Concept of product
Concept map of the EV71 oral vaccine
·Description
Nonproprietary (Generic) Name: EV71 oral vaccine
Dosage: 50ml
Dosage Form: Lactobacillus casei beverage
Color: colorless
Inactive Ingredients: water, fructose, salt, spices, sweetener
Optimum shelf life: 18 months at 32~39.2℉（0～4℃）
·This medicine works for
The drug can be taken orally to vaccinate people mainly infants and young children under 5 ages for preventing hand, foot, and mouth disease (HFMD).
·Before using this medicine
How to take:
Take this medicine by mouth as directed.
This medicine can be taken with or without food.
It should be adjusted according to the disease, age, and symptoms of the patient. Strictly follow the order of your doctor.
Tell your Doctor:
If you ever had any unusual or allergic reaction to any medicines.
If you are using any other prescription or nonprescription (OTC) medicine.
If you are had other vaccines within two weeks.
Patients with: peptic ulcer, abnormality in blood, liver/kidney disease, heart failure, hypertension, asthma.
·Stop taking and seek medical care if you have:
Hoarseness
Cough
Lightheadedness
Drowsiness
Swelling of the face, throat, tongue, lips, eyes, hands, feet, ankles, or lower legs difficulty breathing or swallowing
Rash
Fainting
Bloody nose
Cold sweats
Change in taste bad unusual or unpleasant (after) taste
·Warning
Along with its needed effects, a medicine may cause unwanted effects. Check with your doctor immediately if they occur.
Keep out of reach of children.
When using this product, do not use it in or near the eyes. In case of contact, rinse with water to remove.
·Other information
Store at 32~39.2℉（0～4℃）
May discolor certain fabrics or surfaces
Basic Use flow
Flowchart of the EV71 oral vaccine into the human body and taking effect
Taking the doctor’s or the pharmacist’s suggestions, people can buy our products from drug stores as prescription drugs; then they take the vaccines, following the instructions. If they do not drink the vaccines immediately after buying them, they should store the vaccines at 32~39.2℉. Before drinking, they should also make sure that they have not had any unusual or allergic reaction to any medicines.
After taking the vaccines, L. casei carrying pGEX-6P-1-VP1-LTB will enter people’s GI tract; they will express GST-VP1-LTB proteins there, induce immunological responses, and build up immunity for future EV71 invasion.
Viability test and analysis
·Protein expression in E.coli BL21
Due to the relatively low rate of growth and efficiency of electroporation of L. casei, our team first transformed E.coli BL21, which is commonly used in plasmids transformation, to verify the expression and antigenicity of VP1 and VP1-LTB proteins.
We transformed the plasmids we constructed, pGEX-6P-1-VP1 and pGEX-6P-1-VP1-LTB, into E.coli BL21 respectively, and incubated them. Firstly, we ran a PAGE gel of the whole bacteria, supernate, and sediment of E.coli BL21 and then stained the gel through both coomassie brilliant blue staining and Western Blot. Via the gel after Western Blot, we confirmed that both GST-VP1 and GST-VP1-LTB had been successfully expressed in E.coli BL21. Via the gel after coomassie brilliant blue staining, we confirmed that the proteins existed in the sediments in the form of an inclusion body.
In order to find the optimized condition under which the proteins were expressed the most, we selected bacteria solution of different concentration (OD600=0.5/0.6/0.8/1), and inducted them with IPTG solution of different concentration(IPTG=1mM/10mM). Then we ran a PAGE gel of them and then marked the proteins with coomassie brilliant blue staining solution. To visualize and compare the expression of proteins under different conditions, we used the software ImageJ to quantify specific bands on the gel, collected and arranged the data, and constructed a broken line graph with OD600 the abscissa and the gray value as the ordinate.
Via this graph of the model, we could see that when OD600 was over 0.8, the expression of VP1-LTB proteins was significantly higher than that of VP1 proteins in E.coli BL21. This meant that LTB could promote the expression of VP1 proteins and therefore could enhance the efficacy of our oral vaccine as an adjuvant.
Using E.coli as the expressive system, we tentatively verified the viability of our concept that the bacteria carrying the plasmids we constructed to enter the human GI tract, expressed GST-VP1-LTB and induced immunological response there, and build immunity for humans.
·Protein expression in L. casei ATCC334
The ultimate goal of our concept is to transform the plasmids we constructed into L. casei and take it as the expressive system. We transformed the plasmids we constructed, pGEX-6P-1-VP1 and pGEX-6P-1-VP1-LTB, into L. casei ATCC334 respectively incubated them, and then inducted them with IPTG solution.
To confirm whether pGEX-6P-1-VP1 and pGEX-6P-1-VP1-LTB had been transformed into L. casei, we conducted colony PCR on the bacteria solution and then ran DNA electrophoresis of the fragments. As the graph of the gel showed, we successfully amplified VP1 and VP1-LTB fragments from the plasmids carried in L. casei, which meant that L. casei successfully carried the plasmids.
Next, we ran a PAGE gel of L. casei to further confirm whether they had expressed the plasmids they carried. After staining and scanning the gel, we did not find any obvious bands in certain KD ranges, which meant there was no obvious expression of GST-VP1 and GST-VP1-LTB in L. casei.
In conclusion, through the aforementioned experiments, we confirmed that pGEX-6P-1-VP1 and pGEX-6P-1-VP1-LTB could be carried and expressed by E.coli，and could be carried but still failed to be expressed by L. casei. Currently, we just verified the viability of our concept and determined the optimized condition of protein expression in E.coli. However, because of the difficulty in protein expression in L. casei, we would need to optimize the incubation and electroporation of L. casei, or improve and reconstruct the plasmids, to realize the expression of GST-VP1-LTB in L. casei. If L. casei did express the proteins after the improvement of our design, the next part of our project would be that we let the mice take the transformed L. casei via gavage, and then measured the level of anti-VP1-LTB IgG in the serum of the mice and the level of IgA in their feces. Finally, we could verify the immunogenicity, in another word, viability, of our concept.
Reference
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Expression of VP1 protein in the milk of transgenic mice: A potential oral vaccine protects against enterovirus 71 infection. Hsiao-Ling Chen, Jiun-Yan Huang, Te-Wei Chu, Tung-Chou Tsai, Che-Ming Hung, Chih-Cheng Lin, Fang-Chueh Liu, Li-Chung Wang, Yi-Ju Chen, Ming-Fong Lin, Chuan-Mu Chen. Vaccine. 2008 Jun 2;26(23):2882-9.；
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Optimization of microcarrier cell culture process for the inactivated enterovirus type 71 vaccine development. Suh-Chin Wu, Chia-Chyi Liu, Wei-Cheng Lian. Vaccine. 2004 Sep 28;22(29-30):3858-64.；