Coronavirus (19) Should we use a random live attenuated vaccine for COVID-19 prevention?

My last few blog posts discussed non-specific protection effects by live attenuated vaccines. You may be thinking: if live attenuated vaccines have such properties, can we just take any randomly-chosen live attenuated vaccine (e.g. BCG, OPV, or MMR) for COVID-19 prevention? Why should we bother to test these long-existing and widely-used vaccines before we use them as prophylaxis for COVID-19? Well, there are specific characteristics of the innate immunity system that enable the live attenuated vaccines to provide non-specific protection effects. Let us learn about this "trained innate immunity" today.^1,2

Specific protective effect is attained in adaptive immunity level
You may still remember what you learned from secondary school that vaccines are a harmless forms of virus or bacteria (either killed, greatly weakened, or broken down into small parts) that stimulate the production of neutralizing antibodies that can recognize and bind specifically to the surface structure of the pathogen. Once these antibodies are produced, they are maintained at a low level in the body for the rest of the person's life, so that, should the real pathogen invade, the antibodies can recognize it immediately and trigger the concomitant killing process in a very short time. Neutralizing antibodies are a major part of the adaptive immune system and confer the memory characteristic of this immune system. Therefore, the specific protective effect conferred by a vaccine, including the live attenuated vaccine, is achieved via the adaptive immune system.

Non-specific protective effect is initiated via trained innate immunity
Live attenuated vaccines, such as the BCG, OPV, MMR and measles vaccines, however, also exhibit a broader range of protection which can exist for half a year to 5 years. This non-specificity does not involve the specific priming, and so it is not the result of adaptive immunity. Recently, scientists started to realize that vaccines trigger an immune response, which involves a diversity of cells belonging to the innate immune system, to provide a broader range of protection.¹ The cells of the innate immunity may be primed upon encounter with the live attenuated vaccine, acquiring resistance to a second infection against the same pathogen, or unrelated pathogens with similar molecular patterns associated to the target pathogen. This is the basis of the "trained innate immunity". ^1,2,3

The mechanism of how the innate immunity is being trained by live attenuated vaccines and attains a memory characteristic to react against invading pathogens with similar molecular patterns is not yet fully understood. However, as we can see from the evidence mentioned in my previous blog posts, different live attenuated vaccines induced different trained immunity programmes. For example, vaccination with the BCG vaccine can protect animals against secondary infection with Candida albicans and Schistosoma manasoni,4,5 but the other live attenuated vaccines do not have such heterologous protection effects against these pathogens.

Therefore, when we consider making use of the non-specific protection effects of a live attenuated vaccine for preventing COVID-19, it is important to test if the vaccine also works for the disease.

The molecular mechanisms leading to the trained innate immunity
Although the mechanism of how the trained immunity is attained is not fully understood, scientists have found that it involves multiple regulatory layers at the molecular level. This include changes in chromatin organization, transcription of long non-coding RNAs (lncRNAs), DNA methylation, etc. As the change in the chromatin level that alters gene expression is a relatively long-lasting process, this explains how innate immunity cells display a memory function and are thus able to react spontaneously upon the second infection.³

Moreover, recent work has found that haematopoietic stem cells, the progenitor of innate immune cells, also display a memory function. This solves the question of how the innate immune cells such as myeloid cells, with short-lived and an average half-life of 5-7 days, can display a memory function that can be maintained for several months or even years.³

Based on the above findings, we can understand that the heterologous protection against infections induced by live attenuated vaccine is reversible and can last for up to 5 years but is not retained for a whole lifetime.⁶ However, the duration of the protection varies with the vaccine used and with the pathogen that stimulate the non-specific effects. Therefore, it is important also for a clinical trial to find out how long the live attenuated vaccine can provide the non-specific protection against COVID-19, if the vaccine proved to be efficient against the disease.

What are the benefits from knowing more about the nature of trained immunity?
As trained immunity is initiated via the changes in DNA methylation, examination of the DNA's methylation patterns can provide clues to determine whether people are able to undergo trained immunity to stimuli by vaccination of a live attenuated vaccine. The identification of the genes which are differentially methylated in people who respond to the vaccine versus in people who do not respond, could potentially be used as predictors of responsiveness to stimuli that induce trained immunity.⁷



* The references cited is based on the review paper "Defining trained immunity and its role in health and disease" written by M.G. Netea, et al.

