Coronavirus (21) COVID-19 vaccine candidates in clinical trials: inactivated vaccines (a)

Inactivated vaccines are also known as whole killed virus vaccines. They are prepared by protein denaturation, which is performed by chemicals such as formalin, formaldehyde, or β-propiolactone, or by heat and radiation. This destroys the pathogen's ability to infect and replicate, but still retains its ability to act as an immunogen because the inactivation process keeps the pathogen intact. The inactivated pathogen triggers the production of antibodies against the pathogen when the vaccine is injected into humans.¹

Inactivated vaccines are among the first vaccines invented to act against viruses. The best known traditional inactivated vaccines include the rabies vaccine which was first introduced in 1885,² the polio vaccine which was first widely used in 1955,³ and the hepatitis A vaccine which was first approved in 1991.⁴ The flu vaccines provided by the NHS to adults aged above 18 are also inactivated vaccines.⁵ The fact that the technique in developing inactivated vaccines has long been established makes them one of the attractive types of vaccines prepared in the market today.

There are now 5 inactivated-vaccine candidates for COVID-19 in clinical trials (as of 20th August),⁶ four developed by biotech companies from China and one from India. Let us have a look at the progress of the testing of these vaccine candidates.

Inactivated vaccines in clinical trials
1. CoronaVac by Sinovac Biotech Co. Ltd.*
CoronaVac was developed by inactivating SARS-CoV-2 using formalin. The vaccine uses aluminium sulfate as an adjuvant to help boost the immune response (alum-adjuvanted); results from clinical trials indicated that two injections, 14 days apart, are needed for best results.

Currently the vaccine candidate is in its phase 3 trials to test the efficacy and safety in Brazil (trials started from 20th July) and Indonesia (trials started from early August). The company is partnered with the Butantan Institute, a public research institute recognized in Brazil for work in vaccines, to recruit 9000 healthcare professionals working in COVID-19 specialized facilities.⁷ In around July, Brazil became the second country with the most COVID-19 confirmed cases, thus providing sufficient volunteers to speed up the vaccine's development. Analysts remarked that under the current emergency circumstances, phase 3 trials in Brazil could last less than a semester. CoronaVac might be ready for regulatory approval early next year if all goes well.⁷ Sinovac also partnered with PT Bio Farma, a state-run pharmaceutical company in Indonesia, to recruit up to 1620 volunteers, age between 15 and 59, to run clinical tests of CoronaVac. Indonesia is a country with the world's fourth biggest population and has a high number of COVID-19 infections. The entire process is expected to complete around the coming new year.⁸

Previous phase 2 clinical trials for CoronaVac demonstrated that the vaccine is well tolerated, with relatively lower incidence rate of fever compared with other COVID-19 vaccine contenders, and without any notable dose-related safety concerns from 743 healthy volunteers aged 18 to 59. The result was published and is currently available as a medRxiv preprint.⁹

Sinovac is a Beijing-based pharmaceutical company established in 2001 by its Chief Executive Officer Mr. Weidong Yin and the team at Tangshan Yian Biological Engineering Co. Ltd. According to the Sinovac's website, the company has developed and commercialized 6 human-used vaccines over the past two decades. All are inactivated vaccines. Healive, a vaccine against hepatitis A, is their first vaccine product which was first launched in 2002 and took 20 years to research and develop. With the advancing of their technique, they only used about 6 months to complete clinical trials and obtained a production license from China SFDA for PANFLU.1®, a vaccine against H1N1, when a H1N1 influenza broke out in Mexico in March 2009. This is a good example to show their core R&D competence. Their latest vaccine product, Inlive, is a vaccine against enterovirus 71, which causes severe hand, foot, and mouth disease among children.

