title: Expression of enhancing-activity-free neutralizing antibody against dengue type 1 virus in plasmid-inoculated mice.
abstract: BACKGROUND
  Most candidate dengue vaccines currently under development induce neutralizing antibodies, which are considered important for immunoprotection. However, the concomitant induction of infection-enhancing antibodies is an unavoidable concern. In contrast, a neutralizing antibody developed for passive immunotherapy has been engineered to eliminate its enhancing activity. Therefore, a strategy for the long-term expression of enhancing-activity-free neutralizing antibodies may resolve this concern.
  
  
  METHODS
  A mouse monoclonal antibody, 7F4, of the IgG3 subclass and with no detectable enhancing activity, was selected as the model neutralizing antibody to evaluate the potential of this strategy. Equal amounts of commercial vector (pFUSE)-based plasmids containing 7F4 heavy (H)- or light (L)-chain variable region genes were mixed and used for the cotransfection of 293T cells and co-delivery into ICR and BALB/c mice. The recombinant plasmids were designed to express IgG2b or IgG3 subclass antibodies (p7F4G2b or p7F4G3, respectively).
  
  
  RESULTS
  293T cells transfected with 2 μg of p7F4G2b or p7F4G3 produced approximately 15,000 or 800 ng/ml IgG in the culture fluids, respectively. The dose is expressed as the total amount of H- and L-chain plasmids. Neutralizing antibody was detected dose-dependently in ICR mice inoculated with 50-200 μg of p7F4G2b. A 1:2 dilution of sera from ICR and BALB/c mice inoculated with 100 μg of p7F4G3 showed average plaque reduction levels of >70% on day 3 and >90% on days 5-9. BALB/c mice maintained detectable neutralizing antibody for at least 3 months. The neutralizing antibody expressed by p7F4G3 in mice showed no enhancing activity.
  
  
  CONCLUSIONS
  Although the expression of neutralizing antibodies from immunoglobulin genes is a type of passive immunization, its durability can be utilized as a dengue vaccine strategy. This "proof-of-concept" study using a mouse model demonstrates that the enhancing-activity-free characteristic of this strategy augurs well for dengue vaccine development, although further improvement is required.
authors.affiliation: BIKEN Endowed Department of Dengue Vaccine Development, Faculty of Tropical Medicine, Mahidol University, Bangkok 10400, Thailand; BIKEN Endowed Department of Dengue Vaccine Development, Research Institute for Microbial Diseases, Osaka University, Osaka 565-0871, Japan. Electronic address: knmya@biken.osaka-u.ac.jp.
authors.forename: Atsushi
authors.initials: A
authors.lastname: Yamanaka
authors.affiliation: Center of Excellence for Antibody Research, Faculty of Tropical Medicine, Mahidol University, Bangkok 10400, Thailand; Department of Social and Environmental Medicine, Faculty of Tropical Medicine, Mahidol University, Bangkok 10400, Thailand. Electronic address: pannamthip.pit@mahidol.ac.th.
authors.forename: Pannamthip
authors.initials: P
authors.lastname: Pitaksajjakul
authors.affiliation: Center of Excellence for Antibody Research, Faculty of Tropical Medicine, Mahidol University, Bangkok 10400, Thailand; Department of Social and Environmental Medicine, Faculty of Tropical Medicine, Mahidol University, Bangkok 10400, Thailand. Electronic address: pongrama.ram@mahidol.ac.th.
authors.forename: Pongrama
authors.initials: P
authors.lastname: Ramasoota
authors.affiliation: BIKEN Endowed Department of Dengue Vaccine Development, Faculty of Tropical Medicine, Mahidol University, Bangkok 10400, Thailand; BIKEN Endowed Department of Dengue Vaccine Development, Research Institute for Microbial Diseases, Osaka University, Osaka 565-0871, Japan. Electronic address: ekon@biken.osaka-u.ac.jp.
authors.forename: Eiji
authors.initials: E
authors.lastname: Konishi
mesh_terms: D000818:Animals; D019138:Antibodies, Blocking / Q000276:immunology*; D000911:Antibodies, Monoclonal / Q000276:immunology; D057134:Antibodies, Neutralizing / Q000096:biosynthesis / Q000097:blood / Q000276:immunology*; D000914:Antibodies, Viral / Q000096:biosynthesis / Q000097:blood / Q000276:immunology*; D003470:Culture Media / Q000737:chemistry; D003715:Dengue / Q000276:immunology; D053059:Dengue Vaccines / Q000276:immunology*; D003716:Dengue Virus / Q000276:immunology*; D057809:HEK293 Cells; D006801:Humans; D007116:Immunization, Passive; D008297:Male; D008807:Mice, Inbred BALB C; D008813:Mice, Inbred ICR; D009500:Neutralization Tests; D010957:Plasmids / Q000276:immunology; D013997:Time Factors; D019444:Vaccines, DNA / Q000276:immunology*
chemical_list: D019138:Antibodies, Blocking; D000911:Antibodies, Monoclonal; D057134:Antibodies, Neutralizing; D000914:Antibodies, Viral; D003470:Culture Media; D053059:Dengue Vaccines; D019444:Vaccines, DNA
keywords: DNA vaccine; Dengue; Duration; Monoclonal antibody; Neutralizing antibody; Passive immunization