The present invention provides methods of production of a purified enveloped virus antigen. In particular, it provides purified Ross River Virus (RRV) antigens, and vaccines comprising purified, inactivated Ross River Virus (RRV) antigen.

SUMMARY OF THE INVENTION

It is thus an object of the present invention to provide a method for the production of purified Ross River Virus antigen.

It is also an object of the present invention to provide a method of production of a vaccine comprising purified Ross River Virus being suitable for human administration.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

An object of the invention is to provide a purified RRV antigen substantially free of contaminants derived from the cell culture medium and the cells of the cell culture, such as cellular proteins and cellular nucleic acid, wherein the purified antigen is used in a vaccine particularly suitable for human clinical use in a host protective amount.

The term "cellular nucleic acid" means a heterogeneous DNA or RNA derived from the cells that have been infected with the virus to propagate the virus.

By "purified Ross River Virus antigen" is meant greater than about 97% purity as determined by SDS-PAGE and Western blot analysis with anti-cellular protein specific antibodies and quantification of residual cellular nucleic acid.

The term "substantially free" means that the amount of contaminating proteins derived from the cells or the cell culture or contaminating cellular nucleic said amount being below the detection limit of the state of the art detection method. Westernblot analysis and densitometric determination are used to test the amount of contaminating cellular proteins. A highly sensitive PCR method as described in U.S. Pat. No. 5,858,658 for nucleic acid quantification, particular for genomic VERO cell DNA, is used to quantify residual cellular nucleic acid in preparation.

The term "suitable for human clinical use" means that the endotoxin content for 10 .mu.g antigen is less than about 2 IU, as determined by the chromogenic LAL test. In addition the level of DNA/.mu.g protein antigen in the vaccine dose is less than about 50 pg, preferably less than about 20 pg, more preferably less than about 10 pg/.mu.g antigen. Furthermore, the level of cellular contaminants per dose of virus antigen is less than about 0.1% of the total protein content, preferably less than about 0.05%, preferably below the detection limit of highly sensitive analysis method such as Western blot analysis with specific antibodies or HPLC analysis.

The "host protective amount" means the critical protective dose of viral antigen in the vaccine, wherein said amount is effective to immunize a susceptible mammal against Ross River Virus infection and induces a protective immune response in the host.

In accomplishing these and other objects of the present invention, there are provided in one aspect of the invention a method for the production of purified Ross River Virus antigen. This method comprises the steps of infecting a cell culture of cells with Ross River Virus, incubating said cell culture to propagate said virus, harvesting the virus produced, and filtering the harvested virus.

The cells used for infection of the virus can be any cell which is susceptible to RRV. According to one aspect of the invention the cells are a continuous cell line of monkey kidney cells, such as VERO cells or CV-1 cells. VERO cells are available from the American Tissue Cell Culture, deposited as ATCC CCL81.

Some substances like serum and serum-derived additives that can contaminate the final harvested viral antigen may either be derived from the cell culture medium or the cells. Conventional cell culture media comprise serum or protein additives such as albumin, transferrin or insulin, and other proteins or polypeptides derived from the serum or added to the cell culture medium during cell growth.

According to one embodiment of the invention the cells are grown in a serum free medium. The medium can be a minimal medium, such as DMEM or DMEM HAM's F12 and other minimal media known in the art, such as described in Kistner et al. (1998. Vaccine 16:960 968), which do not comprise any serum additives.

In a preferred embodiment of the invention the cells are grown in a serum and protein free medium prior to infection as described in WO 96/15213, WO 00/0300 or WO 01/23527, whereby said minimal medium can be supplemented with extracts of yeast or soy peptone.