References
1. M.G. Netea, L.A.B. Joosten LA, E. Latz, et al. Trained immunity: a program of innate immune memory in health and disease. Science, 2016, 352 (6284): aaf1098.
2. M.G. Netea, J. Quintin, and J.W.M. van der Meer. Trained immunity: a memory for innate host defense. Review. Cell Host Microbe., 2011, May 19;9(5): 355-361.
3. M.G. Netea, J. Dominguez-Andres, L.B. Barreiro, et al. Defining trained immunity and its role in health and disease. Nat. Rev. Immunol., 2020, Jun;20(6): 375-388.
4. J.W. van't Wout, R. Poell, and R. van Furth. The role of BCG/PPD-activated macrophages in resistance against systemic candidiasis in mice. Scand. J. Immunol., 1992, 36, 713-719.
5. J. Tribouley, J. Tribouley-Duret, and M. Appriou. Effect of Bacillus Callmette Guerin (BCG) on the receptivity of nude mice to Schistosoma mansoni. C. R. Seances Soc. Biol. Fil., 1978, 172, 902-904.
6. V. Nankabirwa, J.K. Tumwine, P.M. Mugaba, et al. Child survival and BCG vaccination: a community based prospective cohort study in Uganda. BMC Public Health, 2015, 15, 175.
7. J. Das, D. Verma, M. Gustafsson, et al. Identification of DNA methylation patterns predisposing for an efficient response to BCG vaccination in healthy BCG-naïve subjects. Epigenetics, 2019, 14, 589-601.

Coronavirus (18) Other live attenuated vaccines that may have protective effects against COVID-19

Although only limited evidence has been observed in support of COVID-19 prevention by non-specific effects of the BCG vaccine, this does not exclude the possibility of the vaccine's non-specific preventive effects against COVID-19. On the other hand, there is also a possibility that other live attenuated vaccines could provide non-specific effects against COVID-19 as well, based on the fact that children are subject to vaccine programmes and are less susceptible to COVID-19 infection. This blog post is going to introduce the other three candidate live attenuated vaccines suggested by scientists: oral poliovirus vaccine (OPV), measles vaccine, and MMR (measles, mumps, rubella) vaccine. I would like to share with you the hypothesis on why and how these three vaccines may also provide protection against COVID-19.

Oral poliovirus vaccine (OPV)
OPV is a live attenuated vaccine that helps to prevent the paralysis caused by the poliovirus infection to the central nervous system. In seven randomized controlled trials from 2002 to 2014 in Guinea-Bissau, a country which did not have previous polio infections, the all-cause mortality decreased 19% since a national immunization campaign with OPV was launched.¹ This indicates that OPV has beneficial non-specific effects. In fact, early clinical studies showed that OPV reduced the amount of other viruses that could be isolated from the immunized group of children, when compared with placebo group.^2,3 Most importantly, clinical studies performed from 1969 to 1971 showed that OPV was effective in preventing influenza and the concomitant acute respiratory disease, with a maximum of 3.1-fold morbidity rate reduction.⁴ This indicates that OPV can stimulate the innate immunity* to protect against respiratory infection, thus suggesting that the vaccine may be able to provide temporary protection against COVID-19, a respiratory infection disease.²

Compared to the BCG vaccine, OPV has some important advantages: the BCG vaccine is a weakened bacteria, while poliovirus and coronavirus are both positive-strand RNA viruses. It is likely that poliovirus and coronavirus may induce and be affected by common innate immunity mechanisms.² Therefore OPV is more likely to confer protective effects against COVID-19 than the BCG vaccine.

Moreover, since OPV has a stronger safety record, the risk of complications due to OPV is extremely low. Vaccine-associated paralytic polio develops in 1 per 3 million vaccine doses, mostly in immunocompromised children. On the other hand, up to 1% of BCG vaccine immunized children need medical attention due to some adverse reactions.²

Furthermore, OPV has more than one serotype, so the vaccines could be used sequentially to prolong protection.^2.4 The lower cost, ease of administration, availability, and the ease to scale up production are other advantages. More than a billion doses of OPV are produced and used in over 140 countries per year, while the supply of BCG vaccine is more limited. Therefore a relatively much smaller fraction of OPV should be enough for clinical trials to test its non-specific effects against COVID-19, without needing to fear that its supply to current polio eradication campaigns in developing countries will be limited.² By contrast, the possible use of the BCG vaccine for COVID-19 has already given rise to concerns over the availability of the vaccine as immunotherapy to patients with bladder cancer, due to its limited availability.

