Vero cell line adapted to grow in suspension

Abstract

A Vero cell line that can be grown in serum-free and protein-free culture and in suspension culture in the absence of supporting materials for its adherence, a process to obtain said Vero cell line and a process for producing viral vaccines with said cell line, a virus obtainable by said process and vaccines formulated by said virus.

Description

This Application claims the benefits of Provisional Application No. 60/723,377 filed on Oct. 4, 2005.

FIELD OF THE INVENTION

The invention relates to a Vero cell line that can be grown in serum-free and protein-free culture and in suspension culture in the absence of supporting materials for its adherence, and to production of viral vaccines using said cell line. More particularly, the present invention relates to establishment of a cell line that can be grown in suspension culture without need of the cells to be adhered to any supporting material. Furthermore, the present invention provides a process to obtain said Vero cell line and a process for producing viral vaccines with said cell line. The present invention further relates to virus obtainable by said process and vaccines formulated by said virus.

DESCRIPTION

The invention resides in a Vero cell line that can be grown in protein-free culture and in suspension culture without a carrier, and to production of viral vaccines using said cell line. More particularly, the present invention relates to establishment of a cell line that can be grown without using fetal calf serum (FCS) and that can be grown in suspension culture without need of the cells to be adhered to any carrier, and a process for producing viral vaccines with said cell line. The present invention further relates to virus obtainable by said process and vaccines formulated by said virus.

BACKGROUND OF THE INVENTION

For production of viral vaccines chicken eggs, mouse brains, primary cells or established cell lines are used in order to propagate the virus (Principles of Virology. Molecular Biology, pathogenesis and control, 2001). These conventional techniques have several problems:

1) The use of chicken eggs requires management of chicken breeding, management of fertilized eggs to be adjusted to a vaccine production schedule, and laborious procedures including extensive purification for completely removing components derived from egg proteins (Tree et al., 2001).

2) Cell lines; in general require the addition of fetal calf serum as a cell growth factor. Therefore, there is an associated risk of contamination with infection agents and prions and high-quality FCS is very expensive, a fact that adds a considerable cost to the vaccine.

• 3) Among established cell lines where various kinds of viruses can be propagated, Vero cells (African Green Monkey Kidney Epithelial Cells) are widely used for production of high quality vaccines for humans and animals (Butler et al., 2000; Franzzati-Gallina et al., 2001; U.S. Pat. No. 4,664,912; Montagnon, 1989; Montagnon et al., 1981). Furthermore, Vero cells are the only recommended cells to prepare viruses for vaccine production by reverse genetics in the document: “WHO guidance on development of influenza vaccine reference viruses by reverse genetics”. 2005.6, page 3. However, Vero cells have a tendency to adhere to surfaces. Thus, the use of Vero cells for large-scale cultures is, due to the need of reactors with very large surface or to the need of introduction of cell-carriers in the culture, extremely costly. Serum is often used for the propagation of mammalian cell lines. However, when mammalian cells are used for the production of recombinant proteins or virus production, there is increasing pressure to remove serum from the manufacturing process. Some of the driving reasons to implement serum-free cell-culture technology are the expense of serum, variation between serum lots and serum quality, regulatory concerns regarding biological agents in serum and the burden of removing serum proteins in downstream processing. There is also a recognized need, for reasons of reduced cost and process scale up, for use of cell lines adapted to grow in suspension. There is an urgent need for more economic processes for quality vaccine production at a low-cost to make them available worldwide.

Many developments have been accomplished to obtain Vero cell lines in suspension to obtain a virus for vaccine manufacturing (U.S. Pat. No. 4,664,912; U.S. Pat. No. 4,525,349: U.S. Pat. No. 5,719,05 1; Higher production of rabies virus in serum-free medium cell cultures on microcarriers. Frazzati-Gallina N M. J Biotechnol. 2001 Dec 14; 92(1):67-72; among other publications referred to and incorporated herein by this reference). However, these documents describe the use of carriers and microcarriers to obtain suspensions of anchorage-depending Vero cell lines. In the state of the art, it has never been reported a Vero cell line to obtain a virus for vaccine manufacturing, adapted to growing in suspension, in the absence of supporting materials for its adherence (carriers or microcarriers), adapted to growth in a culture medium free of fetal calf serum, and capable of growing also in a protein-free medium. On the other hand, there are references to Vero cell lines which may grow in suspensions forming cell aggregates (“The growth of Vero cells in suspension as cell-aggregates in serum-free media”. Cytotechnology. 1992; 10(2):169-74. Litwin J.). Litwin described a method to grow Vero cells in suspension as cell aggregates in serum free media. However, cell aggregates are difficult to infect with viruses and therefore are not adequate for vaccine production (US patent 2005153419-A). There are no reports of Vero cells that may grow as individual cells without forming aggregates in high density cultured.

