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Tuberculosis Vaccine: In the United States and Around the World

There are several reasons it’s not available in the United States

• History and Modern Use

Which Countries Use It?

• In the U.S.

Efficacy and Side Effects

• Vaccine Ingredients

Frequently Asked Questions

The tuberculosis vaccine, also known as the bacille Calmette-Guérin (BCG) vaccine, is used to protect against tuberculosis (TB) and related complications. 

The BCG vaccine is no longer routinely given in the United States and isn’t recommended for the general population by the Centers for Disease Control and Prevention (CDC). However, it’s still given to babies and young children in many countries worldwide. The tuberculosis vaccine may also be considered in the United States for certain people with significant risk factors for prolonged exposure to TB.

In this article, we’ll go over the history and use of the tuberculosis vaccine, including which countries use it, age recommendations, effectiveness, side effects, and more.

Teka77 / Getty Images

History and Modern Use of Tuberculosis Vaccine

Tuberculosis is an infection caused by the bacterium Mycobacterium tuberculosis . Usually, M. tuberculosis bacteria attack the lungs, which causes pulmonary TB. Symptoms of pulmonary tuberculosis include a severe, persistent cough, chest pain, and coughing up blood. Other symptoms of TB may include:

• Night sweats
• Lack of appetite
• Unwanted weight loss

In some cases, M. tuberculosis bacteria can attack other body parts, such as the spine, brain, or kidneys. Many people who are infected with TB don’t have any symptoms. This is known as latent TB infection (LTBI). People with LBTI can’t spread TB to others.

However, about 5 to 10% of people with LTBI develop active TB. People with TB disease have symptoms and can spread M. tuberculosis through actions like coughing, speaking, and singing. Screening for LTBI is recommended for anyone at greater risk of exposure to TB.  

If left untreated, TB can be serious and even fatal. Tuberculosis is especially dangerous for immunocompromised people, including people with human immunodeficiency virus (HIV).

In the early 1900s, the BCG vaccine was developed by researchers Albert Calmette and Camille Guérin to protect against tuberculosis and related complications. It was in wide use by the 1920s but fell out of favor after the Lübeck disaster in 1930 in which 73 infants tragically died in the first year after receiving a contaminated version of the vaccine. More than a decade later, the BCG vaccine came back in response to rising global tuberculosis rates after World War II.  

However, the tuberculosis vaccine is no longer routinely administered in the United States. Studies have shown mixed results in terms of the vaccine’s effectiveness. Some people who have received the vaccine may also get a false positive result on a tuberculin skin test (TST), which can complicate treatment plans and lead to confusion.

While BCG vaccination may cause a false-positive skin test result (when the test indicates that the disease is present when it is not), getting the BCG vaccine will not cause a false-positive TB blood test.

Additionally, the risk of TB in the United States is so low that the benefits of getting vaccinated may not outweigh the potential downsides.

Mandatory BCG vaccination is now somewhat controversial, but many countries with a high incidence of TB cases continue to vaccinate newborns just after birth. Globally, around 2 million–3 million people die from TB disease and related complications yearly. Deaths from tuberculosis are particularly common in developing countries and countries with high rates of HIV, as well as in environments like nursing homes, prisons, homeless shelters, and hospitals.  

In the United States, the CDC recommends that the BCG vaccine be considered only for the following groups:

• Children: Some children with a high risk of developing TB may benefit from BCG vaccination. This includes children who cannot be treated for tuberculosis and who live with adults who have untreated, ineffectively treated, or drug-resistant TB.
• Healthcare workers: Healthcare workers who are employed in settings where a large number of patients have drug-resistant TB and/or where tuberculosis treatments have failed may consider BCG vaccination, if recommended by their healthcare provider.

The BCG vaccine is given to infants on a regular basis in over 180 countries. According to the World Health Organization (WHO), many countries in Southeast Asia, sub-Saharan Africa, and the former Soviet Union have high rates of TB disease. There are also high TB rates in other parts of Europe, Africa, and Asia, as well as parts of the Americas.

Examples of countries where there is a high incidence of tuberculosis include:

• Democratic Republic of the Congo
• Philippines

Tuberculosis Vaccine Travel Restrictions and Requirements

The risk of developing drug-resistant TB disease is extremely rare while traveling internationally. However, your healthcare provider may recommend that your child receive the BCG vaccine if you are planning to travel to a country with high rates of TB if your child is under 5 years old.  If you plan to travel to a country with high rates of tuberculosis, especially drug-resistant tuberculosis, the CDC recommends a tuberculin skin test or blood test first. If you test negative, you should get another test eight to 10 weeks after returning to the United States. Make sure to take any recommended precautions against infection if you spend time in a high-risk environment, such as a healthcare setting.

Age Recommendations

The BCG vaccine is most effective in babies and children under 5. Older children and adults may not benefit as much from receiving it. However, people of all ages may still be considered for the vaccine if they have certain risk factors.