Although OPV seems promising in its ability to protect against COVID-19 and many researchers have suggested the testing of the vaccine,² no clinical trial has yet been launched to investigate this. Not even a registration of a relevant trial is seen in any public clinical trials registry platform.

Measles vaccine
According to the World Health Organization, measles is a highly contagious and deadly disease caused by a virus in the paramyxovirus family. It infects the respiratory tract, then spreads throughout the body. In 2018, it caused more than 140,000 deaths, mainly in children under 5, despite the availability of a safe and effective vaccine. Routine measles vaccination for children, combined with mass immunization campaigns in countries with high case rates and death rates, are key public health strategies to reduce global measles deaths.⁵

The measles vaccine has long been observed in association with pronounced non-specific protective effects against contagious diseases.^6,7 When the measles vaccine was introduced in 7 African countries, the overall mortality in children of those countries declined by 30% to 86%, a reduction far larger than anticipation based on the protection against deaths caused by measles alone.⁶ A random controlled trial found that the measles vaccine was associated with a 30% reduction in overall mortality in children. Among these, only 4% could be explained by prevention of measles infection.⁷

The vaccine is an efficient, live attenuated, replicating virus. It has been safely administered to 2 billion children over the last 40 years, affording life-long protection after a single dose.⁸ These advantages made the vaccine a popular vaccine vector candidate to be genetically engineered. In the last few years, scientists have genetically engineered the measles vaccine and tested the result's protection efficiency against two coronavirus-caused diseases: SARS (Severe Acute Respiratory Syndrome) and MERS (Middle-East Respiratory Syndrome).

In 2014, a recombinant measles vaccine which incorporates and expresses the spike protein (a surface protein mediates attachment of virus) of SARS was constructed.⁹ It was found that the live-attenuated recombinant measles vaccine could induce neutralizing antibodies, and that it protected immunized mice from infection by SARS-CoV.⁹ In 2018, two live-attenuated recombinant measles virus vaccines, either expressing S protein or N protein (structural protein on the surface of virus) of MERS-CoV, were found to induce a robust humoral and cellular immunity response against MERS-S mediating protection in the mouse model.¹⁰

Based on the above experimental results, a genetically-engineered measles vaccine carrying the S or the N protein pf SARS-CoV-2 may be an option to provide protective effects against COVID-19.

MMR (measles, mumps and rubella) vaccine
The MMR vaccine is a combined live attenuated vaccine that helps to prevent measles, mumps and rubella. Research studies of two scientists, Dr Paul Fidel and Prof. Mairi Noverr, in the USA, found that the combined vaccine could induce myeloid-derived suppressor cells (MDSCs). The induction of these cells from bone marrow reduced inflammation and mortality in mouse models.¹¹ Moreover, previous experimental studies showed that MDSCs inhibit septic inflammation.¹² Based on the above findings, the two scientists suggest that the combined vaccine could induce MDSCs in COVID-19 patients and help them to fight the lung inflammation and sepsis which associated with the most serious cases of the disease.¹¹

Adults who received the MMR vaccine as a child will likely still have antibodies against the measles, mumps and rubella viruses, but are unlikely to still have MDSCs, as these cells are not life-long cells. This means they would require a second time injection to reinitiate the MDSCs and obtain the potential benefits against COVID-19.

Besides the experimental evidence, the report from Dr Paul Fidel and Prof. Mairi Noverr cited a recent event that support their hypothesis. The 955 sailors on the U.S.S. Roosevelt who tested positive for COVID-19 had milder symptoms, except for one hospitalization. They suggested that this may have been a consequence of the MMR vaccinations given to all U.S. Navy recruits. However, this may also be the fact that they are young and stronger.¹¹

By genetic data analysis, a team lead by Prof. Robin Franklin at Cambridge University have identified a 29% amino acid sequence homology between the Macro (ADP-ribose-1-phosphatase) domains of SARS-CoV-2 and the rubella virus which is used the MMR vaccine. This provides further preliminary evidence that the MMR vaccine might provide protection against COVID-19.¹³

The MMR vaccine is highly safe. The vaccination of immunocompetent individuals has no contra-indications. A clinical trial of the MMR vaccine in high-risk healthcare workers in New Orleans has been proposed by Dr Paul Fidel and Prof. Mairi Noverr. They have also been awarded a grant to compare the MMR and BCG vaccines in a primate model of COVID-19.¹¹



