A rapid Q-PCR titration protocol for adenovirus and helper-dependent adenovirus vectors that produces biologically relevant results

Abstract

Adenoviruses are employed in the study of cellular processes and as expression vectors used in gene therapy. The success and reproducibility of these studies is dependent in part on having accurate and meaningful titers of replication competent and helper-dependent adenovirus stocks, which is problematic due to the use of varied and divergent titration protocols. Physical titration methods, which quantify the total number of viral particles, are used by many, but are poor at estimating activity. Biological titration methods, such as plaque assays, are more biologically relevant, but are time consuming and not applicable to helper-dependent gene therapy vectors. To address this, a protocol was developed called "infectious genome titration" in which viral DNA is isolated from the nuclei of cells ~3 h post-infection, and then quantified by Q-PCR. This approach ensures that only biologically active virions are counted as part of the titer determination. This approach is rapid, robust, sensitive, reproducible, and applicable to all forms of adenovirus. Unlike other Q-PCR-based methods, titers determined by this protocol are well correlated with biological activity.

Figure 1. Duration of TCID₅₀ Assay Affects Result

Five different stocks of adenovirus were titered according to a standard TCID₅₀ protocol. At multiple points over 20 days, infected wells were identified by the observance of cytopathic effects and used to calculate the titer of each stock. 10 days (indicated by diagonal hash marks) is the standard duration for most protocols. The brackets indicate the change in observed titer between 10 days and 20 days post-infection.

Figure 2. Overview of the Infectious Genome Protocol

Each stock of adenovirus to be titered was used to infect replicate wells of HeLa cell monolayers in 6-well tissue culture plates. 5 μL of vector stock (crude or purified) was diluted into 500 μL of serum-free media and overlaid on the monolayer for three to six hours at 37°C. The inoculant was aspirated, and monolayers were washed twice with PBS. Cell membranes, but not nuclear membranes, were lysed in place by the addition of NP-40 detergent. The nuclei were pelleted, and used to prepare total DNA by means of a commercial kit. The resulting DNA was then used as template in Q-PCR to quantify the number of vector genomes that had translocated to the nucleus during the incubation period. The titer of each stock was then determined relative to a reference standard stock of pre-established titer that was processed in parallel. Additionally, two validation stocks of pre-established titer were processed in parallel in order to identify errors during processing.

Figure 3. Comparison of Q-PCR-based Titration Protocols

Two stocks of a FG-Ad vector, one crude lysate and one purified by CsCl gradient, were subjected to three different protocols designed to determine the vector’s titer. In the IGU protocol, non-infectious and naked DNA were eliminated by selectively harvesting vector DNA capable of infecting HeLa cell nuclei in culture (Figure 2). In the VG protocol, naked DNA was removed by incubating the stocks for 30 minutes at 37°C with DNase I. No effort was made to remove naked DNA in the Total DNA protocol. In all three cases, the vector DNA was liberated from capsids and purified by means of a commercial DNA cleanup kit, and then quantified by Q-PCR. As a control, an unrelated plasmid with an independently assayable template was spiked into the purified FG-Ad stock and processed in parallel. * indicates the result was below the limit of detection (< 5×10⁴ copies). Bars indicate the mean of four replicates ± standard deviation.

Figure 4. Comparison of Physical and Biological Titration Methods

A single stock of purified GFP-expressing FG-Ad vector was titered by five different methods. Each bar indicates the mean of multiple assays ± standard deviation (OD₂₆₀ N=6, TCID₅₀ N=5, GFP Transduction N=5, VG N=8, IGU N=27). Statistical significance was determined by post-ANOVA Bonferroni’s multiple comparison test. * indicates statistical significance with P < 0.05.

Figure 5. Relationship of Q-PCR Titers to Biological Activity

(A) Five different stocks of luciferase expressing HD-Ad vectors were titered by Q-PCR using two different protocols; one including an infection step (IGU/mL) and one that included only a DNase I treatment (VG/mL). Brackets indicate the ratio of VG to IGU. (B) In a series of in vivo experiments, each stock was injected into mice and assayed by bioluminescent imaging. This figure plots the log of the maximum level of luciferase expression against the log of the dose injected as determined by each titration method. Each set of seven data points was used to calculate a curve and to calculate a correlation coefficient. The correlation coefficient was used to calculate two-tailed P-values with five degrees of freedom.

Figure 5. Relationship of Q-PCR Titers to Biological Activity

(A) Five different stocks of luciferase expressing HD-Ad vectors were titered by Q-PCR using two different protocols; one including an infection step (IGU/mL) and one that included only a DNase I treatment (VG/mL). Brackets indicate the ratio of VG to IGU. (B) In a series of in vivo experiments, each stock was injected into mice and assayed by bioluminescent imaging. This figure plots the log of the maximum level of luciferase expression against the log of the dose injected as determined by each titration method. Each set of seven data points was used to calculate a curve and to calculate a correlation coefficient. The correlation coefficient was used to calculate two-tailed P-values with five degrees of freedom.

Figure 6. Titrations of the Adenovirus Reference Material (ARM)

(A) The ARM was titered by OD₂₆₀ (N=4) and by TCID₅₀ (N=3). The results are presented relative to results published by the ARM working group. Bars equal the mean ± the standard deviation. Comparisons between determinations were made by Student’s t-test. * indicates significance with P < 0.05. (B) The ARM was titered by the IGU protocol using duplicate or triplicate Q-PCR determinations of 140 DNA preparations performed on 44 assay days over the course of a year. Each of 300 data points is plotted. The dotted line indicates the mean. The grey area indicates ± one standard deviation.

Figure 6. Titrations of the Adenovirus Reference Material (ARM)

(A) The ARM was titered by OD₂₆₀ (N=4) and by TCID₅₀ (N=3). The results are presented relative to results published by the ARM working group. Bars equal the mean ± the standard deviation. Comparisons between determinations were made by Student’s t-test. * indicates significance with P < 0.05. (B) The ARM was titered by the IGU protocol using duplicate or triplicate Q-PCR determinations of 140 DNA preparations performed on 44 assay days over the course of a year. Each of 300 data points is plotted. The dotted line indicates the mean. The grey area indicates ± one standard deviation.