Titration of rAd with the luciferase marker gene.
For an accurate readout of luciferase transgene expression, Ad was used at a VP/cell ratio that would yield sufficiently high luciferase activity but a suboptimal luciferase level so as not to reach the plateau of transgene expression. On the other hand, high VP/cell ratios require relatively more serum antibodies to demonstrate neutralization, which makes a neutralization assay less sensitive and not suited for small serum samples. Two rAds carrying the luciferase gene were titrated on A549 cells to determine the optimal VP/cell ratio. Figure shows the titration curves of Ad5 and Ad35. The serotypes transduce A549 cells similarly, and for both viruses a concentration of 500 VP/cell, which is in the middle of the linear range, was selected for further experiments.
FIG. 1. rAd Ad5.Luc and Ad35.Luc, each carrying the luciferase marker gene, were titrated on A549 cells. The x axis indicates VP per cell, ranging from 8,000 to 0, added to 104 A549 cells per well in a 96-well plate. Luciferase expression was measured after incubation (more ...) rAd5 neutralization measured by replication versus transgene inhibition.
Two essentially different detection methods for virus neutralization are the Ad replication inhibition assay and the transgene expression assay. Generally accepted replication inhibition assays as described in literature (2
) are performed with a wide array of assay parameters. We have composed one protocol, which in our view is representative of the replication inhibition method and is suitable for comparison with a principally different assay like the luciferase-based assay. To meet the major criteria for a rapid, high-throughput assay, CPE scoring was automated by staining for viable cells and subsequent analysis of optical density (22
To compare replication inhibition with transgene expression, a panel of human serum samples, negative FBS, and a serotype-specific horse serum pool as reference serum were tested for anti-Ad5 antibody titers by both protocols.
The results of the samples are shown in Fig. in ascending order of titer in serum for each method. For the Ad5-specific reference serum, 90 and 50% neutralizing titers yielded 1/4,500 ± 4,300 and 1/20,000 ± 23,000, respectively, by replication inhibition (Fig. , CPE); by luciferase inhibition assays, 90 and 50% neutralizing titers for this serum yielded 1/32,000 ± 4,200 and 1/209,000 ± 36,000, respectively. As shown in Fig. , the 50 and 90% inhibition titers correlate better within the transgene inhibition assay than in the replication inhibition assay (CPE). Both assays correlated well (based on Spearman correlation tests), as shown in Fig. , although one sample scored opposite results, probably reflecting a technical error (Fig. , sample 78). Several of the samples that were negative by measuring replication inhibition scored positive according to transgene expression (samples 73, 79, and 206), indicating that transgene expression is a more sensitive parameter than replication. Thus, based on sensitivity, amount of serum required, and time needed until readout, the transgene expression assay is preferred.
FIG. 2. Serum samples were tested by two different neutralization assays for the presence of neutralizing antibodies against rAd5. Virus infection was read out either by measurement of the luciferase transgene or by virus replication scoring with viable cell (more ...) Selection of the transgene.
One important parameter dictating the usefulness of this assay is the possibility of using small volumes of serum for high-throughput analyses. This result can be achieved by a higher level of sensitivity and by reducing the scale of the assay (384-well plates, for example). Herewith, we determined the detection limits by testing three different transgenes and their corresponding readout systems in combination with low cell numbers (101
cells/well). Since the assays should meet criteria such as the ability for automated readout and suitability for high throughput, Ad5.LacZ-infected cells were automatically quantified by optical density measurements (7
), which proved successful in that obtained results were representative for transduction inhibition as measured by counting infected cells with a microscope. Similarly, GFP expression could be detected by a fluorometer (20
), with results that corresponded to analysis by microscope. The cell monolayer may interfere with the measurements, but this interference is controlled by wells in which cells only are seeded. The cells-only wells represent fluorescent background, whereas wells containing cells and GFP virus but not serum represent the maximum GFP fluorescence. Hence, the negative and positive controls are included in the assays to indicate the window of detection. From the results obtained (Fig. ), it could be concluded that the range between minimum and maximum expression of the LacZ protein and GFP was smaller than the range for luciferase, which makes luciferase more accurate. Furthermore, with LacZ and GFP, neutralizing antibodies could be detected when 104
cells, but not 103
cells, per well were seeded. In contrast, luciferase activity could still be detected when wells were seeded with 103
cells per well, which indicates that luciferase can be used in a smaller-scale (384-well plates) assay than GFP or LacZ. Nevertheless, we prefer to use the 96-well format with 104
cells per well, as relative standard deviations increase and sensitivity decreases (line shifts to the left) with lower cell numbers.
FIG. 3. Neutralization determined by transgene expression inhibition with three different transgenes: LacZ, GFP, and Luc. (A) Virus and serum were incubated in fixed ratios and fixed VP/cell ratios but with various numbers of A549 cells per well. Neutralization (more ...)
We next determined what concentrations of Ad-neutralizing antibodies could be detected with the three different transgenes. Negative human serum was spiked with an anti-Ad5 antibody (Abcam) at different concentrations, which were subsequently tested for neutralization of Ad5.Luc, Ad5.GFP, and Ad5.LacZ recombinant viruses (Fig. ). According to specifications, the spiking agent had a titer of 1/25,000 for 50% replication inhibition of 1,000 VP. The antibody was diluted in negative serum, and the expected titer was calculated (x axis). Results from the three transgene inhibition assays were plotted and showed that similar ranges of neutralizing activity could be detected for all three. GFP results suffered from background interference in the lower range of the antibody dilution, possibly resulting in false positives. Only by using luciferase could both 50 and 90% inhibition titers be obtained, again suggesting this readout to be the most sensitive. In conclusion, all three tested transgenes can be used to determine neutralizing titers, but luciferase is preferred for its suitability for high throughput, a better minimum-maximum ratio, sensitivity, and specificity.