In areas where the BCG vaccine is routinely administered, it’s usually given to newborns. For example, the BCG vaccine is recommended for all newborns as part of the Hong Kong Childhood Immunisation Programme.

How Effective Is the Tuberculosis Vaccine in Children Ages 5 and Up?

Recent research suggests that the tuberculosis vaccine is only significantly effective in preventing severe disease in children under age 5. Among kids age 5 and up who haven’t had a positive TB test, some studies indicate that BCG vaccination doesn’t offer reliable protection against TB disease and related complications.

Tuberculosis Cases in the United States

In the United States, tuberculosis cases are relatively rare. In total, 8,300 TB cases were reported to the CDC’s National Tuberculosis Surveillance System in 2022. Rates of TB disease in the United States decreased consistently from 1993–2019.

They briefly declined sharply (by 19.9%) during the beginning of the COVID-19 pandemic in early 2020 and rose by 9.4% in 2021. Still, the overall number of U.S. TB cases in 2022 was lower than in 2019.

Evidence of the effectiveness of the tuberculosis vaccine is somewhat mixed. According to a 2022 systematic review and meta-analysis, the BCG vaccine was found to be 18% effective overall in protecting against tuberculosis disease and related complications.

It is primarily effective in infants and young children. It is, however, very effective in preventing young children from getting severe forms of tuberculosis like tuberculosis meningitis and miliary tuberculosis.

The most common side effects of the BCG vaccine are:

• Swollen glands in the armpit near the injection site
• A sore at the site of injection, which often releases discharge, scabs over, and leaves behind a scar
• Other skin reactions

Very rarely, BCG vaccination can lead to serious complications, such as abscesses or bone inflammation.

You shouldn’t get the tuberculosis vaccine if you:

• Are pregnant
• Are living with HIV
• Are immunocompromised
• Are allergic to any of the vaccine ingredients

BCG Vaccine Ingredients

The BCG vaccine is a live vaccine . It uses a weakened strain of Mycobacterium bovis (M. bovis), a bacterium closely related to the one that causes TB. Other ingredients include:

• Citric acid
• Magnesium sulfate
• Iron ammonium citrate
• Potassium phosphate

As with other vaccines, the tuberculosis vaccine has been thoroughly tested and vetted for safety.

The bacille Calmette-Guérin (BCG) vaccine, or tuberculosis vaccine, is used in certain countries worldwide to prevent tuberculosis (TB) infection and complications. Typically, the vaccine is given as a shot in the upper arm to infants just after birth. The tuberculosis vaccine is safe, but evidence of its effectiveness in protecting against TB is relatively mixed.

The BCG vaccine is no longer widely used in the United States. However, according to the CDC, the BCG vaccine may be considered for children and adults with a high risk of tuberculosis exposure. Examples include healthcare workers and children who are regularly cared for by adults with drug-resistant tuberculosis or untreated TB.

The TB shot is not given routinely in the United States. However, you can ask your healthcare provider about getting the vaccine if you have a significant risk factor for TB disease.

They may be able to give you the vaccine themselves in their office, or they may recommend that you visit a different clinic or local health agency to be vaccinated. A nearby TB control program may also offer vaccination.

In the United States, the TB vaccine is sometimes considered for people who test negative for TB and are continuously exposed to it regularly. Some examples of high-risk groups include certain healthcare workers and children who live with adults with drug-resistant TB. People who live or work in communal, crowded settings, such as prisons, homeless shelters, and certain hospitals, may also be at risk.

Up to 97% of people who receive the TB vaccine will develop a small scar at the injection site (typically the upper arm). Around two to four weeks after getting the vaccine, you may notice a raised “bubble” on the skin, which usually scabs over and heals within a few months. This is because of the skin’s reaction to the weakened form of Mycobacterium bovis , a bacterium closely related to the one that causes TB disease.

Correction - September 1, 2023: This article was updated to correct the BCG vaccine ingredients.

U.S. Centers for Disease Control and Prevention. Tuberculosis vaccine .

U.S. Centers for Disease Control and Prevention. Clinical overview of tuberculosis .

U.S. Centers for Disease Control and Prevention. Signs and symptoms of tuberculosis .

U.S. Centers for Disease Control and Prevention. Tuberculosis: causes and how it spreads .

US Preventive Services Task Force, Mangione CM, Barry MJ, et al. Screening for Latent Tuberculosis Infection in Adults: US Preventive Services Task Force Recommendation Statement .  JAMA . 2023;329(17):1487-1494. doi:10.1001/jama.2023.4899

World Health Organization. BCG vaccine .

Luca S, Mihaescu T. History of BCG vaccine . Maedica (Bucur) . 2013;8(1):53-8. PMID: 24023600; PMCID: PMC3749764.

Food and Drug Administration. BCG vaccine package insert .

World Health Organization. 2.1 TB incidence .

NSW Health. Overseas travel with children .

U.S. Centers for Disease Control and Prevention. TB risk and people born in or who travel to places where TB is common