The company has developed a SARS vaccine which went into phase 1 trials. Although the project was discontinued due to the sudden disappearance of the disease, the experience from 17 years ago benefits Sinovac in developing the COVID-19 vaccine, given that SARS-CoV-2 is very similar to the SARS virus. Moreover, the company obtained one billion renmibi (approximately USD140 million) from the Chinese government and contributions from international NGOs.¹⁰

However, if CoronaVac is approved to be used, the accessibility of the vaccine is likely to be a big issue. Almost a decade would be needed to vaccinate every person in China, as at least two doses will be required to immunize each person whereas the company can produce only 300 million doses per year.¹⁰

Moreover, it is noteworthy that the company may not be able to run stably. There has been a dispute between the company and its investors since 2018, and its stock in Nasdaq has been frozen since February 2019 and has not since reopened.¹¹



*Information obtained mostly from Sinovac's own website unless otherwise stated. Sinovac's webpage is www.sinovac.com



References
1. Vaccination strategies to combat novel corona virus SARS-CoV-2. Life Sciences 256 (2020), 117956
2. G.L. Geison. Pasteur's work on rabies: Reexamining the ethical issues. The Hastings Center Report. Vol.8, No.2, (April, 1978), p.26-33.
3. Monto AS. Francis field trial of inactivated poliomyelitis vaccine: background and lessons for today. Epidemiol Rev.,1999;21:7-23.
4. P. Vandana, D. Prajakta, and J. Ratnesh. Nanoparticulate drug delivery perspectives on the transition from laboratory to market. Oxford: Woodhead Pub. p212. ISBN 9781908818195.
5. Flu vaccine overview. NHS. https://www.nhs.uk/conditions/vaccinations/flu-influenza-vaccine/
6. Draft landscape of COVID-19 candidate vaccines. World Health Orgainzation. 20 August 2020. https://www.who.int/publications/m/item/draft-landscape-of-covid-19-candidate-vaccines
7. China's Sinovac phase III trials in Brazil could take as little as three months. Sergio Held and Elise Mak. BioWorld, 10 June, 2020. https://www.bioworld.com/articles/436374-chinas-sinovac-phase-iii-trials-in-brazil-could-last-as-little-as-three-months
8. Indonesia joins Sinovac to initiate phase 3 testing of CoronaVac while securing option to purchase 100m doses. Trial Site News, 23 July, 2020. https://www.trialsitenews.com/indonesia-joins-sinovac-to-initiate-phase-3-testing-of-coronavac-while-securing-option-to-purchase-100m-doses/
9. Y.J. Zhang, G. Zeng, H.X. Pan, et al. mmunogenicity and Safety of a SARS-CoV-2 Inactivated Vaccine in Healthy Adults Aged 18-59 years: Report of the randomized, double-blind, and placebo-controlled phase 2 clinical trial. MedRxiv. doi: https://doi.org/10.1101/2020.07.31.20161216
10. "We will share our vaccine with the world." Inside the Chinese biotech firm leading the fight against COVID-19. Charlie Campbell. Time, 27 July, 2020. https://time.com/5872081/sinovac-covid19-coronavirus-vaccine-coronavac/
11. A vaccine with a poison pill. Matt Levine. Bloomberg, 22 May, 2020. https://www.bloomberg.com/opinion/articles/2020-05-22/a-vaccine-with-a-poison-pill

Coronavirus (20) The race to develop COVID-19 vaccines

Five months have passed since the characterization of COVID-19 as a pandemic by the WHO on 11th March. You may wonder how much progress is being made in developing vaccines specifically for COVID-19. Let us have a look at a report by the WHO that provides data on all the COVID-19 candidate vaccines that are being developed in the world.¹

According to the latest data from WHO (20th August), there are 169 candidate vaccines being developed for COVID-19 prevention, of which 139 are in *preclinical evaluation and 30 are in *clinical evaluation. Eleven countries are at the forefront of the race to develop vaccines: among the vaccines in clinical stages, 6 are from China, 4 from the US, 2 from the UK, 2 from India, and one each from Germany, Japan, South Korea, Belgium, Cuba, Singapore, Canada and Russia. The other 8 candidate vaccines which are in the clinical evaluation stages are jointly developed by research institutes and/or pharmaceutical companies from different countries; adding these 8 candidate vaccines causes Italy, France, Australia and Taiwan to be among the contestants in the race to develop a COVID-19 vaccine.

There are some encouraging points to note. Among the 30 candidate vaccines in clinical evaluation, six are already in phase 3, the last phase of clinical trials. China, where COVID-19 first originated, has 3 candidate vaccines already entered into phase 3, plus one vaccine (BNT162b1) which is jointly developed with Pfizer (a worldwide pharmaceutical company with headquarters in the US) is also in phase 3 of clinical trials. The vaccine (AZD1222) co-developed by the University of Oxford and AstraZeneca in the UK, and the mRNA vaccine (mRNA-1273) developed by Moderna in the US, are also in phase 3 of clinical trials.

