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
. 2002 Dec;76(24):12775-82.
doi: 10.1128/jvi.76.24.12775-12782.2002.

Construction and characterization of adenovirus serotype 5 packaged by serotype 3 hexon

Hongju Wu  1 , Igor Dmitriev, Elena Kashentseva, Toshiro Seki, Minghui Wang, David T Curiel
Affiliations

Construction and characterization of adenovirus serotype 5 packaged by serotype 3 hexon

Hongju Wu et al. J Virol. 2002 Dec.

Abstract

Adenovirus serotype 5 (Ad5) has great potential for gene therapy applications. A major limitation, however, is the host immune response against Ad5 infection that often prevents the readministration of Ad5 vectors. In this regard, the most abundant capsid protein, hexon, has been implicated as the major target for neutralizing antibodies. In this study, we sought to escape the host neutralization response against Ad5 via hexon replacement. We constructed a chimeric adenovirus vector, Ad5/H3, by replacing the Ad5 hexon gene with the hexon gene of Ad3. The chimeric viruses were successfully rescued in 293 cells. Compared to that for the control Ad5/H5, the growth rate of Ad5/H3 was significantly slower and the final yield was about 1 log order less. These data indicate that the Ad3 hexon can encapsidate the Ad5 genome, but with less efficiency than the Ad5 hexon. The gene transfer efficacy of Ad5/H3 in HeLa cells was also lower than that of Ad5/H5. Furthermore, we tested the host neutralization responses against the two viruses by using C57BL/6 mice. The neutralizing antibodies against Ad5/H3 and Ad5/H5 generated by the immunized mice did not cross-neutralize each other in the context of in vitro infection of HeLa cells. Preimmunization of C57BL/6 mice with one of the two types of viruses also did not prevent subsequent infection of the other type. These data suggest that replacing the Ad5 hexon with the Ad3 hexon can circumvent the host neutralization response to Ad5. This strategy may therefore be used to achieve the repeated administration of Ad5 in gene therapy applications.