In order to obviate the disadvantages of the prior art as stated above, this invention has now established a novel Vero cell line which can be grown without FCS, without protein and in suspension comprising isolated cells in the absence of cell aggregates. This cell line can be use in large scale production of vaccines in normal fermentation vessels in the absence of supporting materials for its adherence (without the need of any kind of cell carrier or microcarrier in the culture). The individual cells suspensions have known advantages over the aggregates suspension, for those skilled in the art, for example improves of efficiency in virus production systems that involve high density reactors. The present invention also established a process for producing a viral vaccine culturing Vero cells in suspension without FCS and then infecting the cells with the virus to be use in the vaccine production. The vaccine so produced is of low cost and high safety profile

SUMMARY OF THE INVENTION

The Vero cell line, subject matter of the present invention, useful for the production of viruses for vaccine formulation, is adapted to grow in suspension, in the absence of supporting materials for its adherence and in a culture free of fetal calf serum. Furthermore, this cell line is adapted to grow in a protein-free culture. This cell line also is adapted to grow in a suspension as isolated cells, in the absence of cell aggregates. Specifically, this cell line is deposited with the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany) under the name of sVero p66 and whose name assigned by the International Deposit Authority is DSM ACC2791.

The procedure for obtaining said Vero cell line adapted to grow in suspension, in the absence of supporting materials for its adherence and in a culture free of fetal calf serum and free of proteins, another subject matter of this invention, comprises the following steps:

• • a. adapting an anchorage-depending Vero cell line, preferably the Vero E6 cell line (passage 26, Banco Argentino de Células), to a culture with a low content of fetal calf serum by a gradual adjustment of the culture from a high concentration of fetal calf serum of about 7 to approximately 15% to a culture of low concentration of fetal calf serum of about 0 to approximately 3%, preferably about 2%,
  • b. applying the Vero cell line obtained in the previous step “a.” to a culture in suspension of the same culture medium of low concentration fetal calf serum of about 0 to about 3%, preferably 2%, for a time sufficient enough, at least 30 days, for said cell line to grown in suspension in the absence of supporting materials for its adherence; and
  • c. adapting said cell line obtained in step “b” to grow in a medium free of fetal calf serum and free of protein.

In another preferred embodiment of this invention, it is disclosed a procedure to obtain a virus for vaccine formulation, another purpose of this invention, comprising the following steps:

• • a. growing a Vero cell line adapted to grow in suspension, in the absence of supporting materials for its adherence and, preferably, in the absence of proteins, to a concentration of up to between about 1×10⁵ and about 30×10⁶ cells per ml of culture medium,
  • b. infecting the cell line of step “a” with said virus with an multiplicity of infection (m.o.i.) between about 0.001 and about 10, and
  • c. collecting the virus produced during the infection, preferably by centrifugation.

In preferred embodiments of this invention, said virus is selected from the group consisting of the human immunodeficiency virus, such as HIV-1 and HIV-2; polio virus; hepatitis A virus, human coxsackie virus; rhinovirus; echovirus; equine encephalitis virus; rubella virus, dengue viruses, encephalitis virus, yellow fever virus, coronavirus, vesicular stomatitis virus, rabies virus, ebola virus, parainfluenza virus, mumps virus, measles virus, respiratory syncytial virus, influenza virus, Hantaan virus, bunga virus, hemorrhagic fever virus, reovirus, rotavirus, parvoviruses, papilloma virus, polyoma virus, adenovirus, herpes simplex virus (HSV) 1 and 2, varicella zoster virus, cytomegalovirus (CMV), smallpox virus, vaccinia virus, pox viruses, African swine fever virus and the unclassified agent of delta hepatitis, and the agents of non-A, non-B hepatitis. Preferably said virus is selected from the group consisting of polio virus, rabies virus, yellow fever virus, hepatitis A virus and influenza virus.

In other preferred embodiments of this invention, the viruses are the viruses and viral antigenes, also subject matter of the present invention, that can be obtained by the procedure previously described herein.

