[Detection of neutralizing antibody to human adenovirus type 5 in marmosets].
OBJECTIVE
To construct a recombinant human adenovirus type 5 (Ad5) expressing luciferase and GFP reporter gene and detect neutralizing antibodies against adenovirus type 5 in common marmosets (Callithrix jacchus) to provide basic laboratory data for evaluating adenovirus vaccines.


METHODS
Luciferase and GFP reporter genes from plasmid pHAGE-CMV-GFP were inserted into pDC315 to construct the recombinant adenovirus shutter plasmid pDC315-Luc-GFP. The shutter plasmid was co-transduced with pBHGlox(delta)E1,3Cre in 293A cell line to package the recombinant adenovirus rAd5/Luc/GFP. Three rounds of plaque formation experiment were performed to select the monoclonal adenovirus followed by purification with cesium chloride density gradient centrifugation and virus titration with TCID50 method. Chemiluminescence assay and flow cytometry were employed to detect the neutralizing antibody levels in 14 common marmosets.


RESULTS
The shuttle plasmid pDC315-Luc-GFP was successfully constructed and the recombinant adenovirus rAd5/Luc/GFP was packaged with a the titer reaching 6.9×10(11.5) PFU/mL. In the 14 marmosets, chemiluminescence assay identified 4 (28.6%) marmosets that were positive for Ad5-neutralizing antibodies, including 2 with a antibody titer of 1/16 and another 2 with a titer of 1/32; flow cytomery detected Ad5-neutralizing antibodies in 3 marmosets at the titer of 1/16.


CONCLUSIONS
Chemiluminescence assay is a simple, sensitive, and accurate modality for detecting Ad5-neutralizing antibodies. Common marmosets have a very low positivity rate for Ad5-neutralizing antibodies and are therefore promising models for studying adenovirus-based vaccines and therapies.
Department of Transfusion Medicine, Southern Medical University, Guangzhou 510515, China. E-mail: 759921681@qq.com.
Ya-Chun
YC
Sun
Ting-Ting
TT
Li
Yi-Lin
YL
Wang
Ling
L
Zhang
Hai
H
Zhu
Cheng-Yao
CY
Li
D000260:Adenoviruses, Human / Q000276:immunology*; D000818:Animals; D057134:Antibodies, Neutralizing / Q000097:blood*; D000914:Antibodies, Viral / Q000097:blood*; D002144:Callithrix; D002460:Cell Line; D006801:Humans; D056724:Immunity, Humoral; D008156:Luciferases; D010957:Plasmids
D057134:Antibodies, Neutralizing; D000914:Antibodies, Viral; D008156:Luciferases