The cells can be bound to microcarrier during cell culture growth. The microcarrier can be a microcarrier selected from the group of microcarriers based on dextran, collagen, plastic, gelatine and cellulose and others as described in Butler (1988. In: Spier & Griffiths, Animal cell Biotechnology 3:283 303). Therefore, according to one embodiment of the invention the serum free or serum and protein free cells are cultivated and infected on microcarriers. Preferably, the microcarrier is selected from the group of smooth surface microcarriers such as CYTODEX I microcarrier, CYTODEX II microcarrier and CYTODEX III microcarrier, and CYTOPORE microcarrier or CYTOLINE microcarrier (all Pharmacia). Preferably, the microcarrier are selected from the group of smooth surface such as Cytodex I.RTM., Cytodex II.RTM. and Cytodex III.RTM., Cytopore.RTM. or Cytoline.RTM. (all Pharmacia).

The cells bound to microcarrier are infected with RRV at a multiplicity of infection (m.o.i.) between about 0.001 and about 5.

Various conventional methods, such as chromatography, gradient centrifugation etc. are known in the art to remove contaminating proteins from the biological that is desired to be isolated and purified. Efficient purification methods often comprise several steps and combinations of filtration, ion exchange chromatography and gradient centrifugation. However, the various methods may reduce the virus titer and antigen yield during each purification step.

Filtration is used in the art to purify biological material, e.g. to remove contaminating agents, or during preparation of virus-free blood products to remove potential contaminating viruses, whereby viruses, particular enveloped-viruses, remain in the retenate, and the virus titer in the filtrate is reduced.

It has been surprisingly found by the present invention that by filtering the cell culture supernatant derived from cells infected with enveloped viruses (e.g., the Ross River virus), the enveloped virus passes the filter system without reduction of virus titer, while cellular contaminants, like proteins and nucleic acid are efficiently removed. The method of the invention provides purification of a high titer virus preparation by filtration, wherein this method is easily applicable for large-scale purification and efficiently removes most of the protein derived from the host cells as well as of cellular nucleic acid. The method of the invention therefore provides a process of purifying virus antigen by filtering without remarkable loss of virus titer and virus antigen.

The methods of the invention can be used to purify any enveloped viruses. Exemplary viruses include, Alphaviruses (e.g., Ross River Virus, Eastern equine encephalitis Virus, Venezuelan equine encephalitis, Western equine encephalitis, Sindbis Virus, Semiliki Forest Virus); Flaviviruses (e.g., St. Louis encephalitis Virus, Japanese encephalitis Virus, Dengue Virus, Yellow fever Virus, Tick-borne encephalitis Virus); Orthomyxoviruses (e.g., Influenza Virus); and Paramyxoviruses (e.g., New Castle Disease Virus).

According to one aspect of the methods of the invention, the filtering is performed on a filter having a pore size between about 0.3 and about 1.5 .mu.m, which is preferably a filter based on a positively charged matrix. According to another aspect of the invention the filtering is performed on a filter having a pore size between about 0.1 and about 0.5 .mu.m, which is preferably based on a hydrophilic matrix.

According to a preferred embodiment the filtering is performed by a combination of at least two filters, a first having a pore size of between about 0.3 and about 1.5 .mu.m, and a second filter having a pore size of between about 0.1 and about 0.5 .mu.m. The combination of the filtering steps can be performed with a first filtering step on a filter having a pore size of between about 0.3 and about 1.5 .mu.m and the second filtering on a second filter having a pore size of between about 0.1 and about 0.5 .mu.m. The filtering by a combination of filters can be performed either sequentially or in separate steps. The filter can be a filter such as a positively charged depth filter having a pore size of about 0.3 to about 1.5 .mu.m and a hydrophilic filter having a pore size of about 0.22 .mu.m. Any filtration system known in the art can be used as well. By filtering during virus/virus antigen purification, substantially all cellular protein contamination is removed. The cellular contaminating nucleic acid is also efficiently removed by a factor of at least 35, and an intermediate pure preparation having a purity of at least about 97% compared to the starting virus harvest is obtained by this purification step.

