Use of hexon as an antigen for the production of monoclonal antibodies capable of detecting multiple adenovirus types
Introduction
Infectious diseases of viral etiology are one of the most important health problems. Particularly, in Russia, about 50 million cases of infectious diseases are registered annually, with up to 90% falling into the acute respiratory viral infection (ARVI) category. When non-influenza causes of ARVI are analyzed, the main pathogens seen are adenoviruses (AdV), respiratory syncytial virus (HRSV), and parainfluenza viruses, among others [1].
Adenoviral infections usually cause illness affecting the upper respiratory tract, lower respiratory tract, pharynx, conjunctiva, or gastrointestinal tract. Outbreaks of AdV infection can occur throughout the year, not only among those with weakened immunity, but also among healthy children or adults. In particular, people located in dense living conditions, such as military personnel, residents of nursing homes, children in summer camps, and those in specialized medical institutions, are at risk. The contribution of AdV infections in children and young people of draft age to the overall statistical structure of ARVI may be as high as 30.0% and 64.6%, respectively [[2], [3], [4]].
Most of the time, adenovirus caused illnesses resolve uneventfully and further respiratory problems are not seen. However, complications do sometimes occur, including disseminated infections and pneumonias that become fatal. Such outcomes can occur with immunocompromised patients or with immunocompetent patients.
Adenoviruses are divided into 7 groups (A-G) based on their biological, chemical, immunological, and morphological properties. About 50 AdV types are human pathogens and their clinical manifestations are largely dependent on the group to which they belong (Table 1) [5].
The contribution of adenovirus to the overall category of acute respiratory diseases in Russia is about 5% according to the National Centre for Surveillance of Influenza and Acute Respiratory Viral Infections (World Health Organization local partnership). In some age groups, however, this value differs significantly from the overall average for the population [2,4]. Currently, adenoviruses circulating in Russia are not typed in a systematic way. Some researchers are studying adenovirus dynamics, but their results are fragmentary in nature, and the work typically focuses on a limited number of patients with ARVI during limited intervals. It was shown that in the 1997–2011 time period, types seen circulating among military personnel were adenovirus 1, 2, 3, 4, 5, 6, 7, and 21 [6]. Other work, studying the 2010–2014 time frame, showed that adenovirus 1, 2, 3, 4, 6, and 7 were the types circulating in the Moscow population [2].
Currently, DNA detection methods, such as polymerase chain reaction (PCR), as well as viral antigen specific methods (immunofluorescent assays and enzyme-linked immunosorbent assay, ELISA) are used for clinical differential diagnosis of AdV infections. In Russia, polyclonal sera based antibodies are widely used in ELISA and immunofluorescent tests. The main disadvantage of these methods, in terms of the immunoreactive reagents, is inconsistency with regard to antibody composition. Reagent quality depends on the nature of the immune response elicited in the antibody producing animal. The inclusion of monoclonal antibodies (mAbs) targeting specific viral determinants in modern immunological tests permits higher sensitivities, higher specificities, and more appropriately standardized reagents [7].
Adenoviruses are a DNA containing viruses featuring an icosahedron-shaped particle formed by hexon, penton, and fibril proteins. Each virus particle contains 12 molecules of penton and fibril proteins that carry type-specific antigens. Variability of the latter do not permit useful mAbs to be produced for the purpose of subtyping adenovirus members within the larger AdV family. Hexon protein constitutes about 60% of the AdV particle by mass, and it features an extensively conserved region with virus family specific determinants. The amino acid variability in this region is less than 15% [8]. In infected cells, hexon is synthesized in excess, and only 20–30% of the protein is used to assemble viral particles. This makes obtaining hexon in native form and generating mAbs against it relatively straightforward. Anti-hexon mAbs can, in turn, be used for the detection of various AdV types in clinical specimens. A panel of anti-hexon mAbs were generated at the Laboratory of Biotechnology and Diagnostic Reagents (Smorodintsev Research Institute of Influenza, Russian Ministry of Health), and their properties further characterized.
Section snippets
Cell cultures and viruses
The A549 (human lung carcinoma) cell line, the MA-104 (macaque embryonic kidney epithelium) cell line, adenoviruses (types 3, 4, 6 and 19), and human respiratory syncytial virus (HRSV, “Long” strain) were obtained from the Smorodintsev Research Institute of Influenza collection.
Preparation of purified virus concentrates
Viruses were allowed to replicate in cell cultures growing at 37 °C until the appearance of cytopathic effects. Adenovirus was replicated in A549 cells for 72 h, and human respiratory syncytial virus was replicated in
Results and discussion
Adenovirus hexon contains virus family specific antigens and has a fairly conserved amino acid sequence among the various AdV types. In addition, the protein is highly expressed in infected cells and can be obtained in its native form. These features are the reason hexon is promising as an immunogen for the production of mAbs capable of detecting all AdV types. Adenovirus type 6 hexon, in crystalline form (Fig. 1), was generated at the Laboratory of Biotechnology and Diagnostic Reagents
Conclusions
Currently, monoclonal antibodies are made in virtually the same way as described in the original methods authored by G. Köhler and S. Milstein [15], or in alternative methods such as phage display [20]. Monoclonal antibodies are broadly used, both for scientific research and in medical practice. One of the promising areas is the preparation of therapeutic mAbs targeting the viral and bacterial agents that cause infection. Monoclonal antibody based drugs are being successfully used in the
Funding
This work was supported by Russian Ministry of Education and Science, contract number 14.604.21.0180 (project identification number RFMEFI60417X0180).
Acknowledgments
The authors express deep and sincere gratitude to Tatiana Radistovna Tsareva and Evgeny Valentinovich Sorokin for invaluable help in working with mAb producing hybridomas. The authors thank Sergey Anatolievich Klotchenko for his assistance in preparing this text.
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