Although the speed of global vaccine development is already high, the chief medical officer of England, Professor Whitty, said in an interview on 23rd August that he would be "quite surprised if we had a highly effective vaccine ready for mass use in a large percentage of the population before the end of winter, certainly [not] this side of Christmas".² We should not anticipate that the vaccine for COVID-19 will be available this year. This reflects the fact that phase 3 of clinical trials usually takes a much longer time, than the other initial phases, and usually lasts for several years as more volunteers are needed and the safety requirements are more stringent.

An interesting point to note is that a tobacco company, Kentucky Bioprocessing, Inc., in the US is one of the leading contestants in developing a COVID-19 vaccine. The vaccine candidate they developed has already finished phase 1 and is now in phase 1/2 of clinical trials. In fact, when we look at their website, we can understand that this is not a pure tobacco selling company. It plants tobacco to express, extract and purify proteins for use in vaccines and other pharmaceuticals.³

The traditional vaccines developed in the early 20th century are virus-based and are either live-attenuated or inactivated. However, in the race for COVID-19 vaccine development, we can see a much greater variety of vaccine platforms being used. Other than the traditional platform, different viral vectors, nucleic acid-based vaccines, and antigen-presenting cells are also among the vaccine candidates in the clinical stages. I am going to introduce you the 30 candidate vaccines for COVID-19 which are in clinical evaluation, based on their vaccine platforms, in the next blog posts.



*Preclinical evaluation is the analytical results from testing of the toxicity and efficacy of a medication, vaccine in this case, using human cell cultures or animal models in the laboratory.

Clinical evaluation is the assessment and analysis of clinical data collected from clinical trials. Clinical trials are performed, if the result from preclinical research is promising, to see how well the vaccine works in humans. Clinical trials happen in 3 phases, each phase building on the results of previous phases and recruiting more volunteer participants. Clinical studies to demonstrate the safety and efficacy of vaccines must be conducted in humans before a vaccine can be released for widespread use.



References
1. Draft landscape of COVID-19 candidate vaccines. World Health Orgainzation. 20 August 2020. https://www.who.int/publications/m/item/draft-landscape-of-covid-19-candidate-vaccines
2. Coronavirus: Missing school is worse than virus for children - Whitty. BBC news, UK. 23 August, 2020. https://www.bbc.co.uk/news/uk-53875410
3. Home page of Kentucky Bioprocessing, Inc. (KBP). https://kentuckybioprocessing.com/

Western diet triggers trained immunity and causes chronic inflammatory diseases

My last blog post reviewed evidence of the involvement of trained immunity in several chronic inflammatory diseases. You may wonder what are the persistent stimuli that can trigger the innate immunity memory and cause the chronic inflammation that can result in inflammatory-related diseases. The answer is not the live attenuated vaccines, nor any invading pathogens, but the Western style of living. As Prof Netea and others wrote in a review paper, "there is increasing evidence that *sterile inflammation (a pathogen-free inflammation) in response to lifestyle changes in Western societies forms the basis on which chronic inflammatory diseases develop."¹ Let us see the research studies that may give us an idea of how to prevent such chronic inflammatory diseases.

Western diet can induce trained immunity
According to the definition by scientists in the immunity field, the Western style of living includes consumption of a Western-type diet and sedentary behaviour. The Western-type diet, particularly the American diet, contains high calories and is rich in sugars, trans and saturated fats, salt and food additives, but low in complex carbohydrates, fibre, vitamins and minerals.²

In a study aimed at finding out if the Western-type diet can induce trained immunity, mice which were genetically modified so that they are prone to atherosclerosis, had high expression of genes related to inflammation and ^#epigenetic reprogramming in their circulating monocytes and their bone marrow myeloid progenitor cells, after being constantly fed with Western-type diet for 4 weeks. This shows that the Western-type diet induces systemic inflammation and long-lasting innate immune cell reprogramming, which amplifies responses to a secondary immune challenge.³

Even after the mice had been switched to a standard normal diet for another 4 weeks, and the serum cholesterol levels and systemic inflammatory markers returned back to normal, the functional reprogramming in myeloid cells still remained.³ The induction in innate immunity response remaining high indicates that the long-lasting reprogramming of the innate cells triggered by the previous Western-type diet could potentially contribute to chronic inflammatory diseases.

