Cell-derived viral vaccines with low levels of residual cell DNA

Abstract

The present invention relates to vaccine products for the treatment or prevention of viral infections. Further provided are methods of reducing contaminants associated with the preparation of cell culture vaccines. Residual functional cell culture DNA is degraded by treatment with a DNA alkylating agent, such as β-propiolactone (BPL), thereby providing a vaccine comprising immunogenic proteins derived from a virus propagated on cell culture, substantially free of residual functional cell culture DNA.

Claims

1. A vaccine comprising immunogenic proteins derived from two or more influenza virus strains propagated on cell culture, wherein the vaccine comprises 10 pg/mL to less than 20 ng/mL of residual cell culture DNA, wherein the length of the residual cell culture DNA is less than 200 base pairs, and wherein the cell culture is a mammalian cell culture or an avian cell culture.

2. The vaccine of claim 1, comprising less than 0.1% β-propiolactone (BPL).

3. The vaccine of claim 1, wherein the cell culture is selected from the group consisting of Madin Darby canine kidney (MDCK) cells, Vero cells, and human embryonic retinoblasts.

4. The vaccine of claim 1, further comprising an oil-in-water emulsion adjuvant.

5. The vaccine of claim 4, wherein the oil-in-water emulsion adjuvant comprises oil droplets having a sub-micron diameter.

6. The vaccine of claim 5, wherein the oil-in-water emulsion adjuvant comprises squalene.

7. The vaccine of claim 1, wherein the immunogenic proteins are viral antigens selected from the group consisting of hemagglutinin (HA), neuraminidase (NA), nucleoprotein (NP), matrix protein (M1), membrane protein (M2), and one or more of the transcriptase components (PB1, PB2 and PA).

8. The vaccine of claim 7, wherein the immunogenic proteins are NA.

9. The vaccine of claim 7, wherein the immunogenic proteins are HA.

10. The vaccine of claim 9, comprising about 7.5 μg, about 10 μg, or about 15 μg HA per influenza virus strain in a single dosage volume of about 0.5 mL.

11. The vaccine of claim 1, comprising immunogenic proteins derived from three or more influenza virus strains propagated on cell culture.

12. The vaccine of claim 1, wherein the length of the residual cell culture DNA is from 20 to 200 base pairs.

13. The vaccine of claim 1, wherein the length of the residual cell culture DNA is about 72 base pairs.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIGS. 1 and 2 show the results of PCR amplification of 6 named targets in the MDCK genome. Some samples were diluted as indicated, up to 1:10000, prior to PCR.

(2) FIG. 3 shows the results of PCR amplification of SINE sequences in the MDCK genome. As in FIGS. 1 & 2, DNA was sometimes diluted prior to PCR. In the lower panel, the figures are the starting amount of DNA in the PCR (fg).

(3) FIG. 4 shows the effect of BPL treatment on the size of MDCK DNA. Genomic DNA was incubated with BPL at different temperatures and for different lengths of time. FIG. 5 shows similar results. Agarose gels were stained with SYBRGold.

(4) FIG. 6 shows capillary electrophoresis of residual DNA (top line) relative to markers (bottom line) having the indicated sizes.

MODES FOR CARRYING OUT THE INVENTION

(5) Influenza viruses (A/New Caledonia/20/99(H1N1), A/Panama/2007/99(H3N2); B/Jiangsu/10/2003; A/Wyoming/3/2003(H3N2)) were grown in MDCK cells in a suspension culture, following the teaching of WO97/37000, WO03/23025 and WO04/92360. The final culture medium was clarified to provide virions, which were then subjected to chromatography and ultrafiltration/diafiltration. Virions in the resulting material were inactivated using β-propiolactone (final concentration 0.05% v/v; incubated for 16-20 hours at 2-8° C., and then hydrolyzed by incubating at 37° C. for 2-2.5 hours). CTAS was then used to split the virions, and various further processing steps gave a final monovalent bulk vaccine containing purified surface proteins.

(6) MDCK DNA was characterized to evaluate its amount, size, and integrity at three stages of the manufacturing process: (A) after the ultrafiltration/diafiltration step; (B) after β-propiolactone treatment; and (C) in the final monovalent bulk. Capillary gel electrophoresis and nucleic acid amplification were used to investigate the size, the integrity and the biological activity of any residual genomic DNA.

Size Determination

(7) As mentioned above, the size of residual host cell DNA was analyzed at stages (A), (B) and (C) by capillary gel electrophoresis. The analysis was performed on five separate virus cultures.

(8) 500 μl samples were removed at these three points and treated with 10 μl proteinase K at 56° C. for 16 to 22 h followed by a total DNA extraction with the DNA Extractor Kit (Wako Chemicals) following the manufacturer's instructions. DNA was resuspended in 500 μl ultra pure water for electrophoresis on a P/ACE MDQ Molecular Characterization System (Beckman Coulter) at a constant temperature of 20° C. Eleven molecular size markers were used, from 72 to 1353 bp. The nucleic acid detection limit (DL) for this method was 0.7 μg/ml.