Subject matter of this invention are also the vaccines formulated with the viruses that can be obtained by the procedure disclosed in this invention.

DESCRIPTION OF THE DRAWING

FIG. 1 shows growth of Vero E6 AGS according to the present invention.

DETAILED DESCRIPTION OF THE INVENTION

The Vero cell line, subject matter of the present invention, useful for the production of viruses for vaccine formulation, is adapted to grow in suspension, in the absence of supporting materials for its adherence and in a culture free of fetal calf serum. Furthermore, this cell line is adapted to grow in a protein-free culture. This cell line also is adapted to grow in a suspension as isolated cells, in the absence of cell aggregates. Specifically, this cell line is deposited with the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany) under the name of sVero p66 and whose name assigned by the International Deposit Authority is DSM ACC2791.

The procedure for obtaining said Vero cell line adapted to grow in suspension, in the absence of supporting materials for its adherence and in a culture free of fetal calf serum and free of proteins comprises the following steps:

• • a. Adapting an anchorage-depending Vero cell line, preferably the Vero E6 cell line (passage 26, Banco Argentino de Células), to a culture with a low content of fetal calf serum by a gradual adjustment of the culture from a high concentration of fetal calf serum of about 7 to approximately 15% to a culture of low concentration of fetal calf serum of about 0 to approximately 3%, preferably about 2%.
  • b. Applying the Vero cell line obtained in the previous step “a.” to a culture in suspension of the same culture medium of low concentration fetal calf serum of about 0 to about 3%, preferably 2%, for a time sufficient enough, at least 30 days, for said cell line to grown in suspension in the absence of supporting materials for its adherence. That is achieved with an abrupt passage of the cells from T-flasks to spinner flasks, without any carrier, and gradually replacing remaining serum supplementation by serum free media containing proteins and growth factors. During the initial phase of suspension culture adaptation the cells tend to form aggregates of 2-30 cells, especially at cell densities higher than 1.times. 10.sup.6 cells/ml.
  • c. Adapting said cell line obtained in step “b” to grow in a medium free of fetal calf serum and free of protein. After several passages, and replacing step “b” medium by serum free and protein free media, no cell clumping is observed, and Vero E6 is considered adapted to grow in suspension culture in protein free media, and that cellular clone is denominated “Vero E6 AGS”. Vero E6 AGS cells have been shown to maintain an average doubling time of 24 hours in continuous growth cultures over a period of at least one month and have retained the susceptibility to be infected by several viruses.

Additionally, there was not difference in the cellular response (as measured by viability and doubling time) to growth in medium with low concentrations of proteins and growth factors and growth in medium with no proteins (protein free media).

The Vero E6 AGS cell line has been deposited with the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany) under the name sVero p66 and whose number, assigned by the International Deposit Authority, is DSM ACC2791.

Suspension Culture and Infection

Cells are maintained in suspension in 30 to 1,000 ml cultures in 50 to 1,500 ml glass spinner flasks (Techne, UK) agitated at 50-80 rpm in a 5% CO.sub.2 incubator at 33-37.degree. C. During cell maintenance, cultures are kept in exponential phase by diluting cells approximately every second day in order to maintain cell densities between 1.times.10.sup.5 cells/ml and 12.times.10.sup.5 cells/ml. For infection experiments, exponentially growing cells are centrifuged and resuspended in fresh culture media at a cell density of 1.times. 10.sup.6 cells/ml.

Infection is initiated by adding concentrated viruses to the cultures at a multiplicity of infection (M.O.I.) of 0.001 to 10. A 500-1,500 mu.l cell suspension aliquot is transferred in sterile tubes at different times post infection. The different aliquots were stored at −80.degree.C. for further analyses. Viral particles are recovered from the frozen total-suspension samples by 3 freeze-thaw cycles.

According to the present invention cells grow individually in suspension and are well infected by different virus producing a good yield of the antigenic viral particles adequate for vaccine production.