Key substances in a Western-type diet that induce trained immunity
Thus far, research studies have identified several ^@endogenous triggers of innate immunity. These include ^$lipoprotein(a) and ^%oxidized low-density lipoprotein.^4,5 These studies found that oxidized low-density lipoprotein induces long-term cytokine production which promotes inflammation, and foam cell formation which is associated with atherosclerosis. The long-term cytokine production and foam cell formation are induced via epigenetic reprogramming of monocytes.

When ^&oxidized cholesterol in the diet is absorbed, it contributes to the pool of oxidized lipoprotein in human serum.⁶ The high fat Western-type diet thus provides a rich source of endogenous stimulation in form of oxidized low-density lipoprotein that can cause chronic inflammation by triggering a long-lasting innate immunity response via cellular reprogramming.

How does the Western-type diet cause the innate immunity to "memorize"?
The same research study which used mice models to demonstrate the triggering of long-lasting innate immunity by a chronic Western-type diet found that such a diet activates NLRP3 inflammasome, a key innate immune sensor for many environmental danger signals such as uric acid and cholesterol crystals, to induce system inflammation and reprogramming of innate immunity. Therefore, the Western-type diet appears to be mistakenly recognized by the immune system as a threat to the organism.³

What we can learn from these findings?
It is well established that diet high in fat and cholesterol is a risk factor for the development of atherosclerosis, and also for the development of other chronic inflammatory diseases, such as type 2 diabetes, obesity, and chronic liver disease. However, the above studies provide clear evidence that such a diet is mistakenly recognized by innate immunity as a danger signal. The persistent uptake of such a diet could contribute to chronic inflammatory reaction by triggering a long-lasting innate immunity.

Moreover, the fact that studies have shown the reprogramming of innate immune cells occurs via a change in the DNA chromatin level, has caused the emerging concept that ancestral environments and abnormal changes in body functions which are the causes, consequences, or concomitants of disease processes, can be transferred down through generations.

Are the findings from these studies powerful enough to make you determined to make changes to your diet?



*Sterile inflammation: A form of pathogen-free inflammation. This can be caused by mechanical trauma, stress, or environmental conditions. The sterile inflammation referred to in this post is caused by a Western-type diet. These damage-related stimuli induce the secretion of molecular agents, collectively termed danger-associated molecular patterns.

^#Epigenetic reprogramming: A process that involves changing of methylation status of histones on the DNA's chromatin level. This results in a change of chromatin structure and hence of gene expression. Epigenetic reprogramming in innate immune cells usually results in the changing of expression of different inflammatory related genes. The change is reversible but usually can last for a few years. Therefore the epigenetic reprogramming in innate immune cells enables the cells to remember the stimulation and react more quickly next time. However, this can also cause chronic inflammation if the innate immune cells are persistently stimulated.

^@Endogenous triggers: Stimuli from substances inside the body. In this post, these include lipoprotein(a) and oxidized low-density lipoprotein.

^$Lipoprotein(a): This is a plasma lipoprotein composed of apolipoprotein(a) and apolipoprotein B-100 of a low-density lipoproteins-like particle. Lipoproteins are macromolecular complexes of lipid (cholesterol) and proteins that originate mainly from the liver and intestine, and are involved in the transport and redistribution of lipids (cholesterol) in the body.⁷ In other words, cholesterol travels through the blood on lipoproteins.

^%Oxidized low-density lipoprotein: This is a product of chemical reactions between normal low-density lipoproteins and free radicals. It is potentially harmful, because a high level of low-density lipoprotein can lead to a build-up of cholesterol in arteries, which can cause blockages.

^&Oxidized cholesterols: They are mainly generated by frying, heating, and processing of a high cholesterol, fat-rich diet.



References
1. 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.
2. A. Christ, & E. Latz. The Western lifestyle has lasting effects on metaflammation. Nat. Rev. Immunol., 2019, 19, 267-268.
3. A. Christ, P. Gunther, M.A.R. Lauterbach, et al. Western diet triggers NLRP3-dependent innate immune reprogramming. Cell, 2018, 172, 162-175.
4. S. Bekkering, J. Quintin, L.A.B. Joosten. et al. Oxidized low-density lipoprotein induces long-term proinflammatory cytokine production and foam cell formation via epigenetic reprogramming of monocytes. Arterioscler. Thromb. Vasc. Biol., 2014, 34, 1731–1738.
5. F.M. van der Valk, S. Bekkering, J. Kroon, et al. Oxidized phospholipids on lipoprotein(a) elicit arterial wall inflammation and an inflammatory monocyte response in humans. Circulation, 2016, 134, 611–624.
6. I. Staprans, X.M. Pan, J.H. Rapp, et al. Oxidized cholesterol in the diet is a source of oxidized lipoproteins in human serum. Journal of Lipid Research, 2003, 44, 705-715.
7. Nutrition in the prevention and treatment of disease. Jose.M. Ordovas, 2001, p157-182.

