BCG Vaccination for COVID-19 Animal Model Study Proposal

Background

Last month an article was published noting that  a lower percent of the population in Portugal was dying, as compared with the percentage dying in Spain. The author suggested that this might be due to the fact that people in Portugal still receive Bacille-Calmette-Guerin (BCG)  vaccinations for tuberculosis prevention, while the people in Spain do not. Others noticed that there seems to be a tremendous amount of COVID-19-related death in western countries, whereas in less developed countries the percentage of people who are severely affected is much lower. The use of BCG in the less developed countries was again suggested as a possible reason for the difference. There is a large body of literature describing the protective effects of BCG vaccine on reducing infant mortality from all respiratory causes, including respiratory viruses. This generalized type of immunity might be sufficient to prevent the deaths of people at high risk of exposure, such as health care workers. BCG immunization side effects are well known and the vaccine is already approved by the FDA. Over 4000 healthcare workers in Australia received the BCG vaccine in April in a human clinical trial designed to test the benefits of use of this vaccine to keep our front-line workers healthy. Results of that trial are expected in October of this year. Similar trials have begun in the Netherlands and at Texas A&M. 

A recent publication from Israel noted that Israelis who had received BCG 39-41 years ago did not have a lower incidence of COVID19 infection than Israelis 36-38 years old who had not received the vaccine. This study is confounded by the fact that the incidence of having symptomatic COVID19 at age 45 is about double that of having symptomatic disease at age 35. Also, it is not clear if the vaccinated group received any booster vaccinations, as would be the normal protocol for recipients of the BCG vaccine. 

Study proposal

No mammalian trial demonstrating the efficiency of the Bacille-Calmette–Guerin (BCG) vaccine in preventing Covid-19 infection has been published to date. If such a trial was successful, BCG could be administered to humans on a large-scale basis with increased confidence that it would confer immunity. BCG is already approved by the FDA, and its side-effect profile is well established. If BCG is proven to be protective against Covid-19 in an animal model, BCG could be immediately offered in the US, to healthcare workers and/or in the general population. Such a trial could be completed with results by July 2020. BCG trials have begun in humans in 3 clinical trials, but the definitive outcome data in humans is not expected until October 2020. Definitive data on other vaccines is not expected until June 2021. Having vaccinations in place before next fall could be crucial.  This time advantage could save lives. This offering proposes a study to demonstrate the effectiveness of BCG in conferring immunity against COVID-19 in a mammalian model. 

Methods

Golden Syrian hamsters were chosen as the COVID19 animal model based on the work of Chan and Zhang. The protocol for vaccination of the hamsters with BCG was adapted from the protocol used by Oliveira and Lizzi. The protocol for infection of the hamsters was borrowed from the protocol used by Chan and Zhang.1 Determination of efficacy in conferring immunity was borrowed from the work of Oliveira and Lizzi.2 The protocols are summarized below.

Virus and biosafety

SARS-CoV-2 was isolated from the nasopharyngeal aspirate specimens of a laboratory-confirmed COVID-19 patient in (city). The viral isolate was amplified by one additional passage in VeroE6 cells to make working stocks of the virus (1.8 x 107 50% tissue culture infective doses [TCID50]/ml) as described previously. All experiments involving live SARS-CoV-2 followed the approved standard operating procedures of a Biosafety Level-3 facility.,

Animal Model

Female and male golden Syrian hamsters aged between 4 and 6 weeks were randomly allocated into groups of ten animals per group, equally distributed by gender,  Animals were subcutaneously immunized with 106 CFU of rBCG strains with a 21-day interval (first dose at day 0 and second dose at day 21). The animals were transferred to a Biosafety Level-3 facility. Challenge was performed with Dulbecco’s Modified Eagle Medium(DMEM) containing 105 plaque-forming units in 100l of SARS-CoV-2 intranasally under intraperitoneal ketamine(200mg/kg) and xylazine(10mg/kg) anesthesia fifty-one days after the first immunization (day 51). Mock-infected animals were challenged with 100l of Phosphate-buffered saline(PBS). Animals were monitored twice daily for clinical signs of disease. Their body weight and survival were monitored for 14 days post-inoculation(dpi). Five animals in each group were sacrificed at 2dpi, 4dpi and 7dpi for virological and histopathological analyses. Remaining animals were sacrificed at 14dpi. 

Histopathology, immunohistochemical, immunofluorescence, and TUNEL staining

These staining and microscopy procedures on tissue sections were performed as described previously.

Viral load and median tissue culture infectious dose (TCID50) assays

Viral load studies by qRT-PCR assay were conducted on the blood and organ tissues. Quantitation of live infectious virus by TCID50 assay was performed on the nasal turbinate and lung tissues.

Transmission study among close contact animals

Eight BCG-vaccinated and eight BCG-naive hamsters were intranasally challenged with SARS-CoV-2. Twenty-four hours later, each SARS-CoV-2-challenged hamster was transferred to a new cage with each cage containing one naïve hamster as close contact. Five virus-challenged and five contact hamsters in each group were sacrificed for viral load and histopathological studies at 4 days post infection and 4 days post exposure, respectively. The remaining hamsters were kept for clinical observation until 14 days post infection. Surface spike gene of viral isolates from the hamsters was Sanger sequenced as described previously. 

Statistical Analysis

All data were analyzed using statistical software. Weight losses were compared using two-way ANOVA and Student’s T-test was used to determine significant differences in virus titres among the different groups. P<0.05 was considered statistically significant.

Results

This study has not yet been performed. It requires a Biosafety Level 3 lab to which I do not have access. If someone you know has access to a lab like that, perhaps you can forward the study to them. 

This week I learned about a healthy California nurse who performed  CPR on a COVID-19 positive patient and died from the virus 14 days later. I found that quite sobering. 

If BCG vaccines work in hamsters, I know a lot of people who will be signing up to get a BCG immunization. 

There has been interest in the BCG as a vaccine for Covid-19. Clinical trials have begun in Australia. The initial results of the Australia trial are not expected until October 2020. The results of the first Human vaccine NIH trial will not be available till June 2021.  A clinical trial on a manufactured vaccine against Covid-19 has begun at Emory . The trial described in this paper could be completed by July.

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