PubMed Disclaimer

Figures

FIG. 1.
FIG. 1.
Diagram depicting the construction of plasmids pAd5/H3 and pAd5/H5. The plasmid pAd5 containing the GFP and luc (GL) reporter genes in the E1 region was used to create pAd5/ΔH5 by three-piece ligation. A SwaI (Sw) site was introduced into the original site of the H5 gene. Fragments containing H3 or H5 were recombined with the SwaI-linearized backbone pAd5/ΔH5 by homologous recombination in E. coli BJ5183, resulting in the formation of pAd5/H3 and pAd5/H5. S1, the first SfiI site; S2, the second SfiI site. Note: the two SfiI sites in the Ad5 genome are not identical. The different sticky ends allowed for oriented ligation.
FIG. 2.
FIG. 2.
Confirmation of the hexon chimera Ad5/H3. (A) Serotype-specific PCR analysis confirmed that the chimeric virus indeed contained the H3 gene. The specific PCR primers are located in HVR1 (sense) and HVR4 (anti-sense) of H3 or H5. Plasmids pAd5 and pAd5/H3 and purified Ad5/H3 viruses were used as the template. Lanes 1 to 3, PCR with H3-specific primers resulted in specific PCR products of correct size on templates pAd5/H3 (lane 2) and Ad5/H3 (lane 3) but not on pAd5 (lane 1); lanes 4 to 6, PCR with H5-specific primers produced specific product on template pAd5 (lane 4) but not on pAd5/H3 (lane 5) or Ad5/H3 (lane 6); lane L, 100-bp DNA ladder. (B) SDS-PAGE and Western blotting assays demonstrated that, at the protein level, Ad5/H3 contained the Ad3 hexon. In the assays, 1010 VPs of purified viruses was lysed in Laemmli sample buffer with or without boiling and separated on 4 to 15% polyacrylamide gels. The proteins were either stained with Gelcode blue stain reagent (Pierce) (B1) or transferred to a nitrocellulose membrane and then stained with the anti-hexon antibodies AB1056 (B2), MAB8043 (B3), or MAB8047 (B4). Lane M, molecular mass marker; lanes 1 and 5, Ad5; lanes 2 and 6, Ad5/H5; lanes 3 and 7, Ad5/H3; lanes 4 and 8, Ad3. Lanes 1 to 4 were loaded with boiled samples, while lanes 5 to 8 were loaded with unboiled samples.
FIG. 3.
FIG. 3.
Characterization of Ad5/H3. (A) Growth kinetics of Ad5/H3 and Ad5/H5. 293 cells plated in six-well plates were infected with Ad5/H3 or Ad5/H5 at an MOI of 5, and cells including medium were collected daily after infection. The titers of the viruses were determined by plaque assay. (B) Comparison of the gene transfer efficacy of Ad5/H3 and Ad5/H5. HeLa cells were infected with Ad5/H3 or Ad5/H5 at three different MOIs (10, 100, 1,000) in triplicate, and cell lysates were processed for the luciferase activity assay. The RLU showing the activity of luciferase were detected with a luciferase assay kit (Promega) and a luminometer. (C) Thermostability of Ad5/H3. The viruses at an MOI of 100 were incubated at 45°C for different time intervals before infecting HeLa cells. The relative infectivity was obtained by changing the RLU readings of the heat-treated viruses to the percentage of the readings of untreated Ad5/H3 or Ad5/H5 viruses.
FIG. 4.
FIG. 4.
Ad5/H3 and Ad5/H5 were not cross-neutralized in vitro. Ad5/H3 and Ad5/H5 viruses at an MOI of 10 were incubated with 5 μl of sera from PBS-, Ad5/H3-, or Ad5/H5-immunized C57BL/6 mice before infecting HeLa cells plated in 96-well plates. The infectivity was determined by luciferase activity assay. (A) Ad5/H5 infection was blocked by sera from Ad5/H5-immunized mice but not by sera from Ad5/H3-infected mice. (B) In contrast, Ad5/H3 infection was blocked by sera from Ad5/H3-immunized mice but not by sera from Ad5/H5 infected mice. Three mice were used for each group, and all of the infections were done in triplicate.
FIG. 5.
FIG. 5.
Ad5/H3 and Ad5/H5 were not cross-neutralized in vivo. C57BL/6 mice were preimmunized with PBS or 109 VPs of Ad5/H3 or Ad5/H5 per mouse by i.v. injection. One month later, the mice were injected with either PBS or 1010 VPs of Ad5/H3 or Ad5/H5 per mouse by i.v. injection. Three days later, the mice were sacrificed and their livers and hearts were harvested for the luciferase activity assay. In both the liver (A) and heart (B), Ad5/H5 infection was greatly inhibited by Ad5/H5 preimmunization but was retained to a large degree in Ad5/H3-preimmunized mice. Similarly, Ad5/H3 infection was retained in Ad5/H5-preimmunized mice but was blocked by Ad5/H3 preimmunization. Note: it appears that luciferase expression in mice preimmunized with Ad5/H3 was sustained in liver even after 1 month but not in the heart.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Athappilly, F. K., R. Murali, J. J. Rux, Z. Cai, and R. M. Burnett. 1994. The refined crystal structure of hexon, the major coat protein of adenovirus type 2, at 2.9 Å resolution. J. Mol. Biol. 242:430-455. - PubMed
    1. Barnett, B. G., C. J. Crews, and J. T. Douglas. 2002. Targeted adenoviral vectors. Biochim. Biophys. Acta 1575:1-14. - PubMed
    1. Benko, M., B. Harrach, and W. C. Russell. 1999. Family adenoviridae, p. 227-238. In M. H. Van Regenmortel, C. M. Fauquet, D. H. L. Bishop, E. B. Carstens, M. K. Estes, S. M. Lemon, J. Maniloff, M. A. Mayo, D. J. McGeoch, C. R. Pringle, and R. B. Wickner (ed.), Virus taxonomy. Seventh Report of the International Committee on Taxonomy of Viruses. Academic Press, Inc., New York, N.Y.
    1. Crawford-Miksza, L., and D. P. Schnurr. 1996. Analysis of 15 adenovirus hexon proteins reveals the location and structure of seven hypervariable regions containing serotype-specific residues. J. Virol. 70:1836-1844. - PMC - PubMed
    1. Croyle, M. A., N. Chirmule, Y. Zhang, and J. M. Wilson. 2001. “Stealth” adenoviruses blunt cell-mediated and humoral immune responses against the virus and allow for significant gene expression upon readministration in the lung. J. Virol. 75:4792-4801. - PMC - PubMed

Publication types

LinkOut - more resources