The Development of a COVID19 Oral Vaccine Consisting of Bacillus Subtilis Spores

Study Overview
=================
Brief Summary
-----------------
This is a study trial to assess the effectiveness of the immune response stimulated by the genetically engineered Bacillus subtilis which express and display Spike protein of the SARS-COV2 on the spore coat.

Detailed Description
-----------------
Bacillus subtilis is regarded as safe organism by The Food and Drug Administration and it is presented in most food sources. Preliminary experiments have shown that the genetically engineered Bacillus subtilis can express and display receptor binding domain of spike protein of the SARS-COV2 on its spore coat, thus successfully induce the secretion of cytokines of human cells in vitro. Previous experiments also successfully demonstrated that a increased detection of neutralizing IgG and igM levels in mice after oral administrated with the Bacillus subtilis. This suggests that the transgenic spores of Bacillus subtilis have successfully activated the immune system, producing high-affinity neutralizing antibodies and memory B cells. Furthermore, no adverse effects were shown in all the mices. The engineered Bacillus subtilis will be further studied in a human trials through oral administration to test its safety and the immune effect resulted in human bodys.

Official Title
-----------------
The Development of a COVID19 Oral Vaccine Consisting of Bacillus Subtilis Spores Expressing and Displaying the Receptor Binding Domain of Spike Protein of SARS-COV2

Conditions
-----------------
COVID-19 Pneumonia

Intervention / Treatment
-----------------
* Biological: Bacillus subtilis


Participation Criteria
=================
Eligibility Criteria
-----------------
Inclusion Criteria: healthy age over 25 years the outcome of the following examinations should be clinically insignificant: medical and surgical history (hypo-, hypertension, allergy, other diseases, major surgery, micturition, defecation, sleep, illness within the last 4 weeks prior to the start of the trial); participant vaccinated with Sinovac over 4 months anti-SARS CoV 2 neutralizing antibody is negative in serum. Exclusion Criteria: pregnant women history of COVID-19 infection or showing COVID-19 infection symptoms having had contact to people with known COVID-19 infection in the last 14 days having fever (> 37.4oC in the last 24 hours), dry cough or feeling tired and having aches and pains, nasal congestion, runny nose, sore throat and diarrhea. positive real time RT-PCR COVID-19 test. persons with autoimmune diseases allergic diathesis or any clinically significant allergic disease (i.e. asthma) any condition that might impair the immune response recent or current immunosuppressive medication any other vaccine application 30 days before the first dose

Ages Eligible for Study
-----------------
Minimum Age: 25 Years

Sexes Eligible for Study
-----------------
All

Accepts Healthy Volunteers
-----------------
Accepts Healthy Volunteers

Study Plan
=================
How is the study designed?
-----------------

Design Details

Primary Purpose: Basic Science
Allocation: Non-Randomized
Intervention Model: Parallel Assignment
Interventional Model Description: two parallel experimental groups were designed. 1. the vaccinated volunteers and 2. unvaccinated groups.
Masking: None (Open Label)


Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| Experimental: generation of neutralizing antibody for unvaccinated participants<br>participants received vaccine 1 capsule of 1×10^10 CFU of B. subtilis spore at day 0, 14, and 28 respectively. | Biological: Bacillus subtilis<br>* Bacillus subtilis, a harmless intestinal commensal, has earned in recent years, great reputation as a vaccine production host and delivery vector with advantages such as low cost, safe for human consumption and straightforward administration. The technology team has succeeded engineering Bacillus subtilis with spore coat proteins resembling the proteins of the nucleus and spikes of coronal virus. This product could have a vaccine like activity within the intestinal environment.<br>|
| Experimental: neutralizing antibody booster for vaccinated participants<br>participants after 4-month vaccinated with Sinovac received 1 capsule of 1×10^11 CFU of B. subtilis spore | Biological: Bacillus subtilis<br>* Bacillus subtilis, a harmless intestinal commensal, has earned in recent years, great reputation as a vaccine production host and delivery vector with advantages such as low cost, safe for human consumption and straightforward administration. The technology team has succeeded engineering Bacillus subtilis with spore coat proteins resembling the proteins of the nucleus and spikes of coronal virus. This product could have a vaccine like activity within the intestinal environment.<br>|

What is the study measuring?
-----------------
Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Serum Neutralizing Receptor Binding Domain IgG Antibody Concentration | Concentration of neutralizing IgG antibody against receptor binding domain of spike protein in SARS-COV2 | Day 0, 27, 42 post oral administration | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Lentirival Pseudovirus Neutralization Assay (Wild Type of SARS-CoV2) | The ability of neutralization against SARS-CoV-2 was tested by an in vitro pseudo-virus neutralization assay. The lentivirus carrying a GFP gene was pseudotyped with the spike protein from a wild type of SARS-CoV-2. The pseudoviruses were then pre-incubated with serially diluted serum samples from orally vaccinated volunteers before being added to A549 lung carcinoma cells expressing human ACE2 and human TMPRSS2. The percentage of infection rate was measured with a fluorescent plate reader by counting GFP-positive cells. The results were fitted with a non-linear regression model. The dilution of the serum sample resulted in a 50% reduction of infection rate is designated as EC50. The results were presented as NA when the serum samples failed to neutralize pseudovirus infection. | Day 0, 27, 42 post oral administration | 
| Lentirival Pseudovirus Neutralization Assay (D614G SARS-COV2 Variant) | The ability of neutralization against SARS-CoV-2 was tested by an in vitro pseudo-virus neutralization assay. The lentivirus carrying a GFP gene was pseudotyped with the spike protein from a D614G variant of SARS-CoV-2. The pseudoviruses were then pre-incubated with serially diluted serum samples from orally vaccinated volunteers before being added to A549 lung carcinoma cells expressing human ACE2 and human TMPRSS2. The percentage of infection rate was measured with a fluorescent plate reader by counting GFP-positive cells. The results were fitted with a non-linear regression model. The dilution of the serum sample resulted in a 50% reduction of infection rate is designated as EC50. The results were presented as NA when the serum samples failed to neutralize pseudovirus infection. | Day 0, 27, 42 post oral administration | 

 Terms related to the study
=================
Keywords Provided by Centre Hospitalier Valida
-----------------
Covid-19, Vaccine Reaction, Bacillus subtilis