General Adenovirus issues

Q. Is the adenovirus safe to handle?

A. The recombinant adenoviruses made with our expression system kits are defective viruses that are deleted in the E1 and E3 regions; they will not replicate in cells other than complementing cells (293 cells).
According to references issued by the NIH (National Institute of Health) Office of Biosafety, U.S. Department of Health, all serotypes of human adenoviruses have been classified in biosafety level II. Level II consists of agents that are to be considered of ordinary potential harm.

For more information on biosafety levels, please refer to the following CDC publication: Biosafety in Microbiological and Biomedical Laboratories, 4th Edition, May 1999; this publication is also available at http://bmbl.od.nih.gov

Q. What are RCAs and how can I avoid them?

A. One concern when working with Adenoviral vectors is the possible occurrence of replication competent adenoviruses (RCAs) in a population of replication deficient adenoviruses (Ad). RCAs can emerge as a result of a double crossover event between the homologous overlapping sequences present in the recombinant Ad and the 293 genome (Lochmüller, 1994). This event results in the loss of the transgene and its replacement by the E1 region (Zhu, 1999) thus rendering the Ad replication competent without the need of a complementing cell line. Lochmüller et al. demonstrated that Ad stocks contained an increasing amount of RCAs after increasing number of passages in 293 cells.

To avoid the emergence of E1-containing Ad, it is necessary to 1- always plaque purify the adenoviral stocks to be amplified, and 2- keep the passage number in 293 cells as low as possible, ideally no higher than 5.

Included below is a list of references on occurrence, characterization and screening of RCAs in adenoviral stocks.

Hehir, K. M., D. Armentano, et al. (1996). "Molecular characterization of replication-competent variants of adenovirus vectors and genome modifications to prevent their occurrence." J Virol 70(12): 8459-67.

Lochmuller, H., A. Jani, et al. (1994). "Emergence of early region 1-containing replication-competent adenovirus in stocks of replication-defective adenovirus recombinants (delta E1 delta E3) during multiple passages in 293 cells." Hum Gene Ther 5(12): 1485-91.

Louis, N., C. Evelegh, et al. (1997). "Cloning and sequencing of the cellular-viral junctions from the human adenovirus type 5 transformed 293 cell line." Virology 233(2): 423-9.

Zhang, W.-w. K., Patricia E.; Roth, Jack A. (1995). "Detection Of Wild-type Contamination In A Recombinant Adenoviral Preparation by PCR." BioTechniques 18(3): 444-447.

Zhu, J., M. Grace, et al. (1999). "Characterization of replication-competent adenovirus isolates from large-scale production of a recombinant adenoviral vector." Hum Gene Ther 10(1): 113-21.

Q. For in vivo use (animal models), is the cesium chloride purification required?

A. Yes: CsCl purification is essential in order to 1) remove defective particles, 2) remove condition media with its contaminants from the viral preparation, 3) concentrate the virus to a level suitable for injection and 4) resuspend the virus in a buffer suitable for injection.

For in vivo work, the virus must be purified since a cell lysate contains defective particles, a large quantity of fiber and penton proteins which are known to be cytotoxic, as well as media, serum and cellular debris. If the virus is to be injected into animals, the presence of these components will elicit a very strong immune response.

Q. What are the conditions recommended for the storage of recombinant Adenovirus preparations?

A. The viruses should be stored -80°C especially after purification from culture media. In optimal buffer (20mM Tris, pH 8.0 with 25 mM NaCl and 2.5% glycerol) the virus will be stable for 1-2 years; the virus should also be aliquoted to avoid multiple freeze-thaw cycles. Long-term storage at -20°C is not recommended.

Reference:

Hoganson, D.K., et al. Development of a stable adenoviral vector formulation. (2002) BioProcessing 1(1):43-48.

Virus in DMEM supplemented with serum needs to be stored the same way as purified particles but is usually much more stable than in buffer. Storage of virus in DMEM with serum at 4°C for 1 to 2 days to avoid freeze-thaw cycles is acceptable.

Q. What type of buffer is recommended for dialysis?

A. The best buffer to use is 20 mM Tris pH 8.0, 25 mM NaCl, 2.5 % glycerol; this buffer enables one to concentrate the virus to approximately 1 x 10¹³ VP/mL (viral particle/mL) without precipitation and provides very good stability for long-term storage and shipping.

If the virus is to be used for animal studies, a buffer with a high glycerol concentration is not recommended since it would become difficult to inject. PBS buffers can also be used but do not provide very good viral stability and should be avoided if the virus has to be concentrated; the particles will likely precipitate due to the low pH (~7) involved. Using a PBS buffer will enable concentration of the virus up to approximately 5 x 10¹¹ VP/mL without precipitation. Viruses in PBS buffer will also be severely affected by repeated freeze/thaw cycles. For these reasons we recommend the Tris buffer over PBS for all applications.

Reference:

Nyberg-Hoffman, C. and E. Aguilar-Cordova (1999). "Instability of adenoviral vectors during transport and its implication for clinical studies." Nat Med 5(8): 955-7.

Q. What is the capacity of cloning into the Adenovirus as an expression system?

A. The cloning capacity is the maximal length of recombinant DNA that can produce infectious viral particles without reduction in the efficiency of viral replication, and corresponds to 105% of the wild type genome.
The maximal cloning capacity on the other hand is the maximal length of recombinant DNA that can allow production of a recombinant virus. A recombinant virus without gene rearrangement is however difficult to obtain under these conditions. It was demonstrated that the maximum size of DNA that can be packaged is 106.5% of the wild type virus. However the virus produces plaques that are smaller and appear more slowly then usual. The yield of production is reduced 2 - 10 fold compared to a virus that would have 104 - 105 % of the wild type.

For these reasons it is strongly recommended not to exceed a DNA length of 105% of the wild type.

Reference:

Jani, A., H. Lochmuller, et al. (1997). "Generation, validation, and large scale production of adenoviral recombinants with large size inserts such as a 6.3 kb human dystrophin cDNA." J Virol Methods 64(2): 111-24.

Q. What type of cell is used to produce and grow recombinant Adenovirus in your systems?

A. The cells used in the kit for the production of recombinant Adenoviruses are a Human Embryonic Kidney cell line, the HEK-293A cells (A stands for adherent cells). The 293 cells contain the full E1 region of the Adenovirus type 5, from nucleotides 1 to 4344 of Ad5 wt, making these cells suitable for the generation and growth of helper-independent recombinant Adenoviruses.

Our subclone is a very reliable post-crisis cell line that adheres strongly to plastic, grows very easily, and performs very well in plaque assays and in transfection protocols.

References :

Graham, F. L., J. Smiley, et al. (1977). "Characteristics of a human cell line transformed by DNA from human adenovirus type 5." J Gen Virol 36(1): 59-74.

Louis, N., C. Evelegh, et al. (1997). "Cloning and sequencing of the cellular-viral junctions from the human adenovirus type 5 transformed 293 cell line." Virology 233(2): 423-9.