Overexpression and purification of HSV‐2 glycoprotein D in suspension CHO cells with serum‐free medium and immunogenicity analysis

Glycoprotein D (gD2) is the most important candidate antigen for herpes simplex virus type 2 (HSV‐2) vaccine development. Establishment of a stable eukaryotic cell line to overexpress gD2 and an efficient purification process to purify is essential for the development of subunit vaccine against HSV‐2. The DNA sequence of the extracellular epitope‐rich fragment of gD2 was optimized, chemically synthesized, and cloned into plasmid pMD902. The recombinant plasmid pMD902‐gD was stably transfected into CHO‐DG44 cells, and cell lines with high levels of expression of gD2 were established. The recombinant gD2 was purified efficiently using an anion exchange column and a Sephadex G‐25 desalting column. The yield of the purified gD2 was 57 mg/L of serum‐free culture medium, and its purity was determined to be about 95% by HPLC analysis. Finally, the immunogenicity of the purified gD2 was measured and it induced strong and specific humoral immunity and higher level of cellular immune response than gD2 expressed in prokaryotic cells. We established a stable, secretory, and high‐yield gD2‐expression cell line and an easy and efficient gD2‐purification process, which lays the foundation for preparation of large amount of gD2 that is essential for HSV‐2 subunit vaccine development.

[1]  A. Wald,et al.  Current status and prospects for development of an HSV vaccine. , 2014, Vaccine.

[2]  Harvey M. Friedman,et al.  Protection Provided by a Herpes Simplex Virus 2 (HSV-2) Glycoprotein C and D Subunit Antigen Vaccine against Genital HSV-2 Infection in HSV-1-Seropositive Guinea Pigs , 2013, Journal of Virology.

[3]  Y. Qi,et al.  Recombinant protein YbgF induces protective immunity against Rickettsia heilongjiangensis infection in C3H/HeN mice. , 2013, Vaccine.

[4]  A. Wald,et al.  HSV-2: in pursuit of a vaccine. , 2011, The Journal of clinical investigation.

[5]  Scott Rose,et al.  Effect of aciclovir on HIV-1 acquisition in herpes simplex virus 2 seropositive women and men who have sex with men: a randomised, double-blind, placebo-controlled trial , 2008, The Lancet.

[6]  J. Beckmann,et al.  Genetic characterization of CHO production host DG44 and derivative recombinant cell lines. , 2006, Biochemical and biophysical research communications.

[7]  P. Spear,et al.  Different receptors binding to distinct interfaces on herpes simplex virus gD can trigger events leading to cell fusion and viral entry. , 2006, Virology.

[8]  F. Aoki,et al.  Safety and immunogenicity of glycoprotein D-adjuvant genital herpes vaccine. , 2004, Clinical infectious diseases : an official publication of the Infectious Diseases Society of America.

[9]  Martin Fussenegger,et al.  Impact of coexpression and coamplification of sICAM and antiapoptosis determinants bcl-2/bcl-x(L) on productivity, cell survival, and mitochondria number in CHO-DG44 grown in suspension and serum-free media. , 2002, Biotechnology and bioengineering.

[10]  F. Aoki,et al.  Glycoprotein-D-adjuvant vaccine to prevent genital herpes. , 2002, The New England journal of medicine.

[11]  A. Wald,et al.  Herpes simplex virus: the importance of asymptomatic shedding. , 2000, The Journal of antimicrobial chemotherapy.

[12]  A. Adimora,et al.  Recombinant glycoprotein vaccine for the prevention of genital HSV-2 infection: two randomized controlled trials. Chiron HSV Vaccine Study Group. , 1999, JAMA.

[13]  D. Bernstein,et al.  Herpes simplex virus vaccines. , 1999, Vaccine.

[14]  W. Mitchell,et al.  Persistence of Herpes Simplex Virus Type 1 DNA in Chronic Conjunctival and Eyelid Lesions of Mice , 1998, Journal of Virology.

[15]  W. Noé,et al.  Appropriate mammalian expression systems for biopharmaceuticals. , 1998, Arzneimittel-Forschung.

[16]  S. Straus,et al.  The Quantity of Latent Viral DNA Correlates with the Relative Rates at Which Herpes Simplex Virus Types 1 and 2 Cause Recurrent Genital Herpes Outbreaks , 1998, Journal of Virology.

[17]  H. Lönroth,et al.  Identification of Helicobacter pylori by immunological dot blot method based on reaction of a species-specific monoclonal antibody with a surface-exposed protein , 1995, Journal of clinical microbiology.

[18]  R. Burke,et al.  Soluble forms of herpes simplex virus glycoprotein D bind to a limited number of cell surface receptors and inhibit virus entry into cells , 1990, Journal of virology.

[19]  P. Spear,et al.  Initial interaction of herpes simplex virus with cells is binding to heparan sulfate , 1989, Journal of virology.

[20]  D. Johnson,et al.  Herpes simplex viruses lacking glycoprotein D are unable to inhibit virus penetration: quantitative evidence for virus-specific cell surface receptors , 1988, Journal of virology.

[21]  L. Lasky,et al.  Efficacy of recombinant glycoprotein D subunit vaccines on the development of primary, recurrent, and latent genital infections with herpes simplex virus type 2 in guinea pigs. , 1988, The Journal of infectious diseases.

[22]  N. Stow,et al.  Herpes simplex virus immunoglobulin G Fc receptor activity depends on a complex of two viral glycoproteins, gE and gI , 1988, Journal of virology.

[23]  J. Glorioso,et al.  Neutralizing monoclonal antibodies specific for herpes simplex virus glycoprotein D inhibit virus penetration , 1987, Journal of virology.

[24]  J. Kappes,et al.  Antibody response, recurrence patterns and subsequent herpes simplex virus type 2 (HSV-2) re-infection following initial HSV-2 infection of guinea-pigs: effects of acyclovir. , 1986, The Journal of general virology.

[25]  L. Corey,et al.  Risk of Recurrence after Treatment of First‐episode Genital Herpes with Intravenous Acyclovir , 1985, Sexually transmitted diseases.

[26]  R. Eisenberg,et al.  Glycoprotein C of herpes simplex virus 1 acts as a receptor for the C3b complement component on infected cells , 1984, Nature.