METHOD FOR INACTIVATING VIRUSES USING ELECTRON BEAMS

Abstract

The invention relates to a method for inactivating viruses, characterized in that an immunogenic composition or vaccine comprising at least one virus is irradiated with electron beams, said immunogenic composition or vaccine comprising at least one virus (i) being liquid, in particular being a suspension and (ii) comprising at least one viral immunogen, wherein the antigen structure is preferably substantially retained.

Claims

1. A method for inactivating viruses, characterized in that an immunogenic composition or vaccine comprising at least one virus is irradiated with electron beams, said immunogenic composition or vaccine comprising at least one virus (i) being liquid, in particular being a suspension, and (ii) comprising at least one viral immunogen.

2. The method as claimed in claim 1, characterized in that the at least one virus is selected from: (i) an enveloped virus or non-enveloped virus, and/or (ii) a dsDNA virus, dsRNA virus, ssRNA virus or ssDNA virus, and/or (iii) a human pathogenic and/or animal pathogenic virus, the at least one virus preferably being selected from a human pathogenic and/or animal pathogenic enveloped dsRNA virus, enveloped ss()RNA virus or enveloped ss(+)RNA virus, particularly preferably a) that the at least one virus is selected from a human pathogenic and/or animal pathogenic ss(+)RNA virus, very particularly preferably that the at least one virus is selected from a virus of the Arterividae family, and even more preferably that the at least one virus is a porcine reproductive and respiratory syndrome virus (PRRS virus), or b) that the at least one virus is selected from an animal pathogenic virus, the influenza A and B virus, the TBEV virus, the IPV virus and the hepatitis A virus.

3. The method as claimed in either of claim 1 or 2, characterized in that the vaccine or immunogenic composition (i) comprises one virus or (ii) two or more different viruses.

4. The method as claimed in any of claims 1 to 3, characterized in that the electron beams are accelerated at low energy or moderate energy, preferably accelerated with an acceleration energy of between 150 key and 700 keV, more preferably of between 200 keV and 500 keV, even more preferably of between 250 keV and 400 keV and/or are applied essentially under standard atmospheric pressure, preferably wherein the standard atmospheric pressure is present as atmospheric oxygen, nitrogen or carbon dioxide gas.

5. The method as claimed in any of claims 1 to 4, characterized in that the immunogenic composition or vaccine comprising at least one virus is irradiated with an electron beam dose in the range of 50 kGy to 300 kGy, preferably 50 kGy to 200 kGy, further preferably 50 kGy to 150 kGy, more preferably 50 kGy to 120 kGy, even more preferably 50 kGy to 110 kGy.

6. The method as claimed in any of claims 1 to 5, characterized in that the activity of the at least one virus after irradiation, preferably measured as a TCID50 value, is less than 5%, preferably less than 1%, more preferably less than 0.1% of the activity prior to irradiation, even more preferably that no activity of the at least one virus is still detectable after irradiation.

7. The method as claimed in any of claims 1 to 6, characterized in that the at least one virus is an enveloped virus.

8. The method as claimed in any of claims 1 to 7, characterized in that the antigen structure of the viruses of the immunogenic composition or vaccine is substantially retained after irradiation, preferably that the binding of a polyclonal serum directed against the non-inactivated virus to the at least one virus of the irradiated immunogenic composition or vaccine is at least 40%, preferably at least 70%, more preferably at least 80%, even more preferably at least 90% of the binding of the polyclonal serum to the at least one virus of the immunogenic composition or vaccine prior to irradiation, in particular wherein the binding of the polyclonal serum to the at least one virus of the immunogenic composition or vaccine is determined by ELISA.

9. The method as claimed in any of claims 1 to 8, characterized in that the virus structure of the viruses is substantially retained after irradiation.

