Pharmaceutical product containing live, stabilized virus for the therapy of viral and malignant diseases and process for preparing the same

A process for preparing a purified virus vaccine comprises the steps of purifying a fluid containing a virus by centrifugation, ultracentrifuging to pellet the supernatant, purifying the virus by sucrose gradient ultracentrifugation, rehydration and lyophilization. Desirably, a modified starch, such as hydroxyethyl starch having a molecular weight in the range 100,000-300,000, is added as a protective colloid prior to lyophilization. The virus is selected from the group consisting of avian paramyxovirus, avian herpesvirus, avian rotavirus, avian bronchitis, avian encephalitis, avian bursiris (Gumboro) virus, Marek's disease virus, parvovirus, Newcastle disease virus, human paramyxovirus, human parvovirus, human adenovirus, and mixtures thereof. A purified virus vaccine made by the foregoing method is useful for the treatment and control of mammalian disease of viral origin.

The present invention relates to pharmaceutical products containing 
stabilized, live virus for the therapy of viral diseases and malignancies 
and to the process for the production of such products. The present 
invention also relates to a purified virus vaccine and the purification 
procedure therefor. 
Hungarian Patents #197 517 and #197 846 describe the use of certain live, 
apathogenic viruses in the therapy of various human diseases of vital 
origin. Thus, patent #197 517 provides a pharmaceutical product containing 
attenuated Newcastle disease virus suitable for the therapy of herpes, 
rabies, AIDS and malignancies. Patent #197 846 describes a pharmaceutical 
product containing attenuated Gumboro virus suitable for the treatment of 
hepatitis, rabies, and other diseases of viral origin and malignancies. 
Although both Gumboro and Newcastle disease viruses cause poultry 
diseases, the vaccines containing these attenuated viruses are in 
commercial use. The above patents describe the therapeutic application of 
these vaccines. 
Since the purity of veterinary vaccines do not meet human purity 
requirements, infections and complications may result as untoward side 
effects. Moreover, the stability of veterinary vaccines may also be poor. 
The present invention is intended to provide a process to obtain purified, 
apathogenic viruses suitable for human therapy as well as a lyophilized 
product which is stable for long periods without apparent loss of 
effectiveness. 
Recently, it has been found that other apathogenic viruses can also be used 
in the therapy of human diseases of vital origin. It has been proven, 
according to the present invention, that any attenuated virus apathogenic 
for humans can be used, alone or in combination, in the treatment of viral 
diseases. These may be veterinary, in particular, fowl viruses, or human 
viruses; e.g.; arian paramyxovirus, avian herpesvirus, avian rotavirus, 
avian bronchitis, avian encephalitis, avian bursitis (Gumboro) virus, 
Marek's disease virus, parvovirus, Newcastle disease virus as well as 
human paramyxovirus, human parvovirus and human adenovirus. 
The invention relates to attenuated viruses apathogenic to humans which are 
effective in the treatment of diseases of viral origin and malignancies, 
e.g., as follows: AIDS, carcinoma of the rectum, bladder, breast, colon, 
cervix, esophagus, pancreas, bronchus, liver, kidney and stomach, 
gynecological cancers, head and neck cancers, lymphomas, malignant 
melanoma, myeloma, immune deficiency due to irradiation, multiple 
sclerosis, influenza, common cold and related diseases of viral origin, 
herpes genitalis and labialis, warts, collagen diseases, acute and chronic 
hepatitis (B and C), and symptoms following bone marrow transplantation. 
The viruses suitable for the above therapeutic purposes may be obtained as 
usual, e.g., from fibroblast or other cell line cultures or 
allanto-amniotic fluid of egg embryos. The allanto-amniotic fluid can be 
obtained from infected hen eggs. The fluid is purified by centrifugation 
and the supernatant is pelleted by ultracentrifuge. The sediment is 
rehydrated and sedimented over sucrose gradient, ultracentrifuged again 
and the pellet is rehydrated and lyophilized. 
In a preferred embodiment of the invention the allantois fluid is 
centrifuged by approximately 5000.times. g, the pellet is discarded and 
the supernatant is used (if necessary a filtration step can be included). 
The virus is pelleted from this supernatant by ultracentrifugation (the 
ultracentrifugation depends on the r.p.m. and time, and may vary over a 
wide range, usually 35,000.times. g for 1 hour). The supernatant is 
discarded and the pellet is resuspended in a small volume of buffer 
solution. For appropriate homogeneity a relative longer period of mixing 
is required. 
This homogeneous suspension is layered over a high concentration of sucrose 
and ultracentrifuged at 90,000-100,000.times. g (minimal g: 60,000). The 
supernatant is discarded and the pellet is rehydrated and lyophilized. 
Another object of the invention is stabilization of the virus preparation. 
Protective colloids, either alone or in combination, during lyophilization 
are generally used in the production of vaccines. Such colloids are, e.g., 
milk (3-10%), polyvinylpyrrolidone and gelatin (0.1-0.2%), and glucose, 
sucrose or dextran (1-10%). However, for human use, these colloids are 
either unsatisfactory or may cause side effects. 
We have found that modified starch, either alone or in combination, can 
preferably be used as the protective colloid, such as hydroxyethyl starch 
(molecular weight: 100,000-300,000). Hydroxyethyl-starch of an average 
molecular weight of 200,000 is used as plasma expander, but such compounds 
have not been used as protective colloids for vaccine production. 
The new stabilized product according to this invention contains, together 
with other compounds, an effective mount of modified starch as the 
protective colloid. 
The invention will be detailed in the following examples. Newcastle disease 
and Gumboro virus can be purchased from Phylaxia of Budapest, Hungary as 
PHYLAVAC and GUMBOPHYL, respectively.