The viruses that may be produced by application of the present invention are selected from the group consisting of the human immunodeficiency virus, such as HIV-1 and HIV-2; polio virus; hepatitis A virus, human coxsackie virus; rhinovirus; echovirus; equine encephalitis virus; rubella virus, dengue viruses, encephalitis virus, yellow fever virus, coronavirus, vesicular stomatitis virus, rabies virus, ebola virus, parainfluenza virus, mumps virus, measles virus, respiratory syncytial virus, influenza virus, Hantaan virus, bunga virus, hemorrhagic fever virus, reovirus, rotavirus, parvoviruses, papilloma virus, polyoma virus, adenovirus, herpes simplex virus (HSV) 1 and 2, varicella zoster virus, cytomegalovirus (CMV), smallpox virus, vaccinia virus, pox viruses, African swine fever virus and the unclassified agent of delta hepatitis, and the agents of non-A, non-B hepatitis. Preferably the polio virus, the rabies virus, the yellow fever virus, hepatitis A virus and the influenza virus.

The viruses that can be obtained according to the present invention are useful for vaccine formulation using methods known by those skilled in the art of vaccine formulation. Said viruses or the viral antigens are concentrated and purified using methods known in the art such as centrifugation and chromatography. The vaccines that can be obtain by the present invention comprise said viruses or viral antigens, at least one diluent and, optionally, adjuvants, excipients and chemotherapeutic agents known in the art. If necessary, said viruses will undergo an attenuation or inactivation process.

The present invention will now be described in more details through the following application examples showing preferred forms to practice this invention. However, said examples are provided for illustration purposes only, to better understand the present invention, and do not intend to limit the scope of the present invention.

EXAMPLE 1

More specifically, the Vero cell line of the present invention was prepared using the following procedures:

Vero cells from the line Vero E6 (passage 26, Banco Argentino de Células) were used as the starting material. Cells were cultured in a monolayer in a 25 cm.sup.2 culture flask containing MEM (GIBCO BRL, USA) medium supplemented with 10% FCS. Incubation was at 37° C. in a 5% CO2 atmosphere. After sufficient expansion of the cells, the cells were then cultured in the above medium containing 20% serum-free medium and subcultured upon confirming no abnormality in cells. After subculture and sufficient expansion of the cells, the culture medium was replaced with medium containing 40% serum-free medium and sub-cultured upon confirming no abnormality in cells. The procedure was repeated until the content of serum-free medium was 80% (2% FCS). At this point cells were trypsinated and inoculated (2.5.times.10.sup.5 cells/ml) in a spinner flask (Techne, UK) containing the same medium. Agitation was at 50 rpm and incubation was at 37° C. in a 5% CO2 atmosphere. The cells were transfer to fresh medium according to the evolution of the pH of the culture. After 90 days of the initiation of the adaptation procedure, cells were seeded in a medium containing half a volume of MEM supplemented with 0.2% FCS and half a volume of ExCell™ 302 CHO serum-free (JRH Biosciences, USA) medium supplemented with 2 mM glutamine. Duplication period in this medium was about 24 hs and viability 98-100% a week after transfer. Cells were grouped in clusters of less than ten cells when the cell concentration was superior to 1.5.times.10.sup.6 cells/ml. At this point, the half volume of MEM was replaced with the protein free SMIF-6 medium (GIBCO BRL, USA). After this, the proportion of ExCell™ 302 CHO serum-free medium was diminished and 120 days after the initiation of the adaptation procedure, Vero cells (Vero E6) were growing in suspension in 100% protein free SMIF-6 medium. These cells were named “Vero E6 AGS” for Vero E6 adapted to grow in suspension as isolated cells without cells aggregates, without carriers.

For storage, Vero E6 AGS cells in exponential growth period were centrifuge at 1000 rpm and the cell pellet suspended in a solution containing 45% SMIF-6 conditioned medium, ⁴⁵% fresh SMIF-6 medium and 10% dimethylsulfoxide (DMSO). This cell suspension was fractionated in cryotubes containing 1 ml of the suspension each and freeze in steps: 1 h at 4° C., 1 ml at −20° C. in nitrogen atmosphere and finally in liquid nitrogen at −196° C.

EXAMPLE 2

Susceptibility of Vero E6 AGS Cells to Viral Infection

The susceptibility of Vero E6 AGS cells to viral infection was assayed using the vesicular stomatitis virus (VSV, Indiana strain: ATCC VR-1238). Titration of the virus was performed using Vero E6 cells (passage 31). For this, 4.0.times.10.sup.4 cells were seeded in each well of a 96 wells titration plate (GREINER, GERMANY). After 3 hs of incubation at 37° C. in a 5% CO2 atmosphere, cells were infected with different amounts of a viral suspension. After 24 hs viral titer was estimated as the maximal viral dilution with capacity to produce cytopathic in 100% of the cells.

