METHOD FOR PRODUCTION OF pH STABLE ENVELOPED VIRUSES
The present invention provides a method for producing pH-stable enveloped viruses wherein said viruses are used for infection of host cells under low pH conditions and for incubation with cell culture cells under conditions of low pH, as well as influenza viruses obtainable by this method which exhibit a high growth rate in cell culture, increased pH and temperature stability and which have human receptor specificity.
1 . A method for producing an influenza virus, comprising the steps of:
a) diluting influenza viruses in a solution having a pH of between 5.2 and 5.9;
b) infecting host cells with at least one infectious virus particle, wherein:
i) the virus particle is added to said cells; and
ii) said cells and said virus particle are incubated at a pH of between 5.2 and 5.9 to provide a virus/cell complex;
c) cultivating infected host cells to propagate viruses; and
d) harvesting the propagated viruses.
2 . The method of claim 1 , wherein a macrolide polyene antibiotic or a derivative thereof is added to said cells.
3 . The method of claim 1 , wherein the cells are tissue culture cells selected from the group consisting of BSC-1 cells, LLC-MK cells, CV-1 cells, CHO cells, COS cells, murine cells, human cells, HeLa cells, 293 cells, VERO cells, MDBK cells, MDCK cells, MDOK cells, CRFK cells, RAF cells, TCMK cells, LLC-PK cells, PK15 cells, WI-38 cells, MRC-5 cells, T-FLY cells, BHK cells, SP2/0 cells, NS0 cells, and PerC6 cells.
4 . The method of claim 1 , wherein the influenza virus is selected from the group consisting of influenza A, influenza B or and influenza C.
5 . The method of claim 1 , wherein the influenza virus is an attenuated influenza virus.
6 . The method of claim 1 , wherein the influenza virus comprises a deletion or modification within the NS1 gene of the virus.
7 . The method of claim 1 , wherein the influenza virus is a cold adapted virus.
8 . A The method of claim 1 , wherein the viruses are diluted in a buffer is selected from the group consisting of MES (2-(N-morpholino-ethanesulfonic acid) buffer, citric buffer, and acetate buffer.
9 . A The method of claim 1 , wherein in the cultivating step a cultivation medium is used to cultivate the cells, and wherein the cultivation medium is SFM opti-pro™ medium.
10 . The method of claim 1 , wherein the viruses are passaged in the host cells for at least one passage.
11 . An influenza virus produced by the method of claim 1 , wherein the virus retains hemagglutination activity at increased temperature, retains infectivity at a pH range of between 5.5 and 5.8, demonstrates a high growth rate in cell culture, and has human receptor specificity.
12 - 13 . (canceled)
14 . A composition useful for vaccination or therapy of viral disease comprising an influenza virus produced by the method of claim 1 and a pharmaceutically acceptable carrier or adjuvant.
15 . The method of claim 2 , wherein the macrolide polyene antibiotic is amphotericin B.
16 . The method of claim 1 , further comprising the step of inoculating a cell culture using the viruses harvested in step (d).
17 . The method of claim 16 , wherein the cell culture comprises a cell culture medium having a volume of at least 200 liters.
18 . The method of claim 1 , wherein step (a) comprises diluting viruses in a solution having a pH of between 5.5 and 5.8.
19 . The method of claim 1 , wherein the cells and the virus particle are incubated at a pH of between 5.5 and 5.8.
20 . The method of claim 1 , wherein the cells and the virus particle are incubated at a pH of between 5.6.