2.1. Linearization of the recombinant adenovirus plasmid DNA to be used in transfection

Adenovirus packaging systems like HEK293-based systems typically require linear DNA for efficient rescue and packaging of the recombinant virus [13]. Most recombinant adenovirus plasmids are maintained in circular form in bacteria for stability, but they need to be cut at a specific site, often PacI or another unique site, to mimic the natural viral genome structure before being introduced into mammalian cells.

The recombinant adenovirus plasmid usually contains a bacterial backbone used for propagation in E. coli, including antibiotic resistance genes (e.g., ampicillin or kanamycin resistance) and bacterial origin of replication. These elements are non-essential and potentially harmful if carried into mammalian cells. Restriction digestion removes these sequences, ensuring: better biosafety (i.e., antibiotic resistance genes are not introduced into human/animal cells) [14], improved transgene expression since bacterial elements can suppress transcription, and higher purity of viral genome for packaging.

Linearised adenoviral DNA allows the cellular machinery to properly recognize the Inverted Terminal Repeats (ITRs), which are crucial for initiating replication and packaging the genome into capsids [13]. If the plasmid is not linearized, packaging might fail or be highly inefficient [13]. Circular plasmids may lead to non-homologous recombination and rearranged or defective viral genomes [15]. This could result in non-functional or replication-deficient viruses that do not express your gene of interest properly. Therefore, Restriction digestion serves as a quality check to confirm that the plasmid has the correct insert and that no major rearrangements or contaminations have occurred. Maintaining aseptic conditions is essential throughout the process of linearising circular plasmid DNA.

Use the PacI restriction enzyme (NEB, Cat. R0547S) to cleave the recombinant adenovirus plasmid to release the intact adenovirus vector genome. The PacI sites flank both 5′ and 3′ ITRs located at the ends of the adenovirus vector genome. In a 1.5 mL Eppendorf tube placed on ice, set up the digest reaction using 1 µg of recombinant adenovirus plasmid DNA, 0.2 μL of PacI restriction enzyme, 1 μL of CutSmart^TM buffer (10X) and top up the reaction volume to 10 μL using nuclease-free water. Incubate the digest reaction for 60 min at 37°C and terminate the reaction by denaturing the enzymes at 65°C for 10 minutes.

To confirm the successful release of the CAV-2 vector genome from the plasmid backbone, analyse 1 μL of digested plasmid using electrophoresis on a 1% agarose gel.

2.2. Transfection of AD-293 cells with linearised adenovirus vector DNA to express the virus vector

Transfection is the process of deliberately introducing foreign nucleic acids, in this case, the linearised adenovirus vector DNA, into eukaryotic cells [16]. This enables the expression of the recombinant adenovirus in viral particle form. In this protocol, the AD-293 cell line (Cat 240085, Agilent, U.S.A.) was used. AD-293 cells are a derivative of HEK293 cells, optimised for high transfection efficiency and adenovirus production. These cells express adenoviral E1 genes, which are essential for rescuing and propagating E1-deleted adenovirus vectors. Once inside the cells, the linearised viral genome is recognised by the host cell machinery. The virus is replicated, assembled, and released into the culture medium as recombinant adenovirus particles. The step-by-step transfection procedure can be conducted as described below.

One day before transfection, seed AD-293 cells in a 6-well culture plate at 0.5 x10⁶ cells per well in 2 mL of 2% cDMEM and incubate the plate at 37°c and 5% CO_2.

When cell confluency reaches 50% to 70% on the next day, proceed with transfection.

For each transfection, label two 0.5-ml Eppendorf tubes as tubes A and B. Add 1 µg of PacI-digested recombinant adenovirus plasmid DNA to tube A and adjust the total volume to 60 µL with plain DMEM. Add 6.4 µL of Lipofectamine and 53.6 µL of plain DMEM to tube B. Gently mix the contents of each tube separately by swirling the tubes. Do not vortex. Combine the solutions of tubes A and B using a wide-bore pipette and mix them gently by swirling. Allow the mixture to incubate at room temperature for 45 minutes to enable the formation of DNA–liposome complexes.

Five minutes before the incubation ends, gently wash the cells once with 2 ml of plain DMEM pre-warmed to 37°C, then add 3 ml of DMEM without serum and antibiotics. Add the transfection mixture to the cells dropwise, rock the plate gently, and return the cells to the incubator. Three hours later, add 60 µL of FBS and incubate the cultures overnight.

