Assembly activating protein (AAP) and its use for the manufacture of parvovirus particles essentially consisting of VP3
The present invention relates to nucleic acids encoding the novel parvoviral protein “assembly activating protein” (AAP), the encoded polypeptides, methods of producing the polypeptides, antibodies specific for AAP, the use of the nucleic acids for the preparation of the polypeptides, the use of the nucleic acids or the polypeptides for the preparation of the parvoviral particle and methods of producing parvoviral particles essentially consisting of VP3 by providing in addition to the coding sequence of the parvoviral structural protein VP3 a sequence fragment Z/a nucleic acid encoding AAP in the cell and expressing VP3 and fragment Z under control of a rep-independent promoter. Furthermore, the present invention relates to parvoviral particles essentially consisting of VP3 and/oror obtainable by the above method as well as expression cassettes comprising (i) a heterologous promoter and (ii) VP3 coding sequence and/oror fragment Z. The present invention further relates to a medicament, particularly a vaccine, comprising the parvoviral particles or expression cassettes and their use.
1. A parvoviral particle assembled to at least 98% VP3,
i) wherein the VP3 optionally comprises one or more mutation(s),
ii) wherein the VP3 does not contain a heterologous nuclear localization signal, and
iii) wherein the particle does not contain any Rep proteins functional for packaging of virus genomes and unspecific DNA into preformed capsids.
2. The parvoviral particle according to claim 1 , wherein the capsid consists only of VP3.
3. The parvoviral particle according to claim 1 , wherein the mutation(s) of VP3 is/are:
a) one or more mutation(s) selected from the group consisting of one or more deletion(s), one or more insertion(s), one or more substitution(s), and a combination thereof;
b) one or more silent mutation(s);
c) one or more mutations located on the surface of a VP3 virus-like particle (VLP);
d) one or more mutation(s) located at the N-terminus of VP3;
e) one or more insertions at one or more positions selected from the group consisting of I-261, I-266, I-381, I-447, I-448, I-453, I-459, I-471, I-534, I-570, I-573, I-584, I-587, I-588, I-591, I-657, I-664, I-713, and I-716; or wherein two insertions are made at two positions selected from the group consisting of I-261, I-453, I-534, I-570, I-573, and I-587, wherein mutations are made into position 453 in combination with an insertion in position 587 and in combination with an additional mutation;
f) at least one epitope heterologous to the virus, wherein the heterologous epitope is a B-cell epitope and/or wherein the heterologous epitope is inserted into I-453 and/or I-587 of AAV1, AAV2 or AAV4;
g) wherein the VP3 is included in a fusion protein:
h) at least one tag useful for binding to a ligand; and/or
i) at least one further mutation.
4. A pharmaceutical composition comprising the parvoviral particle of claim 1 .
5. The pharmaceutical composition of claim 4 , further comprising one or more excipients.
6. The pharmaceutical composition of claim 4 , wherein the pharmaceutical composition is a vaccine.
7. The pharmaceutical composition of claim 6 , wherein the vaccine further comprises one or more adjuvants.
8. The parvoviral particle of claim 1 , wherein the Rep proteins functional for packaging of virus genomes and unspecific DNA into preformed capsids include Rep40, Rep52, Rep68, and Rep78.