Although no existing live attenuated vaccine, nor specific vaccine has yet found to be effective in preventing COVID-19, the existing vaccines that induce non-specific protection do have theoretical advantages over a vaccine specific to SARS-CoV-2. If proven to be effective against COVID-19, there is a higher chance the existing vaccine will still able to confer protection by its broad protection effects even when SARS-CoV-2 undergoes mutation. This is because the vaccine's non-specific effect works by boosting a person's immune response in general. By contrast, a vaccine specific to SARS-CoV-2 has a higher chance of losing its efficacy if the virus mutates, leading to antigenic drift.²



* Innate immunity: Live attenuated vaccines provide non-specific protection against lethal infections unrelated to the target pathogen by inducing "trained" non-specific innate immune cells. This improves a host's responses against subsequent infections. This type of non-specific immune response is the first line of defence against infection and is called the innate immune response. Recent reports show that COVID-19 may suppress innate immune responses.14 Therefore, stimulation by live attenuated vaccines could increase resistance to infection by SARS-CoV-2, the causal virus.



References
1. A. Andersen, A.B. Fisker, A. Rodrigues, et al. National immunization campaigns with oral polio vaccine reduce all-cause mortality: A natural experiment within seven randomized trials.Front. Public Health, 2018 Feb 2, 6: 13.
2. K. Chumakov, C.S. Benn, P. Aaby, et al. Can existing live vaccines prevent COVID-19. Science, 2020, vol. 368, Issue 6496, 1187-1188.
3. E. Seppälä, H. Viskari, S. Hoppu, et al. Viral interference induced by live attenuated virus vaccine (OPV) can prevent otitis media. Vaccine, 2011, 29, 8615-8618.
4. M.K. Voroshilova. Potential use of nonpathogenic enteroviruses for control of human disease. Prog. Med. Virol., 1989, 36, 191-202.
5. https://www.who.int/news-room/fact-sheets/detail/measles
6. P. Aaby, B. Samb, F. Simondon, et al. Non-specific beneficial effect of measles immunisation: analysis of mortality studies from developing countries. BMJ, 1995 Aug 19, 311(7003): 481-485.
7. P. Aaby and C.S. Benn. Developing the concept of beneficial non-specific effect of live vaccines with epidemiological studies. Clin. Microbiol. Infect., 2019 Dec., 25(12):1459-1467.
8. P.N. Frantz, S. Teeravechyan, and F. Tangy. Measles-derived vaccines to prevent emerging viral diseases. Microbes Infect., 2018, 20:493-500.
9. N. Escriou, B. Callendret, V. Lorin, et al. Protection from SARS coronavirus conferred by live measles vaccine expressing the spike glycoprotein. Virology. 2014, 452-453:32-41.
10. B.S. Bodmer, A.H. Fiedler, J.R.H. Hanauer, et al. Live-attenuated bivalent measles virus derived vaccines targeting Middle East respiratory syndrome coronavirus induce robust and multifunctional T cell responses against both viruses in an appropriate mouse model. Virology, 2018, 521:99-107.
11. P.L. Fidel and M.C. Noverr. Could an unrelated live attenuated vaccine serve as a preventive measure to dampen septic inflammation associated with COVID-19 infection? mBio, DOI:10.1128/mBio.00907-20
12. S.K. Esher, P.L. Fidel, M.C. Noverr. Candida/staphylococcal polymicrobial intra-abdominal infection: pathogenesis and perspectives for a novel form of trained innate immunity. J. Fungi (Basel), 2019 Jun; 5(2): 37.
13. R. Franklin, A. Young, B. Neumann, et al. Homologous protein domains in SARS-CoV-2 and measles, mumps and rubella viruses: preliminary evidence that MMR vaccine might provide protection against COVID-19. MedRxiv, doi: https://doi.org/10.1101/2020.04.10.20053207
14. M. Zheng, Y. Gao, G. Wang, et al. Functional exhaustion of antiviral lymphocytes in COVID-19 patients. Cell. Mol. Immunol., 2020 May, 17 (5), 533-535.

