Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2003 Nov;41(11):5046-52.
doi: 10.1128/JCM.41.11.5046-5052.2003.

Quantifying adenovirus-neutralizing antibodies by luciferase transgene detection: addressing preexisting immunity to vaccine and gene therapy vectors

Mieke C Sprangers  1 , Wandena Lakhai, Wouter Koudstaal, Marielle Verhoeven, Björn F Koel, Ronald Vogels, Jaap Goudsmit, Menzo J E Havenga, Stefan Kostense
Affiliations

Quantifying adenovirus-neutralizing antibodies by luciferase transgene detection: addressing preexisting immunity to vaccine and gene therapy vectors

Mieke C Sprangers et al. J Clin Microbiol. 2003 Nov.

Abstract

The presence of various levels of anti-adenovirus serotype 5 (Ad5)-neutralizing antibodies in humans is thought to contribute to the inconsistent clinical results obtained so far in diverse gene transfer and vaccination studies and might preclude universal dosing with recombinant Ad5. Prescreening of individuals eligible for Ad5 or alternative serotype treatment and subsequently tailoring the vector dose might aid in ensuring the consistency of clinical parameters. For this purpose, a qualified Ad neutralization assay is required. Here we have tested the different protocols used to date to determine anti-Ad neutralizing activity. Based on simplicity, speed, high throughput, sensitivity, and robustness, we propose a qualified assay in which Ad neutralization is monitored by luciferase reporter gene expression.

PubMed Disclaimer

Figures

FIG. 1.
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 for 1 day. For both serotypes, 500 VP/cell in the middle of the linear range was selected for further experimentation.
FIG. 2.
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 staining. Maximum virus infection was determined in control wells without serum. Serum titers were determined by the dilution at which 50 or 90% of cell viability or luciferase expression was observed. (A) The y axes indicate the serum dilution at which 50 or 90% infection inhibition was observed, relative to the maximum control value. Dotted lines indicate the lower limits of detection as defined by the maximum concentration of serum. Individual samples are indicated on the x axis in ascending order of 90% inhibition serum titer for each method. Ad5-positive (Reference) serum and FBS are included as positive and negative controls, respectively. (B) Serum titers obtained by transgene expression inhibition (y axis) and replication inhibition (x axis) are compared and analyzed for correlation coefficients.
FIG. 3.
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 curves calculated from triplicate measurements are shown for each virus and different numbers of cells per well. Different numbers of cells per well resulted in different transgene expression values, which cannot be depicted on the same y-axis scale. To visualize the effect of different cell numbers, the panels decrease in y-axis range from left to right. (B) A standard Ad5-neutralizing polyclonal antibody was diluted in negative human serum at different concentrations and analyzed for Ad5 neutralization with three different transgenes. Transgene inhibition-derived titers in serum were plotted against the expected neutralizing titer based on the polyclonal antibody specification of 1/25,000. For all transgenes, 50% titers are shown, but only the assay using luciferase provided 90% titers also.
FIG. 4.
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 activity. Ad5-positive serum shows a serotype-specific inhibition of Ad vector transduction. (B) Packaged DNA was isolated from A549 cells used in a neutralization assay as in panel A. Isolated DNA was used as a template for Ad-specific real-time PCR. The number of Ad5 genome copies per cell is decreased due to Ad5-specific serum. Ad35 genome copies are stable irrespective of concentrations in serum.
FIG. 5.
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 or negative serum in an rAd5 neutralization assay. (B) IgG isolated from negative or positive serum pools was spiked in negative serum. Inhibition of luciferase activity was detected with increasing IgG concentrations.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Bruna-Romero, O., G. Gonzalez-Aseguinolaza, J. C. Hafalla, M. Tsuji, and R. S. Nussenzweig. 2001. Complete, long-lasting protection against malaria of mice primed and boosted with two distinct viral vectors expressing the same plasmodial antigen. Proc. Natl. Acad. Sci. USA 98:11491-11496. - PMC - PubMed
    1. Chen, Y., D. C. Yu, D. Charlton, and D. R. Henderson. 2000. Pre-existent adenovirus antibody inhibits systemic toxicity and antitumor activity of CN706 in the nude mouse LNCaP xenograft model: implications and proposals for human therapy. Hum. Gene Ther. 11:1553-1567. - PubMed
    1. Crawford-Miksza, L. K., and D. P. Schnurr. 1994. Quantitative colorimetric microneutralization assay for characterization of adenoviruses. J. Clin. Microbiol. 32:2331-2334. - PMC - PubMed
    1. D'Ambrosio, E., N. Del Grosso, A. Chicca, and M. Midulla. 1982. Neutralizing antibodies against 33 human adenoviruses in normal children in Rome. J. Hyg. (London) 89:155-161. - PMC - PubMed
    1. Fallaux, F. J., A. Bout, I. van der Velde, D. J. van den Wollenberg, K. M. Hehir, J. Keegan, C. Auger, S. J. Cramer, H. van Ormondt, A. J. van der Eb, D. Valerio, and R. C. Hoeben. 1998. New helper cells and matched early region 1-deleted adenovirus vectors prevent generation of replication-competent adenoviruses. Hum. Gene Ther. 9:1909-1917. - PubMed

LinkOut - more resources