The filter used can be based a cellulose fiber matrix, hydrophilic filters, such as based on polyvinylidene fluoride membrane, or filters based on polypropylene membrane. Such filters are commercial available, e.g., ZETAPLUS filters (CUNO), DUIRAPORE filters. MILLIPAK filters or MILLIDISK filters (Millipore), or filters from Pall.

The method of the invention as described above therefore provides for a purified Ross River Virus antigen which is substantially free of contaminating proteins and nucleic acid from the cells or the cell culture. The preparation has a purity of at least about 97% compared to the starting material.

Another aspect of the present invention relates to a method for production of a purified Ross River Virus comprising the steps of infecting a cell culture of cells with Ross River Virus, incubating said cell culture to propagate said virus, harvesting the virus produced, filtering the harvested virus, treating the virus filtered with a nucleic acid degrading agent and purifying the virus.

The virus preparation obtained after filtering is treated with a nucleic acid degrading agent to destroy the structural integrity of the nucleic acid by breaking down residual nucleic acid that have not been removed by filtering. The degradation of the nucleic acid ensures that higher molecular weight nucleic acids are broken down to molecules of lower molecular weight, which are then removed during the final purification step.

The nucleic acid degrading agent according to the invention can be an enzyme which degrades nucleic acid, preferably a nucleic acid degradation enzyme, such as a nuclease, having DNase and RNase activity, or an endonuclease, such as from Serratia marcescens, commercial available as BENZONASE endonuclease (Benzon Pharma A/S). Most preferably the nucleic acid degradation agent is BENZONASE endonuclease.

The virus treated with a nucleic acid degradation agent can be further treated with an inactivating agent. The inactivating agent can be any agent with inactivating activity known in the art, such as e.g. formalin, BEI, laser light, UV light, chemical treatment such as methylene blue, psoralen, carboxyfullerene (C60), or a combination of any thereof as described in the prior art (Rowland et al. (1972). Arch. Ges. Virusforsch. 39:274 283; Mowat et al (1973). Arch. Ges. Virusforsch. 41:365 370, Rweyemamu et al. (1989). Rev. Sci. tech. Off. Int. Epiz. 8:747 767 and WO 01/46390). Other known methods in the art for inactivating viruses can be used as well.

The treatment of the virus with the nucleic acid degrading and inactivating agent can be performed by a sequential treatment or in a combined/simultaneous manner, whereby the purified virus antigen is treated with a combination of Benzonase as nucleic acid degrading agent and formalin or UV or BEI or a combination of formalin/BEI or formalin/UV as inactivation agent. The nucleic acid degrading agent is preferably added to the virus preparation prior the addition of the inactivation agent and during the course of the inactivation process, the nucleic acid degrading agent can be further added subsequently, if necessary.

According to another embodiment of the method of the invention the inactivated virus is further purified. The method therefore comprises after the step of nucleic acid degradation/virus inactivation treatment a further step to remove the nucleic acid degrading agent and the inactivating agent from the virus preparation. This can be performed by any method known in the art, such as chromatography, gel filtration or gradient centrifugation. According to one embodiment of the invention gradient purification, such as sucrose gradient centrifugation is preferred. This final purification step also removes the break-down products of the nucleic acid treatment with nucleic acid degrading agent and removes the residual nucleic acid and proteins that have not been removed by the filtering.

The preparation obtained with this method comprising Ross River virus antigen, wherein said preparation is substantially free of contaminating proteins derived from the cells or the cell culture and has less than about 50 pg cellular nucleic acid/.mu.g virus antigen, preferably less than about 20 pg and most preferred less than about 10 pg cellular nucleic acid/.mu.g virus antigen. Purified Ross River Virus antigen obtained is free of contaminating proteins and nucleic acid, suitable for human clinical use and is stable.

Another aspect of the invention provides a method for production of a vaccine comprising purified, inactivated Ross River Virus antigen comprising the steps of infecting a cell culture of cells with Ross River Virus, incubating said cell culture to propagate said virus, harvesting the virus produced, filtering the harvested virus, treating the filtered virus preparation with a nucleic acid degrading agent and virus inactivating agent, purifying the virus and formulating the purified and inactivated virus in a vaccine composition. According to one embodiment of the invention the method provides for a vaccine comprising purified Ross River Virus antigen and being substantially free of contaminating proteins and nucleic acids.

