Enterovirus and rotavirus are the major gastrointestinal pathogens affecting the health of infants and young children. EV71 is a neurotropic virus, which often causes fever, hand-foot-mouth disease, and other clinical symptoms in children. RV mainly causes viral diarrhea in children. The main route of transmission of these two viruses is the fecal-oral transmission.
With the emergence of more and more diseases, the number of Hand-Foot and Mouth disease（HFMD）and Rotavirus(RV) infections are also gradually rising, and the transmission rate is as high as 90%, and most of the infected people are children aged 6-5 months. By the end of 2021, there were 1354548 cases of hand-foot-mouth disease in China, an increase of 593,193 cases over the previous year, a year-on-year increase of 77.91%. In May 2022, a study conducted at the Children's Hospital Affiliated with Soochow University and published in "Pediatr Infect Dis J" showed that more than one-third of pediatric diarrhea is due to rotavirus infection. Nowadays, most of the preventive vaccines against EV71 and RV virus on the market are separate injectable vaccines, either the EV71 vaccine alone or the RV vaccine alone.
Taking one type of vaccine orally can prevent two viruses at the same time, easier and more acceptable for children. The vaccine is suitable for infants above 6 months of age and below 6 years old, which are high-risk groups for HFMD and viral gastroenteritis infection. Our vaccines are also targeting parents who are very cautious about children's health since it doesn't require needles injection and the reaction shall be mild.
The bivalent oral vaccine will be priced at 165 CNY per taking, which is affordable for most parents and can be purchased at hospitals and pharmacies, but with a doctor as supervision. Meantime if some customers who have concerns about our products or do not know much about them can go to the hospital for offline information and directly consult the authoritative doctor.
Bis-Vacc is a bivalent oral live attenuated vaccine against EV71 and rotavirus. In order for easy drinking, our vaccine will design as an oral liquid with certain centration of our live attenuated vaccine.
Product Concept diagram
Bifidobacterium in Lactobacillus is not only an important probiotic to maintain the stability of human intestinal flora, but also has immunomodulatory functions such as presenting antigens. Related studies have found that its preventive effect on gastrointestinal pathogens is more significant, so we choose Bifidobacterium as a carrier for our live vaccines. Our engineered Bifidobacterium will go into the intestines and stomach inside the human body and then express the two antibody proteins, the VP1 protein of enterovirus 71 and the VP7 protein of rotaviruses in order to take immune effect.
Considering the duration of the vaccine in the intestinal tract, the oral vaccine is a better choice compared to injection-based injection. Because oral vaccines could continue express antibody proteins to improve the immune effect which allows self-administration relative to traditional injection vaccines. More importantly, oral vaccines improve safety to avoid the risk of blood-borne infection.
All in all, the Bis-Vacc vaccine provides infants with a great alternative to needle injection vaccines.
| Name | Target Virus | Age For Vaccine | Inoculation process | Price/Dose |
|---|---|---|---|---|
| Bis-Vacc Bivalent Vaccine | Rotavirus + EV71 | 6months-6years | 2 times, 1 month interval | 165 CNY |
In experiments, we constructed plasmids pET28a-VP1-VP2, pET28a-GLBP-VP1-VP7-GFP, and pET28a-GLBP-VP1-VP7-IL12-GFP plasmids. In addition to the antibody proteins (VP1 and VP7) expression systems, the proinflammatory cytokines interleukin-12 (IL-12) were used as a mucosal adjuvant to further enhance the immunome response. And, we used alacto-N-biose/lacto-N-biose I-binding protein (GL-BP) to assist the antigen and IL-12 in expressing on the surface of the membrane.
The bacteria were induced by different concentrations of IPTG to express the protein fused GFP which is able to monitor the protein expression. Finally, we could easily see the proteins were successfully expressed as expected.
Fluorescence microscopy of GLBP-VP7-VP1-GFP.
It’s obvious that the supernatant contains lots of protein tagged GFP after ultrasonic crushing bacteria expressing pET28a-GLBP-VP7-VP1-GFP, which indicates the VP7 and VP1 overexpressed in the bacteria and bond to the cell surface.
● Material and Product Safety
The agents we used are all iGEM compliant, non-toxic, and unharmful. The experiment we conducted was combining the VP7 vaccine and the VP1 vaccine into a bivalent vaccine. As an oral vaccine, it must be safe for infants to take orally and without any endotoxin. Furthermore, we used materials including enzymes, plasmid, and E. coli, which are all safe and low risk during experiments.
● Process Safety
If we performed the bacteria experiments, we must stay on the sterile ultra-clean table to avoid contamination, and the microbes were disposed of in the waste water bucket. Therefore, we always need to sterilize our hands, and the workspace before experimenting. Most importantly, we never use any animal or human genes in our experiments.
● Safety for future industrial production
In the future, we hope to put this vaccine into production after adequate clinical and pathological trials. We will ensure that our products meet the strict requirements of the state for food and medicine throughout the production process, ensure that the production environment meets hygienic standards, and ensure that our operators comply with safe operation specifications.
The experiment proves that it is feasible to express VR-vp7 in tandem with EV71-vp1. In the future, we will fuse the membrane-bound protein GLBP to display on the surface of Bifidobacterium, express the vaccine adjuvant IL-12, and finally, add the green fluorescent protein (GFP) tag to facilitate the detection of vp7-vp1 on Bifidobacterium. Eventually, Bifidobacterium was transferred to make a vaccine that can be taken orally. This method can not only improve the vaccination rate but also facilitate vaccination.
There is currently a lack of vaccines on the market that combine EV71 with RV, especially oral rather than injectable vaccines. So our goal is to become a public company and mass-produce these vaccines and provide personalized services.
Bis-Vacc's ultimate goal is to work with governments to achieve the goal of including our vaccines in health insurance. So that every child can use our vaccine at a low price and safely, and quickly meet the needs of users. Let every child grow up healthily.
[1]Kung, Y.-A., C.-T. Hung, Y.-C. Liu, et al., Update on the development of enterovirus 71 vaccines.[J] Expert Opinion on Biological Therapy, 2014. 14(10):1455-1464.
[2]Vela Ramirez, J.E., L.A. Sharpe, and N.A. Peppas, Current state and challenges in developing oral vaccines.[J] Advanced Drug Delivery Reviews, 2017. 114:116-131.
[3]2022 Analysis on the Current Market Situation and Development Prospect of China's Category II Vaccine Industry（2022年中国二类疫苗行业市场现状及发展前景分析）_ 行业研究报告(n.d.). Retrieved September 14, 2022, from https://www.qianzhan.com/analyst/detail/220/220622-68645c65.html
