Prevalence of neutralizing antibodies to adenoviral serotypes 5 and 35 in the adult populations of The Gambia, South Africa, and the United States - PubMed (original) (raw)

Prevalence of neutralizing antibodies to adenoviral serotypes 5 and 35 in the adult populations of The Gambia, South Africa, and the United States

Edward Nwanegbo et al. Clin Diagn Lab Immunol. 2004 Mar.

Abstract

One of the major limitations of the use of adenoviruses as gene therapy vectors is the existence of preformed immunity in various populations. Recent studies have linked failure of adenoviral gene therapy trials to the presence of antiadenoviral neutralizing antibodies (NAb). Understanding the distribution and specificity of such antibodies will assist in the design of successful recombinant adenoviral gene therapies and vaccines. To assess the prevalence of NAb to adenovirus serotypes 5 and 35 (Ad5 and Ad35), we analyzed serum samples from adult immunocompetent individuals living in The Gambia, South Africa, and the United States by using a neutralization assay. Serum samples were incubated with A549 lung carcinoma cells and adenoviruses encoding enhanced green or yellow fluorescent proteins; results were analyzed by fluorescence microscopy and flow cytometry. Using this technique, we found a high prevalence of NAb against Ad5 in Gambian, South African, and U.S. subjects at both low and high titers. Conversely, all subjects displayed a low prevalence of NAb to Ad35; when present, anti-Ad35 NAb were seen at low titers. Because of the ability of adenoviruses to elicit systemic and mucosal immune responses, Ad35 with its low NAb prevalence appears to be an attractive candidate vector for gene therapy applications.

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Figures

FIG. 1.

FIG. 1.

Infection of A549 cells by recombinant adenoviruses at various Vp/c ratios in the presence of serum. (A) Infectivity profile of Ad5EGFP in the presence of diluted serum containing NAb indicates that a Vp/c ratio of 1,000 (or multiplicity of infection [MOI] of 10) yields optimal cell infection, with a clear inverse relationship between cell infection and serum NAb concentration. At Vp/c = 100 (or MOI = 1), the percentage of infected cells did not change with decreasing serum concentration (increasing titer), whereas at Vp/c = 1,000, cell infection increased with decreased serum concentration. Infection at higher viral doses suggested viral saturation (data not shown). (B) Infection of A549 cells with Ad35EYFP reveals a similar profile as with Ad5EGFP, with Vp/c = 1,000 as the optimal viral dose.

FIG. 2.

FIG. 2.

Fluorescence microscopy and flow cytometry of A549 cells after neutralization assay. (A and B) A549 cells infected with virus Ad35EYFP in the absence of serum exhibit marked fluorescence, as seen in the micrograph (A) and the histogram (B). Similar results were seen in cells infected with Ad5EGFP. (C and D) A549 cells incubated with DMEM but not infected with virus display almost no background fluorescence in either the micrograph (C) or the histogram (D). (E and F) Representative fluorescence micrograph (E) and histogram (F) demonstrating A549 cells incubated with a test serum sample and virus.

FIG. 3.

FIG. 3.

Prevalence of anti-Ad5 and anti-Ad35 NAb in the African and North American populations. (A) Anti-Ad5 NAb is present in The Gambian, South African, and U.S. populations at 84.67, 79.87, and 37.0% prevalence, respectively. (B) There is a very low prevalence of anti-Ad35 NAb in all three populations (<20%).

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