According to another aspect the invention provides for a preparation comprising purified Ross River Virus antigen being substantially free of contamination from the cells or cell culture.

According to another aspect the invention provides for a preparation comprising purified Ross River Virus antigen being substantially free of contaminating proteins from the cells or cell culture. The preparation has less than about 50 .mu.g cellular nucleic acid/pg virus antigen, preferably less than about 10 pg nucleic acid/.mu.g virus antigen. According to one embodiment of the invention the preparation comprises a physiologically acceptable carrier, such as a phosphate or Tris-based buffer.

In accordance with another aspect of the invention, there is provided a vaccine against Ross River Virus infection comprising a host protective amount of Ross River Virus antigen in an amount of between 0.1 to 50 .mu.g antigen/dose, preferably between 0.3 and 30 .mu..g antigen/dose. The antigen can be a whole virus or a fragment of the virus, such as a peptide or polypeptide, having an immunogenic epitope to induce protective antibodies against RRV infection. In a preferred embodiment of the invention the vaccine comprises as RVV antigen as whole inactivated virus.

The vaccine of the present invention comprising highly purified RRV can further comprise an adjuvant. It has been found that the presence of an adjuvant increases the immunogenicity of the purified antigen of the invention by inducing higher titers of neutralizing, protective antibodies when compared to a vaccine composition which does not comprise an adjuvant. The beneficial effect of the present vaccine may be due the higher purity of the RRV antigen used in the vaccine. The adjuvant may be aluminum or a salt thereof, mineral oils, Freund adjuvant, vegetable oils, water-in-oil emulsion, mineral salts, immunomodulator, immunopotentiator or other well known adjuvant know in the art at a desired concentration to increase the immune response e.g. to stimulate the production of neutralizing antibodies. The amount of adjuvant is well within the level of skill.

In a preferred embodiment of the invention, the adjuvant is aluminum or a salt thereof, such as aluminum hydroxide or aluminum phosphate. The aluminum concentration is preferably between 0.001% and 1% (w/v) per dose. The vaccine may be formulated in the most varying manner.

The vaccine may be administered in any known manner e.g. subcutaneously, intramuscularly or intraperitoneally. According to a preferred embodiment the vaccine is administered intramuscularly in a vaccination scheme in humans of 0, 1, 6 or 12 months or in a rapid immunization scheme for travelers of about 0, 14 and 28 days with a higher antigen dose per immunization and booster.

Another aspect of the invention provides for a method of immunizing a mammal against Ross River Virus infection comprising the steps of providing a vaccine comprising a host protective amount of purified Ross River Virus antigen and having an amount of cellular DNA of less than 10 pg/.mu.g antigen, and wherein said vaccine is substantially free of any contaminating protein from the cells or the cell culture and administering said vaccine to a mammal.

According to a further aspect of the invention there is provided a method for the preparation of an immune globulin preparation comprising antibodies specific against Ross River Virus. The preparation comprising the immune globulins is obtained by immunizing a mammal with a Ross River Virus Vaccine as described above and isolating from the serum of the immunized mammal the immune globulin fraction comprising the RRV specific antibodies. The immune globulin fraction can be isolated by conventional method known in the art, such a Cohn fractionation and ion exchange chromatography.

 

Claim 1 of 16 Claims

1. A method for production of purified Ross River Virus antigen, comprising the steps of infecting a cell culture with Ross River Virus; incubating said cell culture to propagate said virus; harvesting the virus produced; filtering the harvested virus with a first filter having a pore size of between 0.3 and 1.5 .mu.m; filtering the harvested virus with a second filter having a pore size of between 0.1 and 0.5 .mu.m; and purifying the virus antigen.
 

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