Trained immunity: a double-edged sword

My last blog post explained that trained immunity, or innate immune memory, provides non-specific protection from secondary infections. The induction of cellular reprogramming in innate immune cells confers an innate immunity with memory characteristics. So far, the only trigger of innate immune memory that we have discussed is microbial triggering, such as triggering from a live attenuated vaccine. But actually the same mechanism of recognition and memory function can also recognize other substances which are not pathogens. In other words, the memory characteristic of the innate immunity mechanism can also be induced by exogenous and endogenous stimuli which are not pathogenic. The fact that trained innate immunity evolved this way, however, does result in harmful effects. Let us have a look at the most recent review paper jointly written by Prof Mihai Netea, who discovered the trained immunity, and the other experts in the field.¹

Netea et al suggested that the reprogramming of the innate immunity and the concomitant increased inflammatory responses to exogenous or endogenous stimuli may have harmful consequences. The induction of trained innate immunity can turn a transient inflammation into a long-lasting effect. Prolonged inflammation is associated with several inflammatory diseases, such as atherosclerosis, and neurodegenerative diseases such as Alzheimer's and dementia, and can also lead to tumour growth and metastasis. The review paper provides evidence from research studies for the association between the innate immunity and the above-mentioned diseases.

Atherosclerosis
Atherosclerosis can lead to heart attack and stroke. Persistent inflammation of vessel wall is characteristic of atherosclerosis. Foam cells, which derived from macrophage*, form a plaque which plays a central role in inflammation during all phases of the atherosclerosis.

A study from Prof Netea found that when *monocytes, an innate immune cell, are stimulated by various micro-organisms, they can develop into *macrophages with persistent ability to promote inflammation, to produce cytokine production and form into foam cells which could lead to atherosclerosis. The stimulated monocytes in the experiment were found to have change at the DNA chromatin level, an indication of the trained immunity process.² The above study indicates that trained immunity stimulated by infections can lead to atherosclerosis.

Alzheimer's disease
Alzheimer's disease is a type of dementia caused by the degeneration of brain cells that process, store and retrieve information, to the point of a loss of function. The disease is characterized by plaques composed of a neurotoxin called β-amyloid. It is hypothesised that the aggregation of the β-amyloid disrupts cell-to-cell communication and activates innate immune cells such as microglia, macrophages which reside in the brain. These immune cells trigger inflammation and ultimately destroy the brain cells.³

In a research study cited by the review, scientists found that peripheral application of inflammatory stimuli in a 3-month old mouse model of Alzheimer's disease, and the second stimulation from β-amyloid plaques at 6-month old leads to a higher β-amyloid plaque load. This indicates a long-lasting training result of microglia.⁴ Moreover, the scientists also found that the functional changes of microglia are accompanied by reprogramming of the cells. This is evidenced by chromatin changes at HIF1α, a gene which usually responds to peripheral trained immunity.^4,5

Cerebral small vessel disease
Cerebral small vessel disease links to systemic inflammation and is a major cause of dementia. According to a research finding cited by the review, in patients with cerebral small vessel disease, peripheral blood-derived monocytes (one type of innate immune cells) have enhanced IL-6 and IL-8 production after **ex vivo stimulation of monocytes with Pam3Cys. Enhanced IL-6 production in the subsequent stimulation is an indication of trained immunity. This shows that the trained immunity contributes to the progression of the disease.⁶ However, a causal link to the pathophysiology of the small vessels in the brain remains to be determined.

Alzheimer's disease and cerebral small vessel disease associated dementia are neurodegenerative diseases occurring generally in older people. In fact, more evidence suggests that there is a link between trained immunity and the inflammatory condition related to an ageing immune system. Therefore, the review suggested that a better understanding of the dark side of trained immunity in ageing populations might help to fight chronic inflammatory diseases such as dementia in elderly patients.

