Team:HZAU-China/Project

• Overview
• The innovation of HZAU-iGEM Team
• The design of experiment
• Rabies
• Flea and Yersinia pestis
• Bacterial Spores as Vaccine Vehicles
• Bacillus subtilis as Probiotics
• Antimicrobial Peptides
• Methods for rearing flea

This picture was designed by our team member Chang Liu.

Rabies is a zoonotic diseases of humans and animals. The rabies virus is transmitted through exposure to the infected saliva during either a bite or direct contact with mucosal tissues. Once identified, the mortality rate is almost 100%. Rabies is widely distributed around the world. Over 55000 people die of rabies per year and about 95% of human deaths occur in Asia and Africa[1]. Dogs are the main carriers of rabies and are responsible for most of the human rabies deaths in China[2]. Once the density of dogs reaches 4.5 per km2, the rabies will spread quickly in population [3]. The density of dogs is about 8-20/km2 in China[2], which is much higher than the minimum threshold. Several ways can be used to control rabies epidemics and the most efficient way is the mass vaccination for dogs[4]. In order to control canine rabies, the vaccination coverage should be over 70% according to the WHO assessment[2]. In China, however, tremendous improvement is needed to reach such a level since the vaccination coverage is lower than 3% in the rural areas of China and almost zero for stray dogs[5,6]. This is mainly because of unawareness of rabies and the great cost of vaccination. In sum, it is a big challenge to control and prevent canine rabies in China because of the rapid spread of endemic rabies among the huge numbers of stray dogs that are very hard to register and regulate.

For this reason, our team has designed a cheap and convenient way to increase the vaccination coverage of dogs by synthetic biology. It is called “safe moving vaccine factory” that can spread Rabies vaccine in the stray dogs rapidly and actively. We hope our design can help in reaching the WHO goal of being free of human rabies by 2020.

Our idea comes from Yersinia pestis and fleas. Blocked flea can transfer Yersinia pestis to mammalian host and the mechanism has been widely researched. Yersinia pestis can build biofilm in the proventricular, which will separate the oesophagus from midgut or stomach, resulting in blockage of fleas. Blocked fleas try to feed repeatedly, causing Yersinia pestis to be regurgitated into blood and thus successfully transferring the bacteria to mammalian host[7,8,9,10]. In our iGEM project, we make use of this interesting mechanism.Bacillus subtilis have already been widely used as vaccine vectors in health and medicine[11]. We chose B. subtilis as our vaccine vector to express the antigen of Rabies. Five proteins are present in rabies virus particles: nucleoprotein, phosphoprotein, matrixprotein, glycoproteinand RNA-dependent RNA polymerase. Because of containing the neutralizing epitopes which are the targets of vaccine-induced immunity, the glycoprotein can stimulate the organism to produce antibody against Rabies[12,13,14]. So once a flea carried our modified B. subtilis which can express glycoprotein to induce immunity of stray dogs, it can be seen as a moving vaccine factory. Taking the safety question of flea into consideration, our flea will be safe enough through the endogenous or exogenous expression of “Antimicrobial Peptides” by B. subtilis[11], which can make sure our flea is free of Yersinia pestis. So it is a safe moving vaccine factory.

1. WHO (2010) Rabies, Available: http://www.who.int/mediacentre/factsheets/fs099/en/ Updated September 2010.Accessed 2011 Jun 1.

2. Tang XC, Luo ML, Zhang SY, et al. Pivotal role of dogs in rabies transmission, China. Emerg Infect Dis, 2005; 11(12), 1970‐2.

3. Cleaveland S, Kaare M, Knobel D, et al. Canine vaccination‐providing broader benefits for disease control. VetMicrobiol, 2006; 117, 43‐50.

4.ZHU Wu,Yang and LIANG Guo Dong. Current Status of Canine Rabies in China.Biomed Environ Sci,2012;25(5):602-605.

5. Zhang YZ, Xiong CL, Xiao DL, et al. Human Rabies in China. Emerg Infect Dis, 2005; 11(12), 1983‐4.

6. Hu RL, Fooks AR, Zhang SF, et al. Inferior rabies vaccine quality and low immunization coverage in dogs (Canis familiaris) inChina. Epidemiol Infect, 2008; 136, 1556‐63.

7. Bacot, A. W. (1915). LXXXI. Further notes on the mechanism of the transmission of plague by fleas. J Hyg (Lond) 14 (Plague Suppl. 4), 744–776.

8. Bacot, A. W. & Martin, C. J. (1914). LXVII. Observations on the mechanism of the transmission of plague by fleas. J Hyg (Lond) 13 (Plague Suppl. 3), 423–439.

9. Hinnebusch, B. J., Perry, R. D. & Schwan, T. G. (1996). Role of the Yersinia pestis hemin storage (hms) locus in the transmission of plague by fleas. Science 273, 367–370.

10. Jarrett, C. O., Deak, E., Isherwood, K. E., Oyston, P. C., Fischer, E. R.,Whitney, A. R., Kobayashi,S. D., DeLeo, F. R. & Hinnebusch, B. J.(2004). Transmission of Yersinia pestis from an infectious biofilm in the flea vector. J Infect Dis 190, 783–792.

11. LU?S C.S. FERREIRA, RITA C.C. FERREIRA1 and WOLFGANG SCHUMANN(2005).Bacillus subtilis as a tool for vaccine development:from antigen factories to delivery vectors.Anais da academia Brasileira de Ciências 77(1): 113-124.

12.Morimoto K, Hooper DC, Spitsin S, et al. Pathogenicity ofdifferent rabies virus variants inversely correlates withapoptosis and rabies virus glycoprotein expression in infectedprimary neuron cultures.J Virol, 1999; 73, 510‐8.

13.Faber M, Faber ML, Papaneri A, et al. A single amino acidchange in rabies virus glycoprotein increases virus spread andenhances virus pathogenicity. J Virol, 2005; 79, 14141‐8.

14.Badrane H, Bahloul C, Perrin P, et al. Evidence of two Lyssavirusphylogroups with distinct pathogenicity and immunogenicity. JVirol, 2001; 75, 3268‐76.