I.Background
HFMD
Hand, foot, and mouth disease (HFMD) is an infectious disease caused by enteroviruses. HFMD occurs in children under 5 years of age. It can cause herpes in the mouth, hands, and feet, or complications such as myocarditis and meningitis.
From July to December in 1997, 11 previously healthy children in Peninsular Malaysia succumbed to an acute severe refractory leftventricular heart failure, following a brief prodromal illness characterized by fever (axillary temperature > 38 degrees C) (100%), oral ulcers (72%), extremity rashes (45%) and significant vomiting (55%); they were in the midst of an outbreak of hand, foot and mouth disease (HFMD). China has the largest number of patients of it (8:87% of the total number of nine foot diseases in 2014), with enterovirus 71(EV71) as its main pathogenic agent. Hence, it is then highly urgent for people to think up a solution to control and forestall the infection of EV71.
Vaccine
The most effective way to deal with the high prevalence of HFMD caused by EV71 is vaccination. The mechanism of the vaccine is to introduce viruses or parts of them to human bodies and to build up specific memory cells, in a word, immunity, for future virus infection.
There are already various types of EV71 vaccines, including live attenuated viruses, inactivated whole viruses, VLPs, and recombinant protein subunits. Nonetheless, these injection-based vaccines are never coming without their limitations--- relatively low coverage rate due to children’s fear of needles, high consumption of time and labor due to requirements of injection, huge production of biohazardous waste, and high incidence of side effects. There isneed to design safer, more convenient and more effective vaccine delivery methods to protect people, especially children, from unaddressed and emerging diseases.
II.General Concept
Vaccinations include injections (hepatitis B, BCG, and influenza, etc.) and oral vaccines (polio, cholera, and rotavirus). Given the prevalent needle fears of children and a series of problems caused by injections, we select oral vaccine as the form of our EV71 vaccine.
Oral vaccine is self-administered, more acceptable to children, and biohazardous-waste-free; additionally, the oral route enables stimulation of humoral and cellular immune responses at both systemic and mucosal sites. It seems that oral EV71 vaccine can compensate for the limitations of traditional injection-based vaccine and provide people with a broader and long-lasting protection from HFMD.
Lactobacillus casei
Lactobacillus casei is an important probiotic to maintain the stability of human intestinal flora. L. casei can tolerate the body's defense mechanisms, including enzymes in the mouth, low pH in gastric juices, and bile acids in the small intestine. Therefore, L. casei can survive in the intestinal tract in large quantities after entering the human body, regulating the balance of intestinal flora, and promoting human digestion and absorption; it is an ideal expression vector for an oral live vaccine. Therefore, after we transform the plasmid into E. coli, we will also transform them into L. casei to express the target proteins.
EV71 vaccine
VP1 is the main antigenic gene of the EV71 virus, which can encode capsid protein and promote the invasion of virus particles into host cells (figure 1). Therefore, we selected VP1 as the target gene for the development of the EV71 vaccine. The B-subunit of heat-labile enterotoxin (LT) in E. coli, called LTB, has strong immunogenicity and adjuvant activity, and will not do harm to the human body. LTB and a variety of unrelated proteins and their non-protein antigens can enhance the mucosal IgA and humoral IgG responses of antigens through different immune pathways. In conclusion, we will combine VP1 and LTB in our vaccine.
Figure-1 VP1 is the main antigen gene of EV71
We use subunit vaccine and adjuvant, VP1-LTB in this case since it includes the immunological part of EV71 but does not confront people with the risk of counteractive effects, such as the virus particles returning back to active stages and infecting people.
Design
First of all, we will manipulate PCR on two types of plasmids: pUC57-VP1 and pUC57-LTB. After each PCR process, we will manipulate electrophoresis on the samples that we have got, in order to examine if our PCR is successful and to extract the DNA fragments we want. Therefore, from the first step, we will obtain DNA fragments VP1, VP1-Lkr, and LTB. With the linker, we are able to manipulate overlap extension PCR (OE PCR) to connect VP1-Lkr and LTB together to form VP1-LTB. After this, we will conduct enzyme digestion on VP1-LTB and VP1, which create sticky ends at both ends of these DNA fragments.