The next day, remove the transfection medium and replace it with fresh complete growth medium (2% cDMEM), then continue incubating the transfected cells. Add 200 μL of fresh Complete growth medium to each well every 3 days and examine the cells for cytopathic effect (CPE). CPE reflects the morphological changes of the cultured cells as infection proceeds. Such distinctive morphological changes result from the accumulation of newly produced virus proteins and progeny virions which disrupt normal cell structure resulting into cell death. CPE is different from necrosis; CPE begins with the rounding of adhered cells, followed by the gradual detachment of the rounded cells from the plate. When the cells detach from the plate, they may form “grape-like” clusters that float in the growth medium (Fig 2: A and B).

Download:

• PNG
  larger image
• TIFF
  original image

Fig 2. Cytopathic effect formation post-transfection of AD-293 cells with CAV-2 vector (A), human adenovirus vector as a positive control (B) and a mock test as a negative control (C).

(Olympus CKX53) at all the three images taken at 100× and 200 × magnifications.

https://doi.org/10.1371/journal.pone.0341642.g002

When 90% of the cell monolayer has shown cytopathic effects (CPE), detach any remaining cells by either gently tapping the plate on the biosafety cabinet surface or by pipetting the medium against the growth surface of the well. Transfer the cell suspension to a 2-ml cryogenic vial and store at −80°C for virus expansion. If the transfected cells do not show any sign of CPE 1 week after the transfection, see the discussion 3.1.

2.3. Expansion of adenovirus vector for large-scale use

After successfully rescuing a recombinant adenovirus vector usually by transfecting linearised vector DNA into packaging cells like AD-293, the next step is virus expansion to produce larger amounts of the virus for experimental use, preclinical studies, or production scale-up. In the case of vaccine development and formulation, sufficient virus titers can be achieved through virus vector expansion, ensuring batch consistency and reproducibility of vaccine evaluation experiments. The adenovirus vector expansion can be conducted as described below.

Seed 10 X 10⁶ AD-293 cells into a T75 cell culture flask in a total volume of 19 ml of 2% cDMEM, a day before viral infection.

Thaw the cell suspension obtained in step 2.2.6 in a 37°C water bath to generate a crude cell lysate. Repeat the freeze–thaw cycle twice more in a dry-ice/ethanol bath and a 37°C water bath. Shake the tube several times after each thaw to ensure that cells do not settle.

Following three freeze-thaw cycles, spin the crude lysate at 3200 rpm for 10 minutes at 4°C, then retrieve the supernatant to infect AD-293 cells cultured in a T75 flask in step 2.3.1.

Add 1 mL of the supernatant directly to 70%–80% confluent T-75 flask of AD-293 cells. Return the cells to the incubator and monitor CPE daily (Fig 2). Usually, CPE will become noticeable 24 hours after infection and will be fully evident within 2–3 days.

Once approximately 90% of the cells show cytopathic effects (CPE), collect them as previously described. With the flask lid secured, gently agitate the culture and use a 10-ml pipette to dislodge any remaining adherent cells. Transfer the harvest into sterile 50-ml conical centrifuge tubes.

Spin the cells at 3200 rpm for 10 minutes at 4°C to form a pellet. Carefully remove the supernatant and resuspend the pellet in 2 mL of 2% complete DMEM.

Freeze-thaw the cell pellet as described in step 2.3.2 to release the cell-bound virus. After three cycles of freezing-thawing, centrifuge the crude cell lysate at 3200 rpm for 10 minutes at 4°C and collect the supernatant. Add this supernatant to the previously collected one and store at −80°C or proceed with the purification method. If the crude viral lysate harvested from the transfection step fails to expand after 1 week, see discussion 3.2.

2.4. Purification of the Adenovirus Vectors using Vivapure® Adenopack™ 20 Kit

Purification of adenovirus vectors is a critical step after virus stock production. It removes host cell proteins, DNA, and debris to yield a high purity. In this protocol, the virus stock was purified using the Vivapure® Adenopack™ 20 kit based on the ion-exchange liquid chromatography principle. This technique offers high specificity and scalability for separating viral particles based on surface charge. Compared to conventional methods such as ultracentrifugation or sucrose/Caesium Chloride (CsCl) density gradient purification, it offers a rapid, reproducible, and scalable method suitable for small-to-medium preclinical vector preparations, and it avoids the extended ultracentrifugation steps required for CsCl gradients [17–20]. Additionally, it reduces the risk of viral particle degradation due to lower shear forces and avoids the use of toxic gradient materials like Caesium Chloride (CsCl) [19]. The Vivapure Q Maxi M spin columns in the Vivapure® Adenopack™ 20 kit has an ion exchange membrane adsorber that binds adenoviral particles. Once bound, virus particles can be further purified by washing away non-specifically bound proteins before elution within one hour. Each Vivapure Q Maxi M spin column is suitable for purifying virus from culture volumes of up to 20 mL. Reagent volumes can be adjusted accordingly for smaller samples. This kit contains sufficient consumables for six such small preparations.