10. The method as claimed in any of claims 1 to 9, characterized in that (a) the irradiation is carried out using a device for generating electron beams, (i) which is operated preferably continuously or rapidly pulsed, and/or (ii) which provides electrons preferably according to the cold or hot cathode principle and/or (iii) which is embodied as an axial emitter (scanner) or linear broadband emitter, and/or (iv) the electrons, after discharge through an emission window of the evacuated generating chamber of the device, preferably are applied to the immunogenic composition or vaccine, wherein the immunogenic composition or vaccine is preferably in a container, and/or (v) the immunogenic composition or vaccine is preferably incorporated statically in the device, or is continuously transported through the electron beam, and/or (b) the dose rate is in the range of 1 kGy/0.1 sec to 150 kGy/1000 sec and/or (c) the irradiation time is between 0.1 sec to 1000 sec, preferably between 1 and 100 sec, and/or (d) the temperature of the immunogenic composition or vaccine prior to irradiation is between 1 C. and 40 C., preferably between 5 C. and 37 C., more preferably between 10 C. and 32 C., even more preferably between 15 C. and 30 C., and/or (e) the temperature increase of the immunogenic composition or vaccine after irradiation compared to before irradiation is between 1 C. and 15 C., preferably between 2 C. and 10 C., and/or (f) the temperature of the immunogenic composition or vaccine after irradiation is between 2 C. and 41 C., preferably between 6 C. and 38 C., more preferably between 11 C. and 33 C., even more preferably between 16 C. and 31 C., and/or (g) the density of the immunogenic composition or vaccine is between 0.9 and 2 g/cm.sup.3, preferably between 1.0 and 1.8 g/cm.sup.3, and/or (h) the immunogenic composition or vaccine comprising at least one virus is a liquid suspension comprising water, preferably is a suspension of the at least one virus in an aqueous solution, wherein the aqueous solution particularly preferably comprises one or more buffer substances.

11. The method as claimed in any of the preceding claims, characterized in that the immunogenic composition or vaccine comprises (a) one or more adjuvants, and/or (b) pharmaceutically acceptable excipients and/or auxiliaries and/or (c) one or more further immunogens.

12. The method as claimed in claim 11, characterized in that one or more further immunogens is selected from (a) an organic substance, particularly a protein, which may be glycosylated or non-glycosylated, a nucleic acid, a toxin, or a sugar molecule which is optionally bound to a support, and (b) a virus or a living organism, particularly a bacterium, wherein the virus or living organism may be active or inactivated.

13. A method for preparing a vaccine comprising at least one viral immunogen, particularly a vaccine comprising a viral whole particle vaccine, characterized in that: (a) the method is carried out as claimed in any of claims 1 to 11, (b1) one or more adjuvants are optionally added to the immunogenic composition comprising at least one virus, and/or (b2) one or more pharmaceutically acceptable excipients and/or auxiliaries are optionally added to the immunogenic composition comprising at least one virus, and/or (b3) one or more further immunogens are optionally added to the immunogenic composition comprising at least one virus, wherein the steps (a) to (b3) are carried out in any sequence.

14. The method for preparing a vaccine as claimed in claim 13, characterized in that the following further steps are carried out: (c) sterilising the immunogenic composition, and/or (d) filling the immunogenic composition in a container, wherein steps (a) to (d) may be carried out in any sequence, and following steps (a) to (d) the vaccine is optionally dried, freeze-dried or frozen.

15. An immunogenic composition or vaccine, preferably vaccine, particularly preferably comprising an inactivated viral whole particle vaccine, which may be prepared by a method of claims 1 to 14.

16. An immunogenic composition or vaccine, preferably vaccine, comprising an inactivated viral whole particle vaccine for an enveloped or non-enveloped virus, characterized in that (a) the activity of the virus in the immunogenic composition or vaccine is less than 10%, preferably less than 1%, more preferably less than 0.1% of the activity of the same number of non-inactivated viruses, and (b) the antigen structure of the inactivated viruses in the immunogenic composition or vaccine is substantially the same compared to the same number of non-inactivated viruses.

