, purified, synthetic or recombinant Chikungunya virus (CHIKV) mutated capsid protein;
 (b) an isolated, purified, synthetic or recombinant CHIKV nascent structural polyprotein comprising a mutated CHIKV capsid protein;   (c) a recombinant CHIKV genome encoding a mutated CHIKV capsid protein;   (d) a recombinant CHIKV comprising a mutated CHIKV capsid protein;   (e) a live attenuated recombinant CHIKV comprising a mutated CHIKV capsid protein;   (f) a chimeric alphavirus comprising a mutated CHIKV capsid protein;   (g) a live attenuated chimeric alphavirus comprising a mutated CHIKV capsid protein;   (h) an inactivated attenuated recombinant CHIKV comprising a mutated CHIKV capsid protein; or   (i) an inactivated attenuated chimeric alphavirus comprising a mutated CHIKV capsid protein,   wherein the mutated CHIKV capsid protein has at least a mutated nucleolar localization region (NoLS) compared with wildtype CHIKV capsid protein and is incapable or substantially incapable of nucleolar localization, and   wherein charged amino acids of the NoLS of wild-type CHIKV capsid protein required for nucleolar transportation are shifted in position, deleted and/or exchanged for one or more different amino acids.   
     
     
         2 . The protein, polyprotein, genome, recombinant CHIKV, live attenuated recombinant CHIKV, chimeric alphavirus, live attenuated chimeric alphavirus, inactivated attenuated recombinant CHIKV, or inactivated attenuated chimeric alphavirus of  claim 1 , wherein for the mutated CHIKV capsid protein:
 (i) positively charged amino acids of the NoLS of wild-type CHIKV capsid protein required for nucleolar transportation are shifted in position, deleted and/or exchanged for one or more different amino acids;   (ii) positively charged amino acids of the NoLS of wild-type CHIKV capsid protein required for nucleolar transportation are replaced with alanine;   (iii) one or more of amino acid positions 62, 63, 65, 66, 68, 69, 84, 85, 95, 96, 101 and 102 of the NoLS of wild-type CHIKV capsid protein are shifted in position, replaced and/or deleted;   (iv) at least amino acid positions 62, 63, 65, 66, 68, 69, 101 and 102 of the NoLS of wild-111999.8009.US01\ 153031782 type CHIKV capsid protein are shifted in position, replaced and/or deleted;   (v) at least amino acid positions 62, 63, 65, 66, 68, 69, 95, 96, 101 and 102 of the NoLS of wild-type CHIKV capsid protein are shifted in position, replaced and/or deleted;   (vi) at least amino acid positions 84, 85, 95, 96, 101 and 102 of the NoLS of wild-type CHIKV capsid protein are shifted in position, replaced and/or deleted;   (vii) the mutated NoLS sequence comprises the sequence of SEQ. ID NO. 4;   (viii) the mutated NoLS sequence comprises the sequence of SEQ. ID NO. 5;   (ix) the mutated NoLS sequence comprises the sequence of SEQ. ID NO. 6; or   (x) the mutated NoLS sequence comprises the sequence of SEQ. ID NO. 7.   
     
     
         3 . The protein, polyprotein, genome, recombinant CHIKV, live attenuated recombinant CHIKV, chimeric alphavirus, live attenuated chimeric alphavirus, inactivated attenuated recombinant CHIKV, or inactivated attenuated chimeric alphavirus of  claim 1 , wherein for the mutated CHIKV capsid protein at least amino acid positions 62, 63, 65, 66, 68, 69, 101 and 102 of the NoLS of wild-type CHIKV capsid protein are shifted in position, replaced and/or deleted. 
     
     
         4 . The protein, polyprotein, genome, recombinant CHIKV, live attenuated recombinant CHIKV, chimeric alphavirus, live attenuated chimeric alphavirus, inactivated attenuated recombinant CHIKV, or inactivated attenuated chimeric alphavirus of  claim 1 , wherein for the mutated CHIKV capsid protein at least amino acid positions 62, 63, 65, 66, 68, 69, 95, 96, 101 and 102 of the NoLS of wild-type CHIKV capsid protein are shifted in position, replaced and/or deleted; 
     
     
         5 . The protein, polyprotein, genome, recombinant CHIKV, live attenuated recombinant CHIKV, chimeric alphavirus, live attenuated chimeric alphavirus, inactivated attenuated recombinant CHIKV, or inactivated attenuated chimeric alphavirus of  claim 1 , wherein for the mutated CHIKV capsid protein the mutated NoLS sequence comprises the sequence of SEQ. ID NO. 6. 
     
     
         6 . The protein, polyprotein, genome, recombinant CHIKV, live attenuated recombinant CHIKV, chimeric alphavirus, live attenuated chimeric alphavirus, inactivated attenuated recombinant CHIKV, or inactivated attenuated chimeric alphavirus of  claim 1 , wherein for the mutated CHIKV capsid protein the mutated NoLS sequence comprises the sequence of SEQ. ID NO. 7. 
     
     
         7 . The protein, polyprotein, genome, recombinant CHIKV, live attenuated recombinant CHIKV, chimeric alphavirus, live attenuated chimeric alphavirus, inactivated attenuated recombinant CHIKV, or inactivated attenuated chimeric alphavirus of  claim 1 , wherein the chimeric alphavirus of (f), (g) and (i) is selected from the group consisting of 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). 
     
