METHOD OF PRODUCING PHARMACEUTICAL COMPOSITIONS COMPRISING IMMUNOGENIC CHIKUNGUNYA VIRUS CHIKV-DELTA5NSP3
The present invention relates to a process for producing an immunogenic live attenuated Chikungunya virus, as well as pharmaceutical compositions comprising the same.
1 .- 35 . (canceled)
36 . A pharmaceutical composition comprising
(i) CHIKV-Δ5nsP3 particles comprising a genome as defined by the polynucleotide sequence of SEQ ID NO: 1;
(ii) CHIKV-Δ5nsP3 particles having genome that differs from SEQ ID NO: 1 by having sequence heterogeneity at nucleic acid positions 8543, 8882, 9119 and/or 9649 corresponding to G55R, E168K, E247K and A423A (silent) point mutations in the E2 structural protein according to SEQ ID NO: 2; and
(iii) optionally a pharmaceutically acceptable excipient,
wherein 1-50% of the CHIKV-Δ5nsP3 particles in the composition having a genome with heterogeneity at nucleic acid 8543, 8882, 9119 and/or 9649 compared with SEQ ID NO: 1.
37 . The pharmaceutical composition according to claim 36 , wherein at least 30% of the CHIKV-Δ5nsP3 particles present in the composition have a genome with heterogeneities at nucleic acid 8543, 8882, 9119 and/or 9649 compared with SEQ ID NO: 1.
38 . The pharmaceutical composition according to claim 36 , wherein less than 40%, less than 25% or less than 10% of the CHIKV-Δ5nsP3 particles present in the composition have a genome with heterogeneities at nucleic acid 8543, 8882, 9119 and/or 9649 compared with SEQ ID NO: 1.
39 . The pharmaceutical composition according to claim 36 , wherein 5-30% or 10-20% of the CHIKV-Δ5nsP3 particles present in the composition have a genome with heterogeneities at nucleic acid 8543, 8882, 9119 and/or 9649 compared with SEQ ID NO: 1.
40 . The pharmaceutical composition according to claim 36 , wherein at least 50%, at least 75% or at least 90% of the CHIKV-Δ5nsP3 particles present in the composition have a genome corresponding to the nucleic acid sequence of SEQ ID NO: 1.
41 . The pharmaceutical composition according to claim 36 , wherein said pharmaceutical composition comprises an effective amount of CHIKV-Δ5nsP3 particles having a genome corresponding to the nucleic acid sequence of SEQ ID NO: 1, wherein said effective amount is defined as at least 10 3 or at least 10 4 CHIKV-Δ5nsP3 particles having a genome corresponding to the nucleic acid sequence of SEQ ID NO: 1.
42 . A process for producing a pharmaceutical composition of claim 36 , comprising the step of growing CHIKV-Δ5nsP3 particles on host cells in such a way as to minimize the presence of sequence heterogeneities in the viral genome.
43 . The process according to claim 42 , wherein said CHIKV-Δ5nsP3 particles are defined by a polynucleotide sequence of SEQ ID NO: 1 and are passaged on host cells in culture less than five times.
44 . The process according to claim 42 , wherein said CHIKV-Δ5nsP3 particles are defined by a polynucleotide sequence of SEQ ID NO: 1 and are passaged on host cells in culture at most three times.
45 . A method of treating or preventing a chikungunya virus infection in a subject in need thereof, comprising administering an effective amount of the pharmaceutical composition according to claim 36 .
46 . A pharmaceutical composition comprising
(i) CHIKV-Δ5nsP3 particles having a genome as defined by the polynucleotide sequence of SEQ ID NO: 1; and
(ii) CHIKV-Δ5nsP3 particles having a genome with sequence heterogeneity at one or more nucleic acid positions in the E2 protein coding region of SEQ ID NO: 1, wherein said genomic sequence heterogeneity results in G55R, G82R, H99Y, E168K, T230I, H232Y, E247K and/or E247D mutations with respect to the E2 structural protein according to SEQ ID NO: 2; and
(iii) optionally a pharmaceutically acceptable excipient,
wherein 1-50% of the CHIKV-Δ5nsP3 particles in the composition having a genome with sequence heterogeneity resulting in G55R, G82R, H99Y, E168K, T230I, H232Y, E247K and/or E247D mutations with respect to the E2 structural protein according to SEQ ID NO: 2.
47 . The pharmaceutical composition according to claim 46 , wherein at least 30% of the CHIKV-Δ5nsP3 particles present in the composition have a genome with sequence heterogeneity resulting in G55R, G82R, H99Y, E168K, T230I, H232Y, E247K and/or E247D mutations with respect to the E2 structural protein according to SEQ ID NO: 2.
48 . The pharmaceutical composition according to claim 46 , wherein less than 40%, less than 25% or less than 10% of the CHIKV-Δ5nsP3 particles present in the composition have a genome with sequence heterogeneity resulting in G55R, G82R, H99Y, E168K, T230I, H232Y, E247K and/or E247D mutations with respect to the E2 structural protein according to SEQ ID NO: 2.
49 . The pharmaceutical composition according to claim 46 , wherein 5-30% or 10-20% of the CHIKV-Δ5nsP3 particles present in the composition have a genome with sequence heterogeneity resulting in G55R, G82R, H99Y, E168K, T230I, H232Y, E247K and/or E247D mutations with respect to the E2 structural protein according to SEQ ID NO: 2.
50 . The pharmaceutical composition according to claim 46 , wherein at least 50%, at least 75% or at least 90% of the CHIKV-Δ5nsP3 particles present in the composition have the genome according to SEQ ID NO: 1.
51 . The pharmaceutical composition according to claim 46 , wherein said pharmaceutical composition comprises an effective amount of CHIKV-Δ5nsP3 particles having a genome corresponding to the nucleic acid sequence according to SEQ ID NO: 1, wherein said effective amount is defined as at least 10 3 or at least 10 4 CHIKV-Δ5nsP3 particles having a genome corresponding to the nucleic acid sequence according to SEQ ID NO: 1.
52 . A process for producing a pharmaceutical composition of claim 46 , comprising the step of growing CHIKV-Δ5nsP3 particles on host cells in such a way as to minimize the presence of sequence heterogeneities in the viral genome.
53 . The process according to claim 52 , wherein said CHIKV-Δ5nsP3 particles are defined by a polynucleotide sequence of SEQ ID NO: 1 and are passaged on host cells in culture less than five times.
54 . The process according to claim 52 , wherein said CHIKV-Δ5nsP3 particles are defined by a polynucleotide sequence of SEQ ID NO: 1 and are passaged on host cells in culture at most three times.
55 . A method of treating or preventing a chikungunya virus infection in a subject in need thereof, comprising administering an effective amount of the pharmaceutical composition according to claim 36 .