17. The immunogenic composition or vaccine as claimed in claim 16, (a) wherein the virus structure of the inactivated viruses is substantially the same compared to the same number of non-inactivated viruses, and/or (b) wherein the immunogenic composition or vaccine has been irradiated with an electron beam, and/or (c) wherein no activity of the at least one virus is detectable in the immunogenic composition or vaccine.

18. The immunogenic composition or vaccine as claimed in any of claims 15 to 17, wherein the immunogenic composition or vaccine has been irradiated with an electron beam dose in the range of 50 kGy to 300 kGy, preferably 50 kGy to 200 kGy, further preferably 50 kGy to 150 kGy, more preferably 50 kGy to 120 kGy, even more preferably 50 kGy to 110 kGy.

19. The immunogenic composition, preferably vaccine, as claimed in any of claims 15 to 18, for use as a vaccine, in particular for preventing or treating viral infections or disorders caused by the virus.

20. The use of electron beams (a) for inactivating viruses in an immunogenic composition or vaccine comprising at least one virus, said immunogenic composition or vaccine (i) being liquid, in particular being a suspension, and (ii) comprising at least one viral immunogen, and/or (b) for preparing an inactivated viral whole particle vaccine.

21. The use as claimed in claim 20, wherein the electron beams are accelerated at low energy or moderate energy, preferably accelerated with an acceleration energy of between 150 keV and 700 keV, more preferably of between 200 keV and 500 keV, even more preferably of between 250 keV and 400 keV and/or are applied essentially under standard atmospheric pressure, wherein the standard atmospheric pressure is present as atmospheric oxygen, nitrogen or carbon dioxide gas and/or the electron beam dose is in the range of 50 kGy to 300 kGy, preferably 50 kGy to 200 kGy, further preferably 50 kGy to 150 kGy, more preferably 50 kGy to 120 kGy, even more preferably 50 to 110 kGy.

22. The use of a device for generating electron beams (a) for inactivating viruses in liquid immunogenic compositions, more preferably liquid vaccines, and/or (b) for preparing an inactivated viral whole particle vaccine.

23. The use as claimed in claim 22, wherein the device a) is suitable for emitting electron beams accelerated at low energy or moderate energy, preferably accelerated with an acceleration energy of between 150 keV and 700 keV, more preferably of between 200 keV and 500 keV, even more preferably are accelerated between 250 keV and 400 keV and/or for applying electron beams essentially under standard atmospheric pressure, and/or b) is suitable for delivering an electron beam dose of 50 kGy to 300 kGy, preferably 50 kGy to 200 kGy, further preferably 50 kGy to 150 kGy, more preferably 50 kGy to 120 kGy, even more preferably 50 to 110 kGy.

24. The use of a method as claimed in any of claims 1 to 14 for preparing an inactivated viral whole particle vaccine.

Description

FIGURES

[0186] FIG. 1: shows the PRRS virus activity after treatment with the electron beam (gray) and without irradiation (black). Stated values are TCID50 per ml of virus solution. Dose: 100 kGy.

[0187] FIG. 2: shows the results for the antigen integrity after treatment. PRRS viruses were treated with an electron beam (gray), formaldehyde (stripes) or untreated (black). The integrity of the antigens was determined via incubation using a polyclonal serum from a PRRSV infected pig. Stated values are OD (optical density) in the ELISA. Dose: 100 kGy.

[0188] FIG. 3: shows the results for the integrity of the virus envelope after treatment. PRRS viruses were treated with an electron beam (gray), peroxide (stripes) or untreated (black). The integrity of the virus envelope was analyzed using an antibody against the capsid (N) protein of PRRSV. Stated values are OD in the ELISA. Dose: 100 kGy.

[0189] FIG. 4: H3N8 viruses were treated with low-energy electrons of 200 kGy (E) or 0 kGy (NC) and their activity measured by TCID50 determination.