For virus production, 3.2 times.10.sup .5 Vero E6 AGS cells were seeded in a spinner flask containing SMIF-6 medium and infected with 0.01 cytopathic units/ml of the viral suspension. Table 1 shows that after 48 hours all the cells have been killed by the virus. Viral titer in the supernatant was 1 cytoathic unit/ml. This result indicates that in average Vero E6 AGS cells are able to amplify 100 times the seeded virus in the conditions here given as an example.

TABLE 1
Time after infection		Viral titer
(hs)	% of viable cells	(cytopathic units/ml)
0	97	0.0001
24	37	ND
48	0	0.01

EXAMPLE 3

The relationship between cell density and viral production was studied using the Vero E6 AGS cell line and the VSV virus. For this, Vero E6 AGS were grown in suspension until density 0.75.times.10.sup.6 or 5.00.times.10.sup.6 cells/ml and infected with VSV virus at a multiplicity of infection (M.O.I.) of 0.1.

Culture medium: SMIF 6

Additives: FCS 2%

Culture condition: 37° C., 5% CO2

Rotation of spinner: 50 rpm

Cellular density at the beginning of culture: about 1.times.10.sup.5 cells/ml

Virus: VSV

MOI: 0.1

Samples were taken 20 hs after infection and viral titer was measured in accordance with a plaque counting method.

The sample dilution that produces cytopathic effect in 50% of the inoculated cultures, contains 1 Tissue Culture Infection Dose 50% (TCID₅₀). The determination of this value was in accordance with the Reed-Muench procedure (Reed and Muench, 1938).

Table.2 shows that the viral titers rose in an approximately proportional form to the cell density.

TABLE 2
Cell Density	Viral Titer	Viral Titer/Cell Density
(cells/ml)	(TCID50/ml)	(TCID50/cell)
0.75.times.10.sup.6	4.64.times.10.sup.8	618
2.44.times.10.sup.6	3.16.times.10.sup.9	1295

EXAMPLE 4

Vero E6 AGS was used to propagate Herpes Simplex (HSV):

Culture conditions were as follows:

Cellular density at infection: 2.0.times.10.sup.5

MOI: 0.002

Culture scale: 100 ml spinner flask

Culture medium: SMIF 6 and SMIF 6/FCS 2%

Rotation of spinner: 50 rpm

Culture conditions: 37° C., 5% CO2

Duration of culture: 2 days

Table.3. shows that the HSV virus, a DNA virus, was produced using Vero E6 AGS in suspension in the absence of microcarriers and in a protein-free culture medium. It was observed a suspension of isolated individual cells without cell aggregates. It is also observed that the viral titration is slightly higher in the experiment conducted in the absence of FCS.

TABLE 3
Culture medium    HSV titer (TCID50/ml)
SMIF6             5.88.times.10.sup.8
SMIF 6 + FCS (2%) 4.64.times.10.sup.8

EXAMPLE 5

Vero E6 AGS was used to propagate Poliovirus (OPV type I Sabin strain).

Culture conditions were as follows:

Cellular density at infection: 3.0.times. 10.sup.5

MOI: 1

Culture scale: 100 ml spinner flask

Culture medium: SMIF 6 and SMIF 6/FCS 2%

Rotation of spinner: 50 rpm

Culture conditions: 34° C., 5% CO2

Duration of culture: 1 day

Table.4 shows that the Polio virus (OPV type I Sabin strain), a RNA virus, was produced using Vero E6 AGS in suspension in the absence of microcarriers and in a protein-free culture medium. It was observed a suspension of isolated individual cells without cell aggregates. It is also observed that the viral titration is slightly higher in the experiment conducted in the absence of FCS.

TABLE 4
Culture medium    OPV titer (TCID50/ml)
SMIF 6            2.5.times.10.sup.8
SMIF 6 + FCS (2%) 1.8.times.10.sup.8

REFERENCES

Patent Documents

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Papers

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• 3. Optimization of virus yield as a strategy to improve rabies vaccine production by Vero cells in a bioreactor. Trabelsi K J Biotechnol. 2006 Jan. 24; 121(2):261-71.
• 4. Inactivated Hantaan virus vaccine derived from suspension culture of Vero cells. Choi Y. Vaccine. 2003 May 16; 21(17-18):1867-73.
• 5. High immunogenic enterovirus 71 strain and its production using serum-free microcarrier Vero cell culture. Liu C C, Vaccine. 2006 Aug. 2.
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Claims

1. A Vero cell line wherein the cell line is adapted to grow in suspension, in the absence of supporting materials for its adherence and in a culture medium free of fetal calf serum.