     
         8 . A pharmaceutical preparation comprising the protein, polyprotein, genome, recombinant CHIKV, live attenuated recombinant CHIKV, chimeric alphavirus, live attenuated chimeric alphavirus, inactivated attenuated recombinant CHIKV, or inactivated attenuated chimeric alphavirus of  claim 1 , or a pharmaceutically acceptable derivative thereof, and at least one pharmaceutically acceptable carrier. 
     
     
         9 . The pharmaceutical preparation of  claim 8 , in the form of a vaccine. 
     
     
         10 . A method of (1) preventing a subject from contracting an alphaviral infection naturally; (2) preventing a subject from developing alphaviral disease; (3) eliciting an alphaviral-protective immune response in a subject; or (4) stimulating an anti-alphaviral immune response in a subject, said method comprising the step of administering to the subject the pharmaceutical preparation of  claim 9 . 
     
     
         11 . A method of (1) treating a subject having alphaviral disease, or (2) reducing the severity of alphaviral disease, said method comprising the step of administering to the subject the pharmaceutical preparation of  claim 8 . 
     
     
         12 . An isolated, purified, synthetic or recombinant nucleic acid encoding the protein, polyprotein, genome, recombinant CHIKV, live attenuated recombinant CHIKV, chimeric alphavirus, live attenuated chimeric alphavirus, inactivated attenuated recombinant CHIKV, or inactivated attenuated chimeric alphavirus of  claim 1 . 
     
     
         13 . A method of preparing a recombinant alphavirus, said method comprising the steps of: (1) mutating a capsid protein of a CHIKV to produce a recombinant alphavirus; and (2) enabling the recombinant alphavirus to replicate, wherein the mutated CHIKV capsid protein has at least a mutated nucleolar localization region (NoLS) compared with wildtype CHIKV capsid protein and is incapable or substantially incapable of nucleolar localization, and wherein charged amino acids of the NoLS of wild-type CHIKV capsid protein required for nucleolar transportation are shifted in position, deleted and/or exchanged for one or more different amino acids. 
     
     
         14 . The method of  claim 13 , wherein for the mutated CHIKV capsid protein:
 positively charged amino acids of the NoLS of wild-type CHIKV capsid protein required for nucleolar transportation are shifted in position, deleted and/or exchanged for one or more different amino acids;   positively charged amino acids of the NoLS of wild-type CHIKV capsid protein required for nucleolar transportation are replaced with alanine;   one or more of amino acid positions 62, 63, 65, 66, 68, 69, 84, 85, 95, 96, 101 and 102 of the NoLS of wild-type CHIKV capsid protein are shifted in position, replaced and/or deleted;   at least amino acid positions 62, 63, 65, 66, 68, 69, 101 and 102 of the NoLS of wild-type CHIKV capsid protein are shifted in position, replaced and/or deleted;   at least amino acid positions 62, 63, 65, 66, 68, 69, 95, 96, 101 and 102 of the NoLS of wild-type CHIKV capsid protein are shifted in position, replaced and/or deleted;   at least amino acid positions 84, 85, 95, 96, 101 and 102 of the NoLS of wild-type CHIKV capsid protein are shifted in position, replaced and/or deleted;   the mutated NoLS sequence comprises the sequence of SEQ. ID NO. 4;   the mutated NoLS sequence comprises the sequence of SEQ. ID NO. 5;   the mutated NoLS sequence comprises the sequence of SEQ. ID NO. 6; or   the mutated NoLS sequence comprises the sequence of SEQ. ID NO. 7.   
     
     
         15 . A vaccine or sub-unit vaccine comprising: a recombinant CHIKV comprising a mutated CHIKV capsid protein; a recombinant CHIKV nascent structural polyprotein comprising a mutated CHIKV capsid protein; or, a recombinant CHIKV genome encoding a mutated capsid protein, wherein the mutated CHIKV capsid protein is a nucleolar localisation region (NoLS) mutant of wild-type CHIKV capsid protein incapable or substantially incapable of nucleolar localisation, and wherein charged amino acids of the NoLS of wild-type CHIKV capsid protein required for nucleolar transportation are shifted in position, deleted and/or exchanged for one or more different amino acids. 
     
     
         16 . The vaccine or sub-unit vaccine of  claim 15 , wherein:
 positively charged amino acids of the NoLS of wild-type CHIKV capsid protein required for nucleolar transportation are shifted in position, deleted and/or exchanged for one or more different amino acids;   positively charged amino acids of the NoLS of wild-type CHIKV capsid protein required for nucleolar transportation are replaced with alanine;   one or more of amino acid positions 62, 63, 65, 66, 68, 69, 84, 85, 95, 96, 101 and 102 of the NoLS of wild-type CHIKV capsid protein are shifted in position, replaced and/or deleted;   at least amino acid positions 62, 63, 65, 66, 68, 69, 101 and 102 of the NoLS of wild-type CHIKV capsid protein are shifted in position, replaced and/or deleted;   at least amino acid positions 62, 63, 65, 66, 68, 69, 95, 96, 101 and 102 of the NoLS of wild-type CHIKV capsid protein are shifted in position, replaced and/or deleted;   at least amino acid positions 84, 85, 95, 96, 101 and 102 of the NoLS of wild-type CHIKV capsid protein are shifted in position, replaced and/or deleted;   the mutated NoLS sequence comprises the sequence of SEQ. ID NO. 4;   the mutated NoLS sequence comprises the sequence of SEQ. ID NO. 5;   the mutated NoLS sequence comprises the sequence of SEQ. ID NO. 6; or   the mutated NoLS sequence comprises the sequence of SEQ. ID NO. 7.