(9) Based on the size markers and relative density of the DNA bands, the distribution and size of the DNA fragments were determined and assigned to four different size categories between ranging from <200 bp to >1000 bp. FIG. 6 shows capillary electrophoresis of a stage (C) sample. The analysis shows that all detectable residual DNA in this sample is substantially below 200 bp in length.

(10) Average results from the five cultures are shown in the following table:

(11) TABLE-US-00001 Stage DNA amount <200 bp 200-500 bp 500-1000 bp >1000 bp A 47 mg 25% 12% 6% 55% B 5 mg 79% 18% 3%  4% C 0.07 mg >99%  <DL <DL <DL

(12) BPL treatment thus causes a ˜10-fold reduction in the amount of DNA, but also shifts the distribution away from long sequences towards small fragments <200 bp. Further processing, between steps (B) and (C), including chromatography and ultrafiltration steps, reduced total DNA levels another ˜70-fold, and removed all detectable DNA ≥200 bp.

DNA Amplification

(13) Neoplastic cell transformation is a phenomenon often associated with modified proto-oncogenes and/or modified tumor suppressor genes. Sequences from several such canine genes were analyzed by PCR before and after BPL treatment i.e. at points (A) and (B). In addition, DNA from uninfected MDCK cells was treated and tested. Proto-oncogenes tested were: H-ras and c-myc. Tumor suppressor genes tested were: p53; p21/waf-1; and PTEN. In addition, repetitive SINE sequences were analyzed by PCR. The high copy number for SINES facilitates sensitive detection.

(14) All samples were spiked with an external control DNA (pUC19 fragment) for monitoring the quality of the sample preparation and the PCR. In all experiments a consistent amplification of the spike control was observed, indicating that residual hydrolyzed BPL and by-products present in the PCR mixtures does not have inhibitory effects on the assay and ensured a sufficient quality of sample preparation and PCR. Samples were diluted in 10-fold steps before amplification, until PCR products could not be detected, thereby indicating the log reduction in DNA levels. The detection limit for the PCR assay is 55 pg.

(15) PCR products were analyzed by agarose gel electrophoresis. FIG. 1 shows results obtained in uninfected MDCK cells, and FIG. 2 shows results obtained during virus culture. All six analyzed genes showed a strong amplification signal before BPL treatment, but signal strength diminished after exposure to BPL. In all tested production lots, residual MDCK genomic DNA was decreased by at least 2 log values after BPL exposure.

(16) Because FIG. 2 shows results at stage (B), before further purification, the results over-represent DNA present in final bulk vaccines. Due to the sensitivity when using SINE sequences, however, PCR was also possible at stage (C), in the final monovalent bulk. Results of this analysis are shown in FIG. 3. DNA amplification in (A) and (B) was relatively similar for the SINE region, indicating that BPL is less active on smaller DNA sequences. Even for these small sequences, however, there was a notable reduction in the amplification signal for all of the analyzed samples. Comparison of the PCR signal intensities between sample collection point (B) and (C) reflects the reduction of residual DNA due to intermediate purification steps.

(17) Extrapolating from the gene-based analysis, and based directly on the SINE-based analysis, a total DNA level of <1 ng per dose, and often <100 pg per dose, is expected in a final vaccine.

Temperature

(18) BPL treatment during virus inactivation had two independent steps: (1) add BPL to the virion-containing mixture at 2-8° C.; then (2) raise the temperature to 37° C. to hydrolyse BPL. The effects of the two BPL steps on MDCK DNA inactivation and fragmentation were investigated.

(19) Purified genomic DNA from uninfected MDCK cells was treated with a final concentration of 0.05% (v/v) BPL at 2-8° C. for 16 hours, or at 37° C. or 50° C. for up to 6.5 hours. Fragmentation of DNA was checked afterwards, and results are shown in FIG. 4. These experiments reveal the activity of BPL against cellular DNA, but uninfected cells do not reflect the state of MDCK after viral growth because cellular DNA is already highly fragmented due to apoptosis.

(20) At 2-8° C., the difference between untreated and treated DNA on an agarose gel was negligible, suggesting that DNA fragmentation by BPL may not substantially occur during under these conditions. In contrast, DNA was greatly modified at both 37° C. and 50° C., with the higher temperature leading to an accelerated reaction kinetic. DNA from untreated cells showed a relatively distinct band in an agarose gel, with a light smear of degradation products (FIG. 5, lane 3). After a short BPL incubation at 37° C., however, MDCK DNA smeared down from the gel slots and band signal intensity decreased (FIG. 5, lane 2). Longer incubation periods resulted in the disappearance of large genomic DNA molecules and enhanced fragmentation of MDCK DNA.

(21) In further experiments, BPL was first incubated with cells at 4° C. for 16 hours. In a first population, the temperature was raized to 37° C. for 2 hours; in a second population, BPL was removed by centrifugal filters and then the temperature was raized. Much lower (>2 log lower) residual DNA levels were seen in the first population.

(22) Thus the DNA fragmentation observed during BPL treatment seems to occur mainly during the BPL hydrolysis step at 37° C. rather than during the virus inactivation step at 2-8° C.

(23) It will be understood that the invention has been described by way of example only and modifications may be made whilst remaining within the scope and spirit of the invention.