2. The cell line of claim 1 wherein the cell line is adapted to grow in a protein-free culture medium.

3. The cell line of claim 1 wherein said suspensions comprises isolated cells in the absence of cell aggregates.

4. The cell line of claim 1 wherein said line is the line deposited with the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany) under the name sVero p66 and whose name assigned by the International Deposit Authority is DSM ACC2791.

5. The cell line of claim 1 wherein said line is useful for virus production for vaccine formulation

6. A procedure to obtain a Vero cell line adapted to grow in suspension, in the absence of supporting materials for its adherence and in a culture medium free of fetal calf serum and free of protein, wherein the procedure comprises the steps of: a. adapting an anchorage-depending Vero cell line to a culture medium of low concentration of fetal calf serum by gradually adjusting the medium from a high concentration of fetal calf serum from about 7 to about 15% to a culture medium from about 0 to about 3%, preferably 2%, b. applying the Vero cell line obtained in step “a” to a culture in suspension with the same culture medium of low concentration of fetal calf serum of about 0 to about 3%, preferably 2% for a period of time sufficient enough for said cell line to adapt to grow in suspension in the absence of supporting materials for its adherence; and c. adapting said cell line obtained in step “b” to grow in a medium free of fetal calf serum and free of proteins.

7. The procedure of claim 6 wherein said sufficient time of step “b” is at least 30 days.

8. The procedure of claim 6 wherein said anchorage-depending Vero cell line is the Vero E6 line (passage 26, Banco Argentino de Células).

9. The procedure of claim 6 wherein said Vero cell line adapted to grow in suspension, in the absence of supporting materials for its adherence and in a culture medium free of fetal calf serum and obtained in step “c” of said Claim is the line deposited with the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany) under the name of sVero p66 and whose number assigned by the International Deposit Authority is DSM ACC2791.

10. A procedure to produce viruses to formulate vaccines wherein the procedure comprises the steps of: a. growing the Vero cell line adapted to grow in suspension, in the absence of supporting materials for its adherence, up to a cell concentration between approximately 1×105 and approximately 30×106 cells per ml of culture medium; b. Infecting the cell line of step “a” with said virus with an infection multiplicity (M.O.I.) of between approximately 0.001 and 10; and c. Collecting the virus production produced during the infection.

11. The procedure of claim 10 wherein said Vero cell line adapted to grow in suspension, in the absence of supporting materials for its adherence is adapted to grow in a culture medium free of fetal calf serum and free of proteins.

12. The procedure of claim 10 wherein said Vero cell line adapted to grow in suspension, in the absence of supporting materials for its adherence is the cell line deposited with the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany) under the name of sVero p66 and whose number assigned by the International Deposit Authority is DSM ACC2791.

13. The procedure of claim 10 wherein said virus is selected from the group consisting of the human immunodeficiency virus, such as HIV-1 and HIV-2; polio virus; hepatitis A virus, human coxsackie virus; rhinovirus; echovirus; equine encephalitis virus; rubella virus, dengue viruses, encephalitis virus, yellow fever virus, coronavirus, vesicular stomatitis virus, rabies virus, ebola virus, parainfluenza virus, mumps virus, measles virus, respiratory syncytial virus, influenza virus, Hantaan virus, bunga virus, hemorrhagic fever virus, reovirus, rotavirus, parvoviruses, papilloma virus, polyoma virus, adenovirus, herpes simplex virus (HSV) 1 and 2, varicella zoster virus, cytomegalovirus (CMV), smallpox virus, vaccinia virus, pox viruses, African swine fever virus and the unclassified agent of delta hepatitis, and the agents of non-A, non-B hepatitis.

14. The procedure of claim 10 wherein said virus is the polio virus.

15. The procedure of claim 10 wherein said virus is the rabies virus.

16. The procedure of claim 10 wherein said virus is the hepatitis A virus.

17. The procedure of claim 10 wherein said virus is the yellow fever virus.

18. The procedure of claim 10 wherein said virus is the influenza virus.

19. The procedure of claim 10 wherein said step “c” comprises a centrifugation.

20. A virus obtained by the procedure of claims 10.

21. A vaccine formulated with a virus of claim 20.