An H5N1-based matrix protein 2 ectodomain tetrameric peptide vaccine provides cross-protection against lethal infection with H7N9 influenza virus

Leung, Ho-Chuen; Chan, Chris Chung-Sing; Poon, Vincent Kwok-Man; Zhao, Han-Jun; Cheung, Chung-Yan; Ng, Fai; Huang, Jian-Dong; Zheng, Bo-Jian

Mouse-adapted A/H7N9/Anhui/01 virus was inoculated in 10-dayold specific pathogen-free (SPF) eggs and the allantoic fluid was collected after three-day incubation at 35 6 C. The allantoic fluid was aliquoted and stored at 280 6 C before use. The virus titer was detected by 50% tissue culture infective dose (TCID 50 ) assay, while the 50% lethal dose (LD 50 ) was determined by challenging BALB/c mice with serial dilutions of the virus as described previously. 20 All experiments involving H7N9 virus were performed in biosafety level 3 (BSL-3) facilities as described previously. 22 Animal experiment Two groups of BALB/c mice (15 mice/group) were subcutaneously (s.c.) vaccinated with H5N1-M2e tetramer peptide pre-mixed, respectively, with Freund's adjuvant (FA, Sigma, St. Louis, SO, USA) and the Sigma adjuvant system (SAS) (Sigma, St. Louis, SO, USA) as described previously. 23 Three other groups of mice (15 mice/ group) were s.c. injected with FA, SAS or phosphate-buffered saline (PBS) alone as negative controls. Briefly, the mice were primaryimmunized with 10 mg of H5N1-M2e pre-mixed with complete FA or SAS, and then boosted with 10 mg of the peptide with incomplete FA or SAS twice in a three-week interval. Mice sera were collected 1 day prior to each vaccination and 10 days after the second booster vaccination for detection of specific antibodies. Ten days after the last vaccination, the mice were intranasally (i.n.) inoculated with 10 LD 50 of the mouse-adapted strain of A/Anhui/01 (H7N9) after anesthetization with ketamine and xylazine. The challenged mice were observed for 21 days or till death. Lung tissues were collected from 5 mice/group at day 6 post-challenge for virological and pathological investigation.