Epitope mapping and cross-reactivity analysis of the monoclonal antibodies against hexon protein of human adenovirus type 3
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.
References (23)
- et al.
Adenovirus infections and lung disease
Curr. Opin. Pharmacol.
(2007) Adenovirus immunoregulatory genes and their cellular targets
Virology
(2001)- et al.
Molecular modeling and epitopes mapping of human adenovirus type 3 hexon protein
Vaccine
(2009) - et al.
Survey on the outbreak of adenovirus upper respiratory tract infection in Yichong City
Zhonghua Liu Xing Bing Xue Za Zhi
(2004) - et al.
Analysis of 15 adenovirus hexon proteins reveals the location and structure of seven hypervariable regions containing serotype-specific residues
J. Virol.
(1996) - et al.
High rate of human bocavirus and adenovirus coinfection in hospitalized Israeli children
J. Clin. Microbiol.
(2008) - et al.
Viral etiological study in children viral pneumonia
Harbin Yi Ke Da Xue Xue Bao
(2001) - et al.
Outbreak of human adenovirus type 3 infection in a pediatric long-term care facility—Illinois, 2005
Clin. Infect. Dis.
(2007) - et al.
Epidemiological study on the outbreak of respiratory infection due to adenovirus
Zhongguo Gong Gong Wei Sheng
(2005) - et al.
New adenovirus species found in a patient presenting with gastroenteritis
J. Virol.
(2007)
Adenovirus serotype 5 hexon is critical for virus infection of hepatocytes in vivo
Proc. Natl. Acad. Sci. U.S.A.
Cited by (7)
Development of a prototype immunochromatographic test for rapid diagnosis of respiratory adenovirus infection
2017, Brazilian Journal of Infectious DiseasesCitation Excerpt :Peroxidase activity was revealed by the addition of ELISA substrate TMB BD OptEIA. The reaction was stopped by adding 50 μL/well of 3 N H2SO4 and the plate read on a TP Reader Thermo Plate at a wavelength of 450 nm.20 Fusion was performed according to the protocol proposed by Dr. Leticia Barboza's group (Bacteriologia – Instituto Butantan).
Mapping the epitope of neutralizing monoclonal antibodies against human adenovirus type 3
2015, Virus ResearchCitation Excerpt :However, among the 52 known adenoviruses, only a few epitopes have been identified so far (Bradley et al., 2012; Yuan et al., 2009; Qiu et al., 2012). In our previous studies, we identified several epitopes of HAdV-3 and HAdV-7 with a series of MAbs (Liu et al., 2014; Tian et al., 2013; Qiu et al., 2009). We also demonstrated with experiments using epitope-incorporated recombinant adenoviruses that HVR1, 2, 5, and 7 of HAdV-3 contain the neutralizing residues (Qiu et al., 2012).
Identification of two novel fowl adenovirus C-specific B cell epitopes using monoclonal antibodies against the capsid hexon protein
2018, Applied Microbiology and BiotechnologyExpression and serological reactivity of hemorrhagic enteritis virus hexon protein
2016, Folia Microbiologica