2.5. Titration of the purified virus stocks using the Improved Kärber method

Titration of purified virus stocks is a crucial step in quantifying the infectious units within a viral preparation. In this protocol, the purified virus stock was titered using the Improved Kärber method, a robust and widely accepted statistical approach for determining the median (50%) tissue culture infectious dose (TCID₅₀) [22]. Unlike plaque assays, which require virus-induced cytopathic effects (CPE) to form discrete plaques, the Improved Kärber method accommodates viruses that produce diffuse or subtle CPE. It is also less time-consuming and easier to interpret in cases where plaque morphology is inconsistent. Furthermore, the Improved Kärber method requires fewer replicates and less precise quantification of individual plaques, making it a practical choice for rapid and reliable viral titration in early-phase studies. This method ensures accurate titration, which is essential for standardising experiments and ensuring reproducibility in virological studies. In this study, we titered the purified CAV-2 vector virus stock.

The Improved Kärber method involves preparing a monolayer of AD-293 cells in 96-well plates, followed by a series of dilutions of the virus stock. The endpoint, where 50% of wells show CPE, helps calculate the tissue culture infectious dose (TCID50), providing a reliable measure of viral infectivity.

2.5.2. Calculation of 50% Tissue Culture Infectious Dose (TCID50) of the titered virus stocks

Calculate the titres by determining the ratio of positive wells per row, as shown in Table 1, and use the Improved Kärber formula to determine the TICD50 [22].

Download:

• PNG
  larger image
• TIFF
  original image

Table 1. Titration of the purified canine adenovirus type 2 (CAV-2) vector virus stock.

https://doi.org/10.1371/journal.pone.0341642.t001

Where:

d_L = lowest dilution at which all wells are positive of which in this case is 10⁻⁷ (Table 1)

d_F = dilution factor of which in this case is 10-fold (Table 1)

= sum of the proportion of positive wells for all dilutions showing CPE of which in this case is 8.8 (Table 1)

3.1. Transfection

In certain instances, AD-293 cells that have been transfected exhibit no observable cytopathic effects even after seven days post-transfection. This can be troubleshooted by repeating the transfection using an alternative transfection reagent like FuGene (Cat. E2311, Promega) and/or extending the incubation time up to 2 weeks post-transfection and/or increasing the amount of transfection DNA [23]. It is essential to include positive and negative controls in the transfection assay to aid accurate interpretation of the observations. A positive control, such as a vector known to express efficiently, confirms that the transfection reagents, cell line, and protocol are working as expected. If the positive control fails, it indicates a problem with the transfection conditions rather than the test vector construct. A negative control, on the other hand (e.g., a mock transfection or a plasmid lacking the gene of interest), helps establish a baseline for background expression or toxicity, ensuring that observed effects are due to the specific vector being tested and not due to non-specific effects or reagent toxicity. Together, these controls provide a reference framework, improve reproducibility, and enhance the reliability of the experimental conclusions.

3.2. Expansion of Virus stocks

The viral lysate may fail to expand in AD-293 cells. Several factors may contribute to this issue. These include cytotoxic effects resulting from transgene expression, which can lead to cell death rather than the development of viral cytopathic effects (CPE) [24]; an oversized transgene cassette [25]; and variations in the type and extent of early gene deletions within the vector backbone (such as deletion of E1 alone, E1/E3, or E1/E3/E4 regions) [26]. Additionally, the expression levels of complementing genes in the host cell line, such as E1 expression in AD-293 cells, used to support replication of replication-defective adenoviral vectors, can influence outcomes [27]. Another contributing factor is asynchronous infection due to a low multiplicity of infection (MOI) [28]. In such cases, especially when cells are infected at low MOIs, serial passaging of the virus may be necessary to achieve complete CPE [26]. Moreover, for vectors that exhibit a slow and delayed infection process, it becomes challenging to distinguish between virus-induced CPE and cell death caused by other factors. To address this issue, several strategies can be employed. First, designing an inducible transgene expression cassette to regulate the expression of potentially toxic transgenes in AD-293 cells, thereby minimising cytotoxicity [24]. Additionally, the expansion process should be slowed down; this involves initiating the expansion from the crude lysate obtained during the initial transfection or rescue phase by infecting AD-293 cells in a T25 flask, and subsequently scaling up to a T75 flask [26]. If, during this expansion phase, full cytopathic effect (CPE) is not observed within one week, the entire infected culture should be harvested, the crude lysate clarified, and used to initiate the next round of infection [26].