Qualification of the transgene expression inhibition assay.
To validate the transgene expression inhibition assay, the intra-assay variation, or the standard deviation of eight measurements within one assay, was determined. The pool of serum tested showed a 90% luciferase inhibition titer of 769 ± 100 (data not shown). The intra-assay variation was thus 13%.
In addition, the assay was performed independently, five times in duplicate, to assess interassay reproducibility. A second Ad5-positive serum pool was aliquoted and stored at −20°C until use, and a neutralization assay was performed with the same virus batch and serum batch by the same operator on five separate days. Given the low standard deviations for the obtained luciferase values, we concluded that the assay is highly reproducible. In this experiment the serum dilution needed for 90% neutralization is 1,260 ± 220, resulting in an interassay variation of 17%. For serum samples measured by the replication inhibition assay, we calculated an interassay variation of 53%. These data show that the luciferase-based assay is highly reproducible with acceptable standard deviations.
The assay is intended to determine the inhibition of virus infection by measuring luciferase activity. To determine whether serum antibodies decreased actual virus entry into target cells, and to exclude that other serum components killed target cells, thereby diminishing transgene expression, we combined transgene detection (measurement of luciferase activity) (Fig. ) with cellular Ad genome detection (by Q-PCR) (Fig. ). Simultaneous detection of the number of virus copies of Ad5 and Ad35 per cell and of luciferase activity showed that transgene expression was correlated with the number of Ad genomes per cell and that addition of other serum components decreased both the amount of luciferase and the number of cellular Ad copies. Serum does not interfere with Q-PCR results, as the positive controls with Ad35 are positive throughout the serum dilution. These results show that neutralization takes place mainly extracellularly, not after virus entry in the cellular vesicles, and that the assay specifically measures the inhibition of virus infection but not the secondary effects of serum.
FIG. 4. Comparison between transgene expression and the number of Ad genomes per cell. A standard neutralization was performed with human Ad5-positive serum in combination with the vectors Ad5.Luc and Ad35.Luc at 500 VP/cell. (A) Cells were analyzed for luciferase (more ...) Standard control samples.
Naturally, qualification of an assay requires the presence of a standard positive control serum, one that is sufficiently characterized and readily available to the scientific and medical communities. One such standard could be the second international standard for antimeasles and antipoliovirus human serum, types 1, 2, and 3 (number 66/202), obtained from the National Institute for Biological Standards and Control, provided that this serum neutralizes Ad5. For this purpose we tested the standard serum and found that, indeed, it contains neutralizing antibodies against rAd5. This positive control serum was titrated by the transgene inhibition assay on neutralizing activity for 1/2,550 (50%) and 1/625 (90%), respectively. In addition, we obtained polyclonal Ad5-neutralizing antibodies (Abcam) with a reported 50% neutralizing activity of 1,000 VP at a dilution of 1/25,000. This serum was tested with the transgene inhibition assay and showed 90% luciferase inhibition at a dilution of 50,000 ± 9,000.
To determine the robustness of the luciferase-based assay, we investigated several factors that may influence the outcomes of the assay. One factor could be the cell line used. The luciferase neutralization assay was performed routinely on A549 cells as this cell line is highly permissive to Ad infection of both Ad5 and Ad35, serotypes that we frequently use. For several cell lines, including 3T3 (mouse fibroblasts), C2C12 (mouse myoblasts), and human and murine dendritic cells, we tested the Ad5- and Ad35-neutralizing activity of Ad5-positive serum (either human or mouse). As Ad vectors had different infectious titers for the different cell lines used, the maximum luciferase activity varied among different cells, as it is receptor dependent. Although each cell line showed that Ad5-positive serum neutralized Ad5 and did not neutralize Ad35, and vice versa (data not shown), 90 and 50% neutralization titers were shifted, depending on the maximum luciferase value.
Furthermore, we tested the effect of the sequence of events, i.e., whether A549 cells should be attached to the bottom of the wells before exposure to serum and virus or whether cells can be added after serum and virus are mixed, but no difference was observed (data not shown). Therefore, the cells can be added after diluting serum and adding VP, which is easier and faster. When large amounts of samples are to be tested, the time between the addition of cells to the virus-serum mix may vary. Therefore, we tested the effect of the incubation time (at room temperature) of serum and virus before cells are added. The incubation of serum and virus was varied from 0.5 to 60 min, but no differences in results were detected, allowing for the flexible timing of subsequent activities in the protocol.
Contribution of serum IgG.
To demonstrate that the neutralizing effect of serum is mainly mediated through antibodies, the assay was performed with isolated IgG. The IgG isolation and purity were confirmed by gel electrophoresis and Coomassie blue staining (data not shown). Figure shows the neutralization capacity of Ad5-positive and -negative serum from which the IgG was isolated and the neutralizing activity of the isolated IgG fractions. Ad5-positive serum and IgG isolated from the same serum batch show neutralization, whereas Ad5-negative serum and cognate IgG do not show neutralization. Figure shows the results obtained when Ad5-negative serum was spiked with IgG isolated from Ad5-positive serum or with IgG isolated from Ad5-negative serum. The results demonstrate that neutralization activity can be transferred from positive to negative serum by using IgG antibodies.
FIG. 5. The role of IgG in Ad neutralization. Results shown are the average of triplicate measurements performed with pooled human serum or isolated IgG and the vector Ad5.Luc. (A) Ad5-positive and -negative sera were compared with IgG isolated from positive (more ...)