[0190] FIG. 5: the integrity of the antigens was determined by incubation with a serum from an influenza-positive human. Values are absorption signals in an ELISA test. E: H3N8 viruses treated with an electron beam dose of 200 kGy; NC: H3N8 viruses treated with 0 kGy (control).

EXAMPLE 1 SUMMARY OF THE EXPERIMENTS

[0191] The experiments were carried out, for example, using the PRRS virus (porcine respiratory and reproductive failure syndrome virus). This virus is a single-stranded, positive strand RNA virus of the Arteriviridae family. The virus affects pigs and causes annual losses in the pig industry in the billions.

[0192] PRRSV in 100 L of liquid medium was conducted through the electron beam and irradiated with 100 kGy. The amount of virus used was 2*10.sup.4 TCID50 per batch. Subsequently, the activity of the pathogens and the conservation of their antigens was investigated.

[0193] For the activity determination, the viruses (and the untreated controls) were added to Marc145 cells and the TCID50 value determined three days later. The irradiation resulted in a reduction of the activity by 2.5 logarithmic steps compared to the control (FIG. 1). The quality of the antigens was investigated by measurements using antibodies. For this purpose, sera from pigs which had been infected with a vaccine strain of PRRSV were investigated. Immunisation with a live virus leads to a comprehensive humoral immune response against the antigens in their original undamaged state. Therefore, the extent of binding of polyclonal antibodies from an animal thus immunized to an antigen is a direct indicator of the integrity of the antigen. FIG. 2 clearly shows that the binding properties of the pig serum to the PRRSV viruses have hardly changed as a result of irradiating the viruses. Consequently, almost all antigens are still in their original undamaged state, although the virus was inactivated by 2.5 logarithmic steps. PRRS viruses were also inactivated with formaldehyde for comparison. This process resulted in a significant decrease in the ELISA signal and therefore a clear destruction of the antigens.

[0194] It was also investigated to what extent the inactivation process affects the virus structure. This was measured using an antibody against the capsid protein (N-protein) of the PRRSV. This protein is protected by the intact virus envelope and is not accessible by the antibody. Therefore, a signal indicates a damaged virus envelope. As FIG. 3 shows, the virus envelope remains intact after irradiation while the peroxide inactivation according to Amanna et al. (supra) leads to significant damage to the structure. These data indicate that during the electron irradiation the inactivation is presumably due mainly to the destruction of the nucleic acids while the antigens and the virus structure remain largely unaltered.

[0195] The method described, therefore, is suitable for preparing inactivated virus particles, e.g. for the preparation of vaccines, where it has clear advantages over formaldehyde: the antigens are far better preserved and the addition of toxic chemicals can be dispensed with.

EXAMPLE 2: MATERIALS AND METHODS

[0196] Virus Culture, Inactivation and Irradiation

[0197] Cell cultures of Marc-145 cells were infected with PRRSV (DV vaccine strain). After three days, the supernatants were removed and centrifuged at 4000g at 4 C. for 15 minutes. The supernatants thus clarified were layered on a 15% sucrose cushion and ultracentrifuged at 100 000g for three hours. This supernatant was removed and the pellet resuspended in sterile PBS (phosphate-buffered saline, pH 7.4). After determining the infectivity, the virus suspension was adjusted to a concentration of 2*10.sup.5 TCID50/mL. Each 100 L of this solution was added to 6-well plates (which had been previously coated with 0.5% agarose) and irradiated at 50, 100 and 200 kGy. Negative controls were treated identically up to the irradiation.

[0198] After irradiation, the virus-containing solution was removed and was further used in TCID50 and antigen measurements. PRRSV was inactivated with 0.3% formaldehyde for 22 hours. The formaldehyde was then removed again from the virus suspension by dialysis. The inactivation by peroxide was carried out according to Amanna et al. (supra) in 3% H.sub.2O.sub.2 for 22 hours, followed by dialysis.