3.3. Purification of adenovirus vector

It’s advisable to first filter the digested supernatant through a 0.22μm filter to remove most of the host cell debris before loading the digested supernatant on a Vivaclear Maxi to avoid clogging the Vivaclear Maxi membrane. Besides the Vivapure® Adenopack™ 20 Kit that purifies 20 mL culture volume, there are other kits, for example, Vivapure Adenopack™ 100, Cat. VS-AVPQ101 and Vivapure Adenopack™ 500, Cat VS-AVPQ501, which can be used to purify bigger culture volumes: 200 ml culture volume and 500 ml culture volume, respectively. In addition to the ion exchange liquid chromatography method of purifying virus stocks, there are also other methods of liquid chromatography, which include normal phase, reverse phase and size exclusion that can be explored [29]. Besides the liquid chromatography principle, density gradient centrifugation is another commonly used principle for purifying virus stocks: these include rate zonal centrifugation and isopycnic equilibrium gradient centrifugation [30]. While several reports indicate that membrane-based ion-exchange purification can yield high-quality adenoviral preparations with reduced processing time [17,31,32], we acknowledge that direct, side-by-side purity comparisons between Adenopack-purified CAV-2 and CsCl-purified CAV-2 are limited.

We used the Vivaspin 20 units because our workflow required a rapid buffer exchange into the physiological storage buffer and a small final volume compatible with downstream analyses (i.e., the achieving the high dose in small volume). While alternative approaches (e.g., diafiltration, desalting columns) can also perform buffer exchange, they either require larger starting volumes, introduce longer processing times, or provide less precise control over final concentration [33]. For these reasons, the Vivaspin concentrators were the most practical choice for our small-scale preclinical preparations.

3.4. Titration

During the titration process, in some cases, no CPE may be observed in all the wells two weeks post-inoculation, even in the wells containing the highest concentration of virus stock (10^-1 dilution). This could be due to low virus stock titre or the virus stock being non-infectious. Therefore, it is advisable that the titration experiment be repeated using a lower dilution factor instead of a 10-fold dilution factor or troubleshooting the virus stock by running a positive control virus of known infectious stock and a mock control on the same plate [34]. This immediately tells you whether the assay/cells are working. Check cell confluency, passage number, viability and health by testing for mycoplasma; unhealthy/contaminated cells often fail to show CPE [35–37]. On the other hand, all wells may show CPE, causing uncertainty on the dilution endpoint. Therefore, it is advisable that the titration experiment be repeated with a high dilution factor instead of a 10-fold dilution factor [38].

3.5. Biosafety guidelines

The NIH Guidelines for Research Involving Recombinant DNA Molecules classify both wild-type and replication-deficient human adenoviruses as biohazard risk group 2 agents [39]. The human diseases caused by these biohazard agents are preventable, treatable, and generally mild [40]. Handling of adenoviral vectors should occur within a Biosafety Level 2 (BL2) facility and must be approved by the Institutional Biosafety Committee (IBC) of the respective institution.

Personnel should use appropriate personal protective equipment (PPE), including lab coats, gloves, and eye protection. Work should be performed inside a certified biological safety cabinet (Class II BSC) to minimise the risk of aerosol exposure. Special attention should be paid to preventing spills and accidental release of the virus, and any spills must be immediately disinfected using an approved disinfectant effective against adenoviruses, such as a freshly prepared 10% bleach solution.

During purification procedures, particularly when using high-speed centrifugation (e.g., density gradient ultracentrifugation), additional precautions should be taken to avoid the formation of infectious aerosols. Centrifuge rotors and buckets should be loaded and unloaded within a BSC, and rotors should be sealed during centrifugation. After centrifugation, equipment and surfaces must be carefully disinfected.

In the titration and handling of concentrated viral stocks, it is critical to minimise potential exposure risks. All viral stocks should be clearly labelled, securely stored within secondary containment, and waste must be decontaminated before disposal, typically by chemical disinfection or autoclaving. Accidental exposure incidents should be immediately reported and medically assessed according to institutional policies. Additionally, training in adenovirus handling, awareness of the symptoms of accidental exposure (e.g., conjunctivitis, respiratory symptoms, etc), and having an established medical surveillance plan for laboratory workers handling adenoviral vectors is highly recommended.