Elsevier

Virus Research

Volume 146, Issues 1–2, December 2009, Pages 58-65
Virus Research

Epitope mapping and cross-reactivity analysis of the monoclonal antibodies against hexon protein of human adenovirus type 3

https://doi.org/10.1016/j.virusres.2009.08.011Get rights and content

Abstract

Human adenovirus 3 (HAdV-3) occurs epidemically in China in recent years. It can cause life-threatening infection in young children and immunocompromised patients. Development of reliable diagnostic reagents for adenovirus infection is important in surveillance and control of the infection. In this study, three monoclonal antibodies (MAbs) 5D4, 7B2 and 3D10 were generated against the recombinant HAdV-3 hexon protein. Western-blots analysis showed that all three MAbs recognized the native HAdV-3 hexon protein, but only 5D4 reacted with the native HAdV-3 virus particles by indirect enzyme-linked immunosorbent assay (ELISA). The epitopes recognized by these MAbs were further investigated by epitope analysis with a series of peptides of the hexon protein. The epitope for 3D10 was located to the sequence 339GVLAGQASQLNA350 and present in all the currently recognized serotypes of human adenovirus. The MAb 7B2 showed broad reactivity with the peptides from group B, group E, and most serotypes of group D, but not with those from group A, C and F adenoviruses. The epitope of 5D4, located in the hypervariable region 3 (HVR3), was only shared by HAdV-3 and HAdV-7. Further ELISA demonstrated that 5D4 only recognized HAdV-3 and HAdV-7 virus particles. The MAbs and the identified epitopes may play roles in clinical serotype-specific diagnosis, viral structure analysis, as well as in the identification of new serotypes of human adenovirus.

Introduction

Human adenovirus (HAdV) was first isolated from adenoids surgically removed from children with acute respiratory illness in 1953 (Rowe et al., 1953). To date, 52 human adenovirus serotypes have been identified that were divided into 6 groups (A to F) based on their DNA sequence homology, fiber and hexon protein characteristics, and biochemical properties (Jones et al., 2007). More than one third of the adenoviruses may cause human diseases such as respiratory infections (group B1 HAdV-3, -7, -21, group B2 HAdV-14, and group E HAdV-4), gastroenteritis (group F HAdV-40 and -41), ocular infections (group D HAdV-8, -9, and -37), pneumonia (group B1 HAdV-3, -7, group C HAdV-1, 2, and group E HAdV-4), keratoconjunctivitis (group B2 HAdV-11, and group D HAdV-8, -19 and HAdV-37), menigoencephalitis (group A HAdV-12, group B1 HAdV-7, and group D HAdV-32) and urinary infections (group B2 HAdV-11) (Hayashi and Hogg, 2007, Horwitz, 2001, Jones et al., 2007). Of these, the acute respiratory diseases induced by the subgroups B1 species including HAdV-3, HAdV-7 and group E HAdV-4 have occurred epidemically and caused outbreaks in Asia, Europe, and the North America (Chen et al., 2004, Hindiyeh et al., 2008, James et al., 2007, Purkayastha et al., 2005).

HAdV-3 has been one of the major pathogens for respiratory infections in China. A total of 871 cases of acute respiratory tract infection were found induced by HAdV-3 in Dongtai, Jiansu Province in 2004 (Jiang et al., 2005). In the same year, 468 cases of pharyngoconjuctival fever caused by HAdV-3 were reported in Hohhot, Inner Mongolia (Liu et al., 2007). HAdV-3 outbreaks were also reported in other provinces in China in the last few years (Ou et al., 2008, Zhu et al., 2005). Infants, children and immunocompromised individuals were most especially susceptible to adenovirus infection. About 5% of acute respiratory tract infections in children younger than 5 years old are caused by adenoviruses in China (Huang et al., 2001). Among them, pneumonia was the most serious manifestation.

However, currently there is no specific antiviral therapy against adenoviruses. And the diseases caused by adenoviruses are difficult to distinguish from other infections due to a variety of RNA viruses. It is therefore necessary to develop rapid and reliable tests for the diagnosis of adenovirus infection. Pan-adenovirus and sero-specific monoclonal antibodies to adenovirus have extensive applications in developing such immunological assays.

The adenovirus capsid is an icosahedron composed of three structural proteins: hexon, penton base and fibre, and hexon protein is the major antigenic determinant (Pichla-Gollon et al., 2007, Roberts et al., 2006, Sumida et al., 2005). We generated three MAbs (5D4, 7B2 and 3D10) against the hexon protein and analyzed the epitopes recognized by these three MAbs, and their cross-reactivity among different serotypes of adenoviruses. This study will be useful for developing reliable and stable serotype-specific diagnosis for adenovirus infection.

Section snippets

Animals, virus strains, cell and plasmids construction

Female BALB/c mice (6–8 weeks) were purchased from the Animal Center at Sun Yat-sen University, and housed in pathogen-free conditions. HAdV-3 strains GZ1 (strains GZ1) and HAdV-7 were isolated originally from children with acute respiratory diseases (Zhang et al., 2006). HAdV-5 was provided by Dr Lu Junpeng at South China Agricultural University. HAdV-3, HAdV-5 and HAdV-7 were cultured in HEp-2 cells. The cells were cultured in Dulbecoo's modified Eagle's medium (DMEM) supplemented with 100 IU 

Generation and identification of MAbs against HAdV-3 hexon

Hexon is the major antigenic protein of HAdV-3. To generate monoclonal antibodies against hexon protein, we tried to express the full length recombinant hexon protein in E. coli system, and were unsuccessful. Therefore, three overlapping protein fragments P1, P2 and P3 (Table 1, animo acid residues 1–356, 350–677, and 671–944) fused with the His tag were produced. The purified protein fragment mixtures were used to immunize BALB/c mice (Fig. 1A). Three monoclonal antibodies (5D4, 7B2 and 3D10)

Discussion

HAdV-3 contributes greatly to respiratory diseases. The identification of epitopes of HAdV-3 capsid protein hexon is, therefore, important for the development of virus-specific serological diagnostic reagents and subunit vaccines. In this paper, we used three truncated overlapping hexon fragments as mixed antigens to produce three MAbs 5D4, 7B2 and 3D10 that recognized different epitopes in hexon protein. Epitope mapping, sequence and mutagenic analysis revealed that the three MAbs might show

Acknowledgements

This work was supported by Science and Technology Planning Project of Guangdong Province and The National Natural Science Foundation of China Grant 30770102. We are grateful to Drs. Zhuang Yan and Xu Xiuling for helpful advice of this manuscript.

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