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Merck
  • Generation of oligomers of subunit vaccine candidate glycoprotein D of Herpes Simplex Virus-2 expressed in fusion with IgM Fc domain(s) in Escherichia coli: A strategy to enhance the immunogenicity of the antigen.

Generation of oligomers of subunit vaccine candidate glycoprotein D of Herpes Simplex Virus-2 expressed in fusion with IgM Fc domain(s) in Escherichia coli: A strategy to enhance the immunogenicity of the antigen.

3 Biotech (2020-10-14)
Vikas Kumar Singh, Sandeep Kumar, Rajeev Kumar Dhaked, Abdul S Ansari, Nirmal K Lohiya, Suman Tapryal
ABSTRACT

Glycoprotein D (gD) of Herpes Simplex Virus-2 is used as an antigen in various anti-herpes subunit vaccines owing to its involvement in binding the host cell receptors for host infectivity. However, most of these monomeric protein based candidates have shown low immunogenicity in animal models. To enhance the immunogenicity of gD, a fresh approach of fusing its ectodomain with the Fc domain(s) of IgM has been adopted to oligomerize the viral antigen and to exploite the immune-modulating potential of IgM Fc. Six vaccine constructs, generated by fusing three gD-ectodomain-length-variants with the Ig µ-chain domain 4 (µCH4) and µCH3-CH4 fragment, were cloned in Escherichia coli using pET28b( +) vector. The vaccine proteins were expressed in the form of inclusion bodies (IBs) and were in vitro refolded into protein oligomers of high stoichiometries of ~ 15-24, with 70-80% refolding yields. The conformations of gD and Fc components of the refolded oligomers were analyzed by ELISA and CD spectroscopy and were found to be native-like. The sizes and profiles of the size-distribution of oligomers were determined by dynamic light scattering (DLS). The candidate C2 (gD-μCH3-CH4), showing the most compact oligomer size and uniform distribution of its particles was chosen as the suitable candidate for mice immunization studies to assess the immunogenicity of the antigen gD. The C2 oligomer stimulated a strong anti-gD humoral response with an antibody titer of 102,400 and a strong, biased Th1 immune response in C57BL/6 mice, indicating its potential as a strong immunogen which may serve as an effective vaccine candidate.

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