Study Of An Animal Model Infected With Repeated Low-dose Influenza Virus And Development Of A Novel H7N9 Vaccine

Influenza is an acute respiratory disease caused by influenza virus.Influenza virus spreads among humans through the droplets,direct or indirect contact with influenza virus contaminated goods.Influenza pandemics caused huge threat to human public health due to the high contagious and high incidence of influenza infection,which resulting in 50 million global infection annualy,of which about 500,000 people died.Influenza virus exhibits rapid drug-resistant characters because of its high mutation rate and high recombination rate.The most effective way to combat with influenza virus is vaccination.In traditional vaccine research and development,a single high-dose virus challenge of animals is used to evaluate vaccine efficacy.This type of challenge model may have limitations.During the natural infection with influenza virus,people may get infected for several times at low lose in a confined space.In order to understand the immune responses involved in the natural influenza infection and to establish a more stringent challenging model for influenza vaccine research,we conducted a repeated low-dose influenza infection in mice,and explored the immune responses involved in this model.In the current clinical study,the vaccine effect was evaluated by a single high-dose influenza virus attack by infecting the animals immunized with the vaccine.However,in the natural infection process,individual cold may be in a specific time and specific space repeated several times after low doses of exposure to influenza virus caused by viral infection.In this process,the body’s antiviral immune response is not clear.In order to study the mechanism of immune response in the process of repeated infection of influenza virus,we restablished the mouse model of repeated low-dose of influenza virus infection for the first time,and systematically studied the pathologic reaction and immune responses involved in this infection model,and explored the potential of using this infection model for the evaluation of influenza vaccine efficacy and anti-influenza drug screening.In this study,8 groups of 6-8-week old female C57BL/6 mice were infected with different doses of A/PR8 influenza virus and PBS by nasal infection.Some groups of mice were got repeated infection with A/PR8 virus for three times at same dose every 24 hours.Mice body weight and survival rate were mornitored every day.Virus titers in lung tissues of infected mice were titrated and the pathological changes of lung tissues were evaluated.The results showed that repeated low-dose challenges caused more severe disease in mice compared with a single high-dose infection,manifested chiefly by that the mice received repeated low-dose challenges showed earlier morbidity,high mortality,high virus titer and serious damage in lung.Virus-speicific antibodies and CD8+ T cells were detected 7 days after the first infection.We found that antibody responses were rarely activated in the repeated low-dose infection groups,while the magnitude of CD8+T cell activation did not correlate with virus dose or the number of infections.In addition,we measured the levels of NLRP3 inflammasome related cytokines IL-1β and IL-18 on the 7th day after the initial infection of influenza virus to explore the pathogenesis of the repeated low-dose infection animal model.The production of cytokines IL-1β and IL-18 in the animals that received repeated low-dose infection was higher.Therefore,we speculated that the serious damage in mice may be caused by a "Cytokine Storm" response after influenza virus repeated low-dose infection.We also found that commercial inactivated trivalent influenza vaccines can protect mice from single high-dose of influenza virus infection,but was inefficacious against repeated low-dose influenza virus infection.This result indicated that repeated low-dose challenges may provide a more stringent mode to evaluate vaccine efficacy.Taken together,we established a mouse model of repeated low-dose influenza virus infection for the first time.The pathologic and immune responses of mice in the process of repeated infection of influenza virus were studied.The novel influenza infection model is a more realistic model for the evaluation of influenza vaccine and drug development.Meanwhile,the establishment of the repeated low-dose infection animal model has a profound significance in the influenza prevention and treatment.In the spring of 2013,a novel H7N9 avian influenza virus emerged in Shanghai and Anhui provinces and was found to infect humans.The novel H7N9 virus posed a huge threat to human health and daily life.Since 2013,H7N9 influenza epidemic has not been in effectively controlled.Although H7N9 influenza virases have not evolved the ability of human-to-human transmission,reports of poultry-to-human appeared now and then.Human infections of H7N9 virus showed common cold symptoms at first,and then developed to serious pneumonia and acute respiratory distress syndrome and death.During the winter of 2016 and the spring of 2017,the fifth outbreak of human infection with avian influenza A(H7N9)virus is more serious than previous outbreaks over the past few years.The number cases of infection with the H7N9 suddenly increased from early time in the winter of 2016.The virulence of novel H7N9 virus has increased and the drug-resistant influenza strain has been isolated.According to the National Health and Family Planning Commission of China,298 cases of H7N9 infection(including 99deaths)were reported in December 2016 and January 2017.Now a variety of H7N9 vaccine developments are in progress.H7N9 inactivated vaccine and live attenuated vaccine have been evaluated in animal models and show good effectiveness.Some of the vaccines have been in the phase 1 trial.However,preparing H7N9 viruses is a high-risk task and must be performed in a BSL3 lab,which made the vaccines expensive.In addition,adjuvants are needed due to the poor immunogenicity of the inactivated influenza vaccines.Whafs more,the stability of these vaccines is a problem,and a little improper preservation will invalidate the vaccine.The development of novel H7N9 vaccine is in extremely urgent.Viral vector has been proven to be a good foreign gene carrier in both vaccine development and cancer therapy.Various vaccines and gene therapeutic agents have been lisenced in clinical use.In this study,we adopted chimpanzee adenovirus AdC68 to express H7N9 HA(AdC68-H7HA)as a novel influenza vaccine.The HA gene was amplified from the virus RNA and then cloned into the El-deleted chimpanzee adenoviral vector AdC68.AdC68-H7 HA vector was linearized by Pac I digestion and then transfected into human embryonic kidney cells(HEK293)for virus package.Rescued adenoviruses were fiirther expanded and purified by caesium chloride density-gradient centrifugation.Expression of the target protein was identified by Western Blot and FACS.A DNA vaccine pCAGGS-H7 HA was constructed for comparision.A DNA-prime-Adenovirus-boost regimen was also evaluated in this study.The mice were divided into four groups,which were immunized intramuscularly with AdC68-H7HA(5× 1010vp),pCAGGS-H7HA(50μg),pCAGGS-H7HA(50μg),or AdC68-empty(5×1010vp).Two weeks after the prime,only the second group was boosted with AdC68-H7 HA at a dose of 5×1010 vp,which was designated as the prime-boost group.H7 HA-specific antibodies and CD8+T cells in the mice were detected after the immunization.Mice were further challenged with lethal H7N9 virus to evaluate the effectiveness of the vaccines.Serum-transfer experiments and the CD8+T depletion experiments were performed to study the mechanism of the vaccines.We also found that the recombinant adenovirs vaccine can induced high hurmol immunity in guinea pigs.Taken together,the recombinant adenovirus vaccine AdC68-H7HA and Prime-boost regimen can activate robust immune responses in both mice and guinea pigs,and protected mice from lethal H7N9 virus infection.