title: A novel high-throughput vaccinia virus neutralization assay and preexisting immunity in populations from different geographic regions in China.
abstract: BACKGROUND
  Pre-existing immunity to Vaccinia Tian Tan virus (VTT) resulting from a large vaccination campaign against smallpox prior to the early 1980s in China, has been a major issue for application of VTT-vector based vaccines. It is essential to establish a sensitive and high-throughput neutralization assay to understand the epidemiology of Vaccinia-specific immunity in current populations in China.
  
  
  RESULTS
  A new anti-Vaccinia virus (VACV) neutralization assay that used the attenuated replication-competent VTT carrying the firefly luciferase gene of Photinus pyralis (rTV-Fluc) was established and standardized for critical parameters that included the choice of cell line, viral infection dose, and the infection time. The current study evaluated the maintenance of virus-specific immunity after smallpox vaccination by conducting a non-randomized, cross-sectional analysis of antiviral antibody-mediated immune responses in volunteers examined 30-55 years after vaccination. The rTV-Fluc neutralization assay was able to detect neutralizing antibodies (NAbs) against Vaccinia virus without the ability to differentiate strains of Vaccinia virus. We showed that the neutralizing titers measured by our assay were similar to those obtained by the traditional plaque reduction neutralization test (PRNT). Using this assay, we found a low prevalence of NAb to VTT (7.6%) in individuals born before 1980 from Beijing and Anhui provinces in China, and when present, anti-VTT NAb titers were low. No NAbs were detected in all 222 samples from individuals born after 1980. There was no significant difference observed for titer or prevalence by gender, age range and geographic origin.
  
  
  CONCLUSIONS
  A simplified, sensitive, standardized, reproducible, and high-throughput assay was developed for the quantitation of NAbs against different Vaccinia strains. The current study provides useful insights for the future development of VTT-based vaccination in Beijing and Anhui provinces of China.
authors.affiliation: Department of Cell Biology, National Institutes for Food and Drug Control, Key Laboratory of the Ministry of Health for Research on Quality and Standardization of Biotech Products, Beijing, China.
authors.forename: Qiang
authors.initials: Q
authors.lastname: Liu
authors.forename: Weijin
authors.initials: W
authors.lastname: Huang
authors.forename: Jianhui
authors.initials: J
authors.lastname: Nie
authors.forename: Rong
authors.initials: R
authors.lastname: Zhu
authors.forename: Dongying
authors.initials: D
authors.lastname: Gao
authors.forename: Aijing
authors.initials: A
authors.lastname: Song
authors.forename: Shufang
authors.initials: S
authors.lastname: Meng
authors.forename: Xuemei
authors.initials: X
authors.lastname: Xu
authors.forename: Youchun
authors.initials: Y
authors.lastname: Wang
mesh_terms: D000293:Adolescent; D000328:Adult; D000818:Animals; D057134:Antibodies, Neutralizing / Q000097:blood; D000914:Antibodies, Viral / Q000097:blood; D002681:China; D003430:Cross-Sectional Studies; D005260:Female; D057166:High-Throughput Screening Assays / Q000379:methods; D006801:Humans; D049409:Luciferases, Firefly / Q000235:genetics; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008875:Middle Aged; D009500:Neutralization Tests / Q000379:methods*; D011817:Rabbits; D012900:Smallpox Vaccine / Q000276:immunology; D013997:Time Factors; D014611:Vaccination; D014616:Vaccinia virus / Q000235:genetics / Q000276:immunology*; D055815:Young Adult
chemical_list: D057134:Antibodies, Neutralizing; D000914:Antibodies, Viral; D012900:Smallpox Vaccine; D049409:Luciferases, Firefly