Coronavirus (17) Limitations of the evidence of COVID-19 prevention from non-specific effects of BCG vaccine

My last blog post introduced the BCG vaccine and its non-specific protective effects. However, we should not be too optimistic about it until a clinical result from a prospective random clinical trial comes out to prove the non-specific protection of BCG against COVID-19. This is because the evidence suggesting BCG vaccine's preventive effect against COVID-19 was drawn mainly from retrospective studies and speculative reports. The claims that universal BCG vaccination in a country results in lower morbidity and mortality rate of COVID-19 in that country, were based on observation and did not account for several factors that could also explain the discrepancies between different places.^1-5 These factors include:

1. Public awareness:
The places with universal BCG vaccination policy such as South Korea, Taiwan and China have also been stricken by different epidemics in recent decades. The SARS outbreak in 2003 affected China, Taiwan and South Korea, while the MERS outbreak in 2015 seriously affected South Korea. Therefore the people and the governments of these countries have more experience and could act quickly in tackling the new infectious disease. This helps in slowing down the spread and lowering the severity of COVID-19.

2. Cultural differences which affect the effectiveness of the non-pharmaceutical interventions being adopted:
Most of the developed countries that do not have universal BCG vaccination, such as USA, UK and Italy, implemented quarantine, social distancing and isolation measures to contain and mitigate COVID-19. However, there is a higher proportion of civilians in these countries that did not obey and even protested against the measures. These reactions will have made the measures less effective.
On the other hand, the higher obedience among Asian people, such as Japanese, Korean and Chinese people, make it easier for the government to enforce the measures to contain the disease. Moreover, most of the people in these areas are conscientious and more willing to put on a face mask whenever they are outside their homes. According to WHO, the use of face masks is one of the measures that can limit the spread of COVID-19.⁶

3. Testing rate:
Within the first month or so from the start of the pandemic, Korea was already able to expand the detection of SARS-CoV-2, with a sensitivity rate of over 95%, to more than 10,000 people a day. This can quickly identify clusters of infections and quarantine the infected individuals.

4. Genetics of the population:
Super-spreaders can ignite a large-scale transmission. The low number of cases and deaths per population in some countries might be due to fewer super-spreaders, which may reflect genetic differences.⁷ Further study is needed to clarify this assumption.

5. Different countries had different onset times, consequently having different positions on the epidemic curve:
The observational studies correlating the BCG coverage with the low morbidity and mortality rate of COVID-19 were made based on the first three months of the outbreak of the disease (January to March). During that period, India and Ethiopia, which have long-standing BCG vaccination policies, were not yet affected by COVID-19. However, these two countries have been hit seriously by the pandemic since May. Therefore it was too early to correlate the BCG coverage with the low severity of COVID-19. The different onset in a pandemic between countries is largely due to the difference in frequency of travelling from the disease's outbreak epicentre, China. More people from China travel to Italy, the UK and the USA than to India and African countries, which had a later onset of the epidemic.

6. The differences in diagnosing and reporting COVID-19 cases:
Developing countries, which usually have BCG vaccination policies against tuberculosis, such as India, Indonesia and the Phillipines, have relatively less advanced medical systems. They have lower testing ability and less efficient reporting systems. This can result in lower morbidity and mortality rates on paper, and does not reflect the real figure.

7. The beneficial off-target effects of the BCG vaccine might be altered by subsequent administration of a different vaccine:⁸
Children are found to be less susceptible to the COVID-19 infection than adults, and their symptoms are generally milder.^9-12 This may be due to the required vaccine programmes for every child having non-specific effects against COVID-19, but not necessarily the BCG vaccine. On the other hand, the beneficial off-target effects of the BCG vaccine might be altered by subsequent administration of different live attenuated vaccines.^8,13

Moreover, it is unlikely that a BCG vaccine given decades ago in childhood could help in preventing COVID-19 nowadays. Whether older people could maintain a pool of trained monocytes many years after BCG vaccination is still questionable. A possible explanation for the observation is that children who have been vaccinated with BCG are less susceptible to infection with SARS-CoV-2 and therefore less likely to spread it to older populations.²

Conclusion
There is evidence that the BCG vaccine has non-specific effects against respiratory infections. However, there is currently no direct evidence that BCG vaccine protects against COVID-19. Hopefully, with the ongoing random clinical trials mentioned in my previous blog post, we can find out the answer in the near future.