We will then carry out enzyme digestion on the plasmid pGEX-6P-1 to cut off the DNA fragment. Then, we will ligate link VP1 and VP1-LTB fragments respectively with the plasmid pGEX-6P-1. Finally, we will get two plasmids: pGEX-6P-1-VP1 and pGEX-6P-1-VP1-LTB.
Next, we will transform these two plasmids into chemically competent cells, E. coli DH5α, and allow these bacteria to grow and replicate. After this step, a massive number of plasmids pGEX-6P-1-VP1 and pGEX-6P-1-VP1-LTB could be extracted from the bacteria. A small amount of this sample will be sent to genetic sequencing agencies to test if the transformation of the plasmid has been successful. The rest of the sample will be processed in another transformation process. pGEX-6P-1-VP1 and pGEX-6P-1-VP1-LTB will be transformed into two other types of bacteria: E. coli BL21 and L. casei ATCC 334. Then, we will carry out IPTG induction for E. coli BL21 and L. casei ATCC 334. After this, we will manipulate SDS-PAGE, Coomassie brilliant blue staining, and Western blot to detect the expression of VP1 and VP1-LTB proteins.
All the above processes are shown in figures 2 and 3.
Figure-2 General concept of the experiments (1)
Figure-3 General concept of the experiments (2)
III.Expected Result
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The plasmid pGEX-6P-1-VP1 and pGEX-6P-1-VP1-LTB are successfully constructed, which are identified by enzyme double digestion and enzyme single digestion.
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VP1 protein products with biological functions could be obtained from E. coli BL21 and L. casei ATCC 334. SDS-PAGE, Coomassie brilliant blue staining, and Western blot analysis show that the protein has good antigenicity.
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We hope that our project could pave the way for a much safer, more convenient and efficient oral vaccine for children to defend against HFMD.
IV.Reference
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Buch MH, Liaci AM, O'Hara SD, Garcea RL, Neu U, Stehle T (October 2015). "Structural and Functional Analysis of Murine Polyomavirus Capsid Proteins Establish the Determinants of Ligand Recognition and Pathogenicity". PLoS Pathogens. 11 (10): e1005104. doi:10.1371/journal.ppat.1005104
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Ramqvist T, Dalianis T (August 2009). "Murine polyomavirus tumour specific transplantation antigens and viral persistence in relation to the immune response, and tumour development". Seminars in Cancer Biology. 19 (4): 236–43. doi:10.1016/j.semcancer.2009.02.001
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Nassef, C., Ziemer, C., & Morrell, D. S. (2015). Hand-foot-and-mouth disease: a new look at a classic viral rash. Current opinion in pediatrics, 27(4), 486–491. https://doi.org/10.1097/MOP.0000000000000246
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Who.int. 2021. How do vaccines work?. [online] Available at: <https://www.who.int/news-room/feature-stories/detail/how-do-vaccines-work?gclid=EAIaIQobChMIn4OC7YOh8gIVsG1vBB0wYgcmEAAYAyAAEgIBFvD_BwE> [Accessed 8 August 2021].
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Yee, Pinn & Poh, Chit. (2015). Development of Novel Vaccines against Enterovirus-71. Viruses. 8. 1. 10.3390/v8010001.
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Orlando, A.; Refolo, M. G.; Messa, C.; Amati, L.; Lavermicocca, P.; Guerra, V.; Russo, F. (October 2012). "Antiproliferative and Proapoptotic Effects of Viable or Heat-Killed IMPC2.1 and GG in HGC-27 Gastric and DLD-1 Colon Cell Lines". Nutrition and Cancer. 64 (7): 1103–1111. doi:10.1080/01635581.2012.717676