EXAMPLE #1 
Purification of Newcastle disease virus from allantois fluid 
Three liters of allanto-amniotic fluid containing the virus were 
centrifuged at 5000.times. g for 1 hour. The supernatant was faltered 
through multiple layers of gauze. The virus was pelleted from the 
supernatant by ultracentrifugation (SCP 85 H2 ultracentrifuge, RP 19 
rotor, 18,500 rpm (35,000.times. g, 4.degree. C., 1 h)). After discarding 
the supernatant, the pellet was resuspended in 30 ml NTE buffer (0.15M 
NaCl, 0.001M EDTA, 0.05M TRIS; pH 7.4). The suspension was gently mixed 
for 24 hours in an ice bath. 
The suspension was further purified by sucrose gradient 
ultracentrifugation. Thirty ml of 30% (w/w) (=33 % w/v) sucrose in NTE 
buffer was placed into centrifuge tubes and 5 ml of suspension was layered 
onto the sucrose. The tubes were ultracentrifuged in an SRP rotor at 
95,000 x g (27,500 rpm) for 80 min. 
After discarding the supernatants, the pellets were resuspended in NTE 
buffer (0.5 ml/tube). The collected supernatants were gently mixed for 24 
hours in an ice bath. 
The concentration of virus during the purification procedure was checked by 
neuraminidase activity, hemagghtination and ELISA. The infectivity of the 
virus was measured by the inoculation of preincubated eggs. The protein 
concentration was measured by-the method of Spector. The purity of the 
product was checked by SDS gel electrophoresis; except for HN, NP and M 
proteins no other bands (contaminants) should be seen. 
The above method displayed the following features: 
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Volume ELISA (HI) yield % 
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Original material 
3 l 154 100 
Supernatant 3 l 1 0.06 
Resuspended pellet 
42 ml 9531 87 
Supernatant over sucrose 
310 ml 467 31 
Purified virus 11 ml 20803 50 
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EXAMPLE #2 
Purification of Gumboro virus from Vero cell culture 
2300 ml supernatant of Vero cell culture was centrifuged for 30 min at 
5000.times. g at 4.degree. C. Virus was pelleted from the supernatant by 
ultracentrifugation (SCP 85 H2 ultracentrifuge, RP 19 rotor, 18,500 rpm 
(35,000.times. g, 4.degree. C., 1 h)). After discarding the supernatant, 
the pellets were resuspended in 23 ml NTE buffer (1% of the original 
volume). The suspension was gently mixed for 24 hours in an ice bath. 
The suspension was further purified by sucrose gradient 
ultracentrifugation. Thirty ml of 30% (w/w) (=33 % w/v) sucrose in NTE 
buffer was placed into centrifuge robes and 5 ml of suspension was layered 
onto the sucrose. The robes were ultracentrifuged in SRP 288A rotor at 
95,000.times. g (27,500 rpm) for 80 min. 
After discarding the supernatants, the pellets were resuspended in NTE 
buffer (1 ml/tube), then washed with 1 ml buffer. The collected 
supernatants were gently mixed for 24 hours in an ice bath. 
The concentration of virus during the purification procedure was checked by 
ELISA. The infectivity of the virus was measured by its cytopathogenic 
effect. The protein concentration was measured by the method of Spector. 
The above described method displays the following features: 
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volume ELISA (HI) yield % 
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Original material 
2300 ml 171 100 
After centrifugation 
2300 ml 133 78 
Supernatant 2300 ml 48 28 
Resuspended pellet 
28 ml 3621 26 
Supernatant over sucrose 
180 ml 212 10 
Purified virus 13 ml 5271 17 
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EXAMPLE #3 
Stabilized virus for human therapeutic use 
2-2 % (v/v) glucose, sucrose and hydroxyethyl-starch (mw: 200,000) (ISOHES, 
HES 200/0.5) were added to the virus suspension obtained from example #1, 
then lyophilized. After reconstitution, even after prolonged storage, the 
original ELISA titre was obtained.