If the BCG vaccine could really help in preventing COVID-19, it is important to know 1) which BCG strain is the most effective, as there are about 8 strains of BCG vaccine in the world,^14,15 2) if the BCG vaccine will exacerbate COVID-19 in a minority of patients with severe disease,³ 3) how long could the heterologous protective effect conferred by BCG last after vaccination,¹⁶ and 4) the optimal time in the life to vaccinate.¹⁷

*Examples of countries which do not have a universal BCG vaccination policy: Italy, the USA, Spain, Germany, and the UK. Italy has never had a national BCG programme; BCG is not recommended for generalized use in the US; and others phased theirs out as TB became less of a concern - Spain in 1981, Germany in 1998 and the UK stopped in 2005. ¹⁸ Examples of countries with long-standing universal BCG vaccination policies: South Korea, Japan, India, Ethiopia.¹⁸

References
1. Does BCG vaccination protect against acute respiratory infections and COVID-19? A rapid review of current evidence. CEBM, April 24,2020. https://www.cebm.net/covid-19/does-bcg-vaccination-protect-against-acute-respiratory-infections-and-covid-19-a-rapid-review-of-current-evidence/
2. L.A.J. O'Neill, and M.G. Netea. BCG induced trained immunity: can it offer protection against COVID-19? Nature Reviews Immunology,2020, 20, 335-337.
3. N. Curtis, A. Sparrow, T.A. Ghebreyesus, et al. Considering BCG vaccination to reduce the impact of COVID-19. Lancet, 2020 May 16;395(10236):1545-1546.
4. L. Faust, S. Huddart, E. MacLean, et al. A. Universal BCG vaccination and protection against COVID-19: critique of an ecological study. April 1, 2020. https://naturemicrobiologycommunity.nature.com/users/36050-emily-maclean/posts/64892-universal-bcg-vaccination-and-protection-against-covid-19-critique-of-an-ecological-study
5. C. Ozdemir, U.C. Kucuksezer, and Z.U. Tamay. Is BCG vaccination affecting the spread and severity of COVID-19? Allergy. 2020 Jul;75(7):1824-1827.
6. Advice on the use of masks in the context of COVID-19. Interim guidance. 6 April 2020. World Health Organization. https://apps.who.int/iris/bitstream/handle/10665/331693/WHO-2019-nCov-IPC_Masks-2020.3-eng.pdf?sequence=1&isAllowed=y
7. Akiko Iwasaki, & Nathan D Grubaugh. Why does Japan have so few cases of COVID-19? EMBO Molecular Medicine, 2020, 12: e12481. https://doi.org/10.15252/emmm.202012481
8. A.J. Pollard, A. Finn, and N, Curtis N. Non-specific effects of vaccines: plausible and potentially important, but implications uncertain. Arch Dis Child 2017; 102: 1077-1081.
9. J. Zhang, M. Litvinova, Y. Liang, et al. Changes in contact patterns shape the dynamics of the COVID-19 outbreak in China. Science, 2020 Jun 26;368(6498): 1481-1486.
10. D.F. Gudbjartsson, A. Helgason, H. Jonsson, et al. Spread of SARS-CoV-2 in the Iceland population. N. Eng. J. Med, 2020 Jun 11;382(24): 2302-2315.
11. COVID-19 National Emergency Response Center, Epidemiology and Case Management Team, Korea Centers for Disease Control and Prevention. Coronavirus disease-19: The first 7,755 cases in the Republic of Korea. Osong Public Health and Research Perspectives, 2020 Apr;11(2):85-90.
12. CDC COVID-19 Response Team. Coronavirus disease 2019 in children-United States, February 12-April 2, 2020. Morbidity and Mortality Weekly Report. 2020 Apr;69(14):422-426.
13. N. Curtis, A. Sparrow, T.A. Ghebreyesus, et al. Considering BCG vaccination to reduce the impact of COVID-19. Lancet, 2020 May 16;395(10236): 1545-1546.
14. N. Ritz, W.A. Hanekom, R. Robins-Browne R, et al. Influence of BCG vaccine strain on the immune response and protection against tuberculosis. FEMS Microbiol Rev 2008; 32: 821-841.
15. M. Miyasaka. Is BCG vaccination causally related to reduced COVID-19 mortality? EMBO Molecular Medicine, 2020, Jun 8;12(6):e12661. doi: 10.15252/emmm.202012661.
16. J. Kleinnijenhuis, J. Quintin, F. Preijers. et al. Long-lasting effects of BCG vaccination on both heterologous Th1/T H 17 responses and innate trained immunity. J. Innate Immun., 2014, 6, 152-158.
17. M.G. Hollm-Delgado, E.A. Stuart, & R.E. Black. Acute lower respiratory infection among Bacille Calmette-Guerin (BCG)-vaccinated children. Pediatrics, 2014, 133, e73-e81.
18. A. Zwerling, M.A. Behr, A. Verma, et al. The BCG World Atlas: a database of global BCG vaccination policies and practices. PLoS Med 2011;8(3):e1001012.