[0199] TCID50 Measurements

[0200] In order to investigate the activity of the irradiated viruses, serial dilutions (each in steps of 1:10) of the virus suspensions in Marc-145 cells were added to 96-well plates. Three days later, the cytopathic effect (CPE) was determined. The TCID50 corresponds to the dilution at which 50% of the infected cell culture wells still have a CPE.

[0201] Antigen Measurements

[0202] 1.5 L of the virus suspension (irradiated and control samples) were incubated overnight at 4 C. in 96-well microtitre plates. The next day, an ELISA (enzyme-linked immunosorbent assay) was carried out according to a standard protocol. To detect the antigens, serum from a PRRSV-infected (DV vaccine strain) pig was used (dilution 1:100). For the detection, a secondary anti-pig IgG antibody was used (conjugated with horseradish peroxidase, Zymed) at a 1:5000 dilution.

[0203] Irradiation with Electrons

[0204] The composition comprising PRRSV viruses was thinly applied to a large agarose surface for the irradiation. In detail, the following was carried out:

[0205] 1.) Preparation of 0.5% agarose gels in PBS,

[0206] 2.) Pouring the gels into gel pouring apparatus for 1 mm layer thickness,

[0207] 3.) Cutting out the gels in circular form of 3.5 cm diameter,

[0208] 4.) Drying the gels (ca. 14 h under continuous sterile work bench) in petri dishes (3.5 cm diameter), the dosimetry negative controls were then dried with a dosimeter foil already inserted,

[0209] 5.) Dispensing 100 L of virus suspension (pure PBS for dosimetry negative controls, ca 15 min. exposure)

[0210] 6.) Packaging with PET/PE film,

[0211] 7.) Irradiation under the conditions stated below.

[0212] The irradiation was carried out by quasi-stationary irradiation of 100 ml of medium in each case in air.

[0213] A continuously operating electron beam emitter (Navarone type, manufacturer: COMET) was used. The electrons were accelerated to 150 keV, the beam current was 5 mA. Distance in air between the composition comprising PRRSV viruses and the electron emission window: 45 mm.

[0214] The application of energy doses of 50, 100 and 200 kGy was performed in single steps of 25 kGy each (corresponding to 2, 4 or 8 cycles of the samples or linear passages each of 115 mm/sec). The target doses could be achieved under standard atmospheric pressure air with an accuracy of ca. 10%.

[0215] The documentation of the applied dose was performed spectrometrically using pararosaniline cyanide dosimeter films and the Risscan system at a measuring wavelength of 554 nm. For irradiation at 100 kGy, the dosimeter film was changed after 50 kGy since the dosimeter type mentioned has a measuring range of up to a maximum of 80 kGy. For the target dose of 200 kGy, the dosimeter film was correspondingly changed after 50, 100 and 150 kGy.

[0216] The blank sample showed no dose input, i.e. the applied dose is due exclusively to the electron beam treatment and the contact with PBS and the agarose gels did not cause any change to the dosimeter strips.

EXAMPLE 3: IRRADIATION OF INFLUENZA VIRUSES

[0217] Influenza A viruses (equine influenza H3N8, strain A/equine 2/Miami/1/63) were propagated in MDCK cells and concentrated by ultracentrifugation analogously to PRRS viruses (Examples 1 and 2).

[0218] The irradiation with electron beams was also conducted analogously to Examples 1 and 2 but at a dose of 0 kGy (control) and 200 kGy.

[0219] The activity measurements were performed via a TCID50 endpoint determination. The antigens were assayed in the ELISA format using the serum of an influenza A-infected human.

[0220] As with PRRSV, inactivation of the influenza viruses is shown. At a dose of 200 kGy, no active viruses were still detectable in the cell culture (FIG. 4). Nevertheless, antigens are still present and largely unchanged (FIG. 5).