FAQ – Recombinant Adenovirus

What’s the difference between AAV and Adenovirus?

You can read about the primary differences between the two viral vectors here.

What is the required biosafety level for using recombinant adenoviruses?

The recombinant adenoviruses we produce are replication deficient due to deletions in the E1 and E3 regions. According to references issued by the NIH Office of Biosafety, recombinant human adenoviruses are classified as biosafety level II for agents considered ordinary potential harm.  You need a BL-2 level facility to work with them. It should be noted that cell culture facilities in most institutes are certified as BL-2 level.

Wild type, replication competent adenoviruses provoke cold symptoms and strong immune responses in healthy individuals but generally do not cause serious illness.

For more information on biosafety levels, please read the NIH Biosafety Guidelines.

How can I find out whether adenoviruses will work well in my cell models?

The adenovirus has a very broad host range; it can infect human and other mammalian cell lines or primary cells, including replicative as well as non-replicative cells. In fact, there are very few cell lines that cannot be infected. Some lymphoid cell lines may be more resistant to adenoviral infection, and may need higher viral quantities to achieve sufficient infection levels.

For your convenience, we offer some marker adenoviruses, such as Ad-CMV-ß-Gal or Ad-CMV-GFP to allow you to conduct pilot testing in your systems.

What’s the optimal concentration of viruses that I should use for infection?

The appropriate amount of active/infectious viruses used for infecting cells is very important for the outcome of your experiments. If not enough virus is used, you will not reach 100% infection. If too much is used, it will result in cytotoxicity or other undesired effects. You should use the minimum viral concentration that will result in 100% gene delivery. This optimal concentration differs dramatically between cell types. To determine this optimal viral concentration, you could conduct pilot testing in your system by using marker adenoviruses, such as Ad-CMV-ß-gal.

We have tested numerous cell types by exposing cells to virus-containing media for 6-8 hours or overnight. For most cell types, viral concentrations of 2 x 10⁵ – 1 x 10⁶ IFU/PFU (infectious unit)/ml of media results in 100% infection without visible side effects. However, we recommend you test your cell system first by using marker viruses.

How much media should I use during infection?

For your reference, we recommend the following amount virus-containing media for infection:

• 10-cm plate: 8-10 ml per plate
• 6-well plate: 1.0 ml per well
• 12-well plate: 0.5 ml per well
• 24-well plate: 0.2 ml per well

This roughly reflects the surface area of each well or plate.

What are the differences between Viral Particle (VP), Plaque Formation Unit (PFU) and Infectious Unit (IFU)? Which one of these better reflects the amount of active virus used?

Viral Particles (VP) represent the total number of both live and dead viral particles. Due to variations in virus preparations, the ratio of live/dead varies significantly. Therefore, VP does not reflect the amount of active virus in the preparation.

Plaque Formation Unit (PFU) represents the number of infectious or live viruses. It reflects the amount of working viruses in the preparation.

Infectious Unit (IFU) is equivalent to PFU.

For most virus preparations , the VP/PFU ratio is 20:1 to 50:1.

Using Viral Particles (VP) as measurement will result in significant variations in the amount of actual live viruses present, whereas using IFU or PFU as the viral unit will give more consistent outcomes.

How are virus titers determined?

There are 3 commonly used protocols for determining adenovirus titer: (1) OD260 Assay, (2) Plaque Formation Assay, and (3) End-point Dilution Assay.

OD260 assay measures the concentration of viral DNA and protein. It does not distinguish between intact, infectious viruses and damaged, non-infectious viruses. It is a physical assay measuring the concentration of total viruses, both live and dead. Based on OD260, the concentration of viral particles (VP) could be obtained. To measure OD260, the virus stock has to be purified first.

On the other hand, Plaque Formation Assay measures the concentration of infectious viruses, and therefore is a biological assay. Essentially, a mono-layer of HEK293 cells are infected with a series of virus dilutions. Viruses will propagate in the infected cells, and eventually cause cytotoxicity effects before being released. The released viruses will infect neighboring cells, and the whole process will be repeated, eventually leading to the formation of holes (or plaques) on the cell monolayer. In order to prevent the diffusion of viruses and plaque formation, top agarose is layered on top of cells after the initial infection.

The biological principle for End-Point Dilution Assay is similar to the Plaque Formation Assay, although the procedure and measurement is different, and the formula for calculating the virus titer is more complicated.

Although both the Plaque Formation Assay and End-Point Dilution Assay gives the titer of infectious or working viruses, they are scored by the human eye and subject to human and procedural variations. For the same virus stock, it is not uncommon that two different people will get significantly different titer readings.

For in vitro use (cell culture studies), is CsCl- or chromatography purification required?

No. If the viruses will be used in in vitro cell cultures, double CsCl purification is not required. For in vivo studies (i.e. animal studies), purification is essential in order to remove defective particles, cell debris, and small amounts of media components, as these contaminants induce significant immune responses. In addition, CsCl purification will concentrate the virus to a level suitable for in vivo injections.

What are the recommended storage conditions of recombinant adenoviruses?

For long-term storage, the virus should be kept at -80°C, especially after CsCl or chromatography purification. At -80°C, the virus should be stable for 6 months to a year (and in some cases, up to 2 years).

If the virus is in DMEM supplemented with serum or BSA and stored at -80°C, it will remain stable for longer periods of time and through several freeze-thaw cycles.

On the other hand, if the virus CsCl-purified and kept in PBS or Tris solution (20mM Tris PH8.0, 200 NaCl, 2-3% glycerol or sucrose), repeated freeze-thaw cycles should be avoided, as it will cause a significant decrease in titer.

What is the capacity of cloning into the adenovirus as an expression system?

The cloning capacity for the transgene, by using (DE1/E3) adenovirus type 5, is about 8 Kb in length.

There are many adenovirus serotypes, which one is the most commonly used for gene delivery?

The most commonly used adenovirus for gene delivery is human adenovirus serotype 5 (DE1/E3), which we produce.

What are RCAs?

One concern when working with adenoviral vectors is the rare occurrence of replication-competent adenoviruses (RCAs) in a population of replication-deficient viruses. RCAs can emerge from a rare double crossover through overlapping sequences present in the recombinant adenovirus and the genome of HEK293 cells. This results in replacement of the transgene by the E1 region. Once this happens, the adenovirus can replicate without a complementing cell line. To detect RCA, non-complementary cells (such as A549 cells) are incubated with the viral stocks (1E4-1E6 PFU) and monitored for cytopathic effects (CPE) and/or plaque formation. According to NIH guidelines, less than 1 plaque in about 1E4 viruses is considered safe to use. To avoid the occurrence of RCAs, viruses should be produced and amplified in low passage packaging cells.