Production of a monoclonal antibody against SARS-CoV spike protein with single intrasplenic immunization of plasmid DNA

Abstract

Severe acute respiratory syndrome (SARS) is a highly infectious disease caused by a novel coronavirus (SARS-CoV). Specific monoclonal antibodies (mAbs) against the SARS-CoV are vital for early diagnosis and pathological studies of SARS. Direct intrasplenic inoculation of plasmid DNA encoding antigen is an effective and fast approach to generate specific mAb when the protein antigen is difficult to prepare or dangerous in use. In this study, we selected one fragment of SARS-CoV spike protein (S1-(3)) as antigenic determinant by immunoinformatics. Single intrasplenic immunization of plasmid DNA encoding S1-(3) induced anti-spike protein antibodies. We established one hybridoma cell line secreting specific mAb and evaluated this mAb with murine leukemia virus pseudotyped with SARS-CoV spike protein (MLV/SARS-CoV). The mAb could recognize the spike protein on the MLV/SARS-CoV-infected Vero E6 cells albeit with no neutralizing effect on the infectivity of the pseudotype virus. Our results show that a single-shot intrasplenic DNA immunization is efficient for the production of specific mAb against SARS spike protein, and a linear epitope of the spike protein is recognized in this study.

DNA immunization as a technology platform for monoclonal antibody induction

“…38,39 Additional immunogen manipulations include the production of a 'mini-gene insert' to express a short peptide sequence to cover a receptor-binding domain. 40 In this case, antigenic determinants in the angiotensinconverting enzyme 2 binding domain of the severe acute respiratory syndrome spike protein, which does not closely match other coronaviruses, were predicted using software PROTEAN to induce anti-spike protein antibodies. Alternatively, a transmembrane anchor sequence can be added to non-membrane-associated antigens.…”

“…Abbreviation: enzyme-linked immunosorbent assay, ELISA; fluorescence-activated cell sorting, FACS; immunohistochemistry, IHC. Extracellular domain Single transmembrane [33][34][35][36][37] Fragment/subunit Any type 25,38,39,55 Mini-gene insert Any type 40 Novel format, vaccibody Secretory 56 Immunogen-transmembrane domain fusion Viral non-structure 41 Immunogen-Fc fusion Intracellular 57,58 Abbreviation: glycophosphatidylinositol, GPI.…”

“…30 There have also been reports of producing mAbs with a single intrasplenic injection. 40,54 The author generated hybridomas by fusing spleen cells at 2, 3, 5, 10 and 25 days after a single intrasplenic injection of DNA vaccine plasmids. The highest number of specific hybridomas was generated at day 5 after a single initial injection.…”

Positive reactions on Western blots do not necessarily indicate the epitopes on antigens are continuous

“…There are multiple other examples of epitopes determined by Western blots to be continuous without any confirmative evidence that was so. [19][20][21][22][23][24][25][26][27][28] In characterizing monoclonal antibodies (mAbs) that reacted with hepatitis E virus (HEV) capsid protein on Western blots, we tried to determine the epitopes using random peptide libraries; however, no reactive peptide was identified. This led us to question whether Western blot-reactive antibodies exclusively recognize continuous epitopes.…”

Mouse monoclonal antibodies in biological research: strategies for high-throughput production

“…With this approach, specific hybridomas secreting antibodies against a 18-kDa protein from Brucella abortus were generated only 25 days after single-shot (Velikovsky et al 2000). Therefore, the intra-splenic immunisation of DNA encoding for the antigen of interest has been proposed as a helpful tool for high-throughput production of monoclonal antibodies (Yu et al 2005;Velikovsky et al 2000).…”