Tumour growth and metastasis
According to the review, chronic inflammation can provide fuels to sustain disease progression and neoplastic transformation, in particular tumour entities. Studies found that trained immunity and the metabolic processes in cancer cells are both reliant on glycolytic metabolism and the up-regulation of the expression and activity of transcription factors such as HIF1α. These findings provide the basis for the interplay between trained immunity and the tumour cells.

Moreover, the review mentioned that innate immune cells infiltrating the tumours' microenvironments can undergo a reprogramming process that leads to the development of maintained inflammatory responses that can boost up the infection-associated proliferation of lymphocytes, impair apoptosis, promote mitochondrial dysfunction and increase oxidative stress. This ultimately promotes the progression of the tumour. Trained cells produce cytokines such as IL-6 and tumour necrosis factor (TNF) that are associated with increased tumorigenicity and the spread of metastases in specific types of tumours. These include oral squamous cell carcinoma and lung, kidney and breast cancer.^7,8

On the other hand, the review mentioned that innate immune cells can be reprogrammed by tumour cells to acquire a more anti-inflammatory situation. This can be exemplified by the low levels of cytokine production in monocytes from chronic lymphocytic leukaemia patients.⁹ Cancer cells also produce a series of soluble mediators that can induce direct epigenetic and metabolic reprogramming in immune cells and can in turn contribute to the progression of the tumour.¹⁰

Conclusion
The reprogramming of the innate immune cells is beneficial when trained immunity results in a fast response to the heterologous pathogen with similar molecular characteristics as the first stimuli. However, from the above examples, we can see that innate immune cells can also be harmful. The resulting prolonged inflammation may also lead to various chronic inflammatory related diseases.



* Monocytes and macrophages are mononuclear cells composing innate immunity. Monocytes are bone-marrow derived leukocytes that circulate in the blood and spleen. Once recruited to tissues, monocytes can differentiate into macrophages and dendritic cells. Macrophages are generally considered terminally-differentiated cells that "engulf" pathogens or toxins and secrete chemokines to recruit other immune cells. The engulfed pathogen is processed by the macrophage and presented to the adaptive immune cells such as B lymphocytes. ** Ex vivo refers to experiments done in cells or tissue from an organism in an external environment with minimal alteration of natural conditions. In the experiment mentioned, the ex vivo stimulation of monocytes were achieved by first isolation of monocytes from blood samples.



References
1. 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.
2. J. Leentjens, S. Bekkering, L.A.B. Joosten, et al. Trained innate immunity as a novel mechanism linking infection and the development of atherosclerosis. Circ. Res., 2018,122, 664-669.
3. Beta-amyloid and the amyloid hypothesis. Alzheimer's Association. https://www.alz.org/national/documents/topicsheet_betaamyloid.pdf
4. A.C. Wendeln, K. Degenhardt, L. Kaurani, et al. Innate immune memory in the brain shapes neurological disease hallmarks. Nature, 2018, 556, 332-338.
5. S.C. Cheng, J. Quintin, R.A. Cramer, et al. mTOR- and HIF-1-mediated aerobic glycolysis as metabolic basis for trained immunity. Science, 2014, 345, 1250684-1250684.
6. M.P. Noz, A. tel Telgte, K. Wiegertjes, et al. Trained immunity characteristics are associated with progressive cerebral small vessel disease. Stroke, 2018, 49, 2910-2917.
7. S.H. Lee, H.S. Hong, Z.X. Liu, et al. TNFα enhances cancer stem cell-like phenotype via Notch-Hes1 activation in oral squamous cell carcinoma cells. Biochem. Biophys. Res. Commun., 2012, 424, 58-64.
8. D.R. Hodge, E.M. Hurt, & W.L. Farrar. The role of IL-6 and STAT3 in inflammation and cancer. Eur. J. Cancer, 2005, 41, 2502-2512. 9. T. Jurado-Camino, R. Cordoba, L. Esteban-Burgos, et al. Chronic lymphocytic leukemia: a paradigm of innate immune cross-tolerance. J. Immunol., 2015, 194, 719-727.
10. K. Rabold, M.G. Netea, G.J. Adema, et al. Cellular metabolism of tumor-associated macrophages-functional impact and consequences. FEBS Lett., 2017, 591, 3022-3041.

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.