Rotavirus
Rotavirus is a double-stranded RNA virus, the genome of which is composed of 11 segments — each coding for a single protein. The virus spreads through fecal-oral transmissions such as from the waste of an infected person to another individual and is the leading cause of diarrhea in children younger than five. Common symptoms of Rotavirus including watery diarrhea, fever, abdominal pain, and vomiting. In severe cases, these symptoms can develop into threatening complications. Rotavirus is also a triple-layered particle that typically comprises 3 layers: outer capsid, inner capsid, and core. There are 9 species of the genus (A, B, C, D, F, G, H, I, and J) and Rotavirus A is known to be the most common type causing 90% of human infections. The outer layer consists of two vital proteins — VP7 (G protein for glycoprotein) and VP4 (P protein for protease-cleaved protein). These two proteins play important roles in vaccine development as they are the primary antigens to which neutralizing antibodies are directed.
Insipration
According to the National Institute of Health, rotavirus infections result in greater than 200,000 deaths annually, mostly in low-income countries. Despite the fact that children are threatened by rotavirus disease globally, the parents of those children being infected by rotavirus do not clearly realize the existence and detriments of rotavirus—they usually ignore it or simply regard it as watery diarrhea. This phenomenon aroused our attention and intrigued our desire to study it. Therefore, our purpose is to improve the vaccine meantime increase public awareness of rotavirus.
Figure above shows the overall prevalence of rotavirus diarrhea hospitalization after being vaccinated. The number of hospitalizations decreased after the introduction of vaccinations. This figure informs our team that the rotavirus vaccine is of great importance in decreasing the epidemicity. While ensuring the safety and efficacy of the existing rotavirus vaccine, we plan to improve the vaccine by making it more suitable for infants, such as adding flavor by mixing it with daily drinks. Also, we are going to give speeches about our project and make the public realize the jeopardize of rotavirus disease and the advantages of our product.
Goal and Design
Considering our primary audience are children younger than five, we aim to develop an oral vaccine that can be vaccinated in a mixture form with daily drinks. In this way, the fierce resistance that might adopt through drug injection might be avoided. Among the three types of vaccines, live-attenuated vaccines and inactivated vaccines use the complete form of the virus as the antigen, which could potentially be harmful to a vulnerable immune system. Since children have an immature immune system, we do not consider those two kinds of vaccines.
However, nucleic acid vaccines can also lead to an overwhelming immune response, which can be exampled by the case of people contracting severe fever after COVID vaccination. Thus, subunit vaccine with adjuvant is what we should consider, as it guarantees safety through the usage of only a fraction of a virus as an antigen for antibodies development. The adjuvant is an extra protein integrated alongside the antigen that functions to enhance the immune system and allows for fewer doses or lesser quantities of the vaccine required to obtain the desired effect.
This said, we are going to produce a vaccine that includes VP7, a surface protein of rotavirus, and adjuvant LTB, a subunit of heat-labile enterotoxin B. VP7, being the most abundant surface protein of rotavirus, is the primary antigen detected by antibodies when rotavirus enters the body. This makes it as the optimal protein candidate for the vaccine. Heat labile Enterotoxin B is a harmful bacterium that can cause excessive secretion of water and electrolytes from mucosal cells and lead to diarrhea, triggering a strong immune response during the process. LTB is one of its subunits that does little harm to the body, but at the same time, it can trigger the same level of response when detected by antibodies. Based on this mechanism, we want to integrate LTB with VP7 to develop a safe and effective rotavirus vaccine.
In order to evaluate the possibility of successful protein expression, we selected E. coli as the test bacteria. The reason we chose E.coli was that this strain is known to be the most common bacteria for rapid expression, inexpensive cultivation, and high yield. Nevertheless, E. coli can be harmful to the human body if it is used excessively (endotoxin accumulation). Meanwhile, its proteins can only be released when the bacteria is fully destroyed. Aside from this, we also used Bacillus subtillus for protein expression. Bacillus subtillus has the ability to secrete numerous enzymes (subtilisin and gramicidin) to degrade a variety of substrates, enabling it to survive under continuously changing environments. Furthermore, Bacillus consumes free oxygen in the intestines which promotes the growth of beneficial anaerobic bacteria and enhances the development of immune organs. At the same time, it has excellent protein secretion abilities and prevents potential inflammations by strengthening the gut barrier.
Reference
Department of Health, Au. https://www.health.gov.au/health-topics/rotavirus
WebMD. https://www.webmd.com/children/guide/what-is-rotavirus
Different Types of Vaccines. https://www.historyofvaccines.org/content/articles/different-types-vaccines
Liya Hu,Sue E Crawford,Joseph M Hyser,Mary K Estes,BV Venkataram Prasad. Rotavirus non-structural proteins: structure and function[J]. Current Opinion in Virology,2012,2(4).
Isanaka Sheila,Djibo Ali,Grais Rebecca F. Heat-Stable Oral Rotavirus Vaccine.[J]. The New England journal of medicine,2017,377(3).
Bernstein David I. Rotavirus Vaccines-Going Strong After 15 Years.[J]. JAMA pediatrics,2021.
Carl D. Kirkwood,Lyou-Fu Ma,Megan E. Carey,A. Duncan Steele. The rotavirus vaccine development pipeline[J]. Vaccine,2019,37(50).
C.A. Perez,C. Eichwald,O. Burrone,D. Mendoza. Rotavirus vp7 antigen produced by Lactococcus lactis induces neutralizing antibodies in mice[J]. Journal of Applied Microbiology,2005,99(5).
Alexander Falkenhagen,Corinna Patzina-Mehling,Ashish K. Gadicherla,Amy Strydom,Hester G. O’Neill,Reimar Johne. Generation of Simian Rotavirus Reassortants with VP4- and VP7-Encoding Genome Segments from Human Strains Circulating in Africa Using Reverse Genetics[J]. Viruses,2020,12(2).
Offit Paul A. Challenges to Developing a Rotavirus Vaccine.[J]. Viral immunology,2018,31(2).
刘洋. 轮状病毒和诺如病毒重组抗原的表达、纯化及免疫原性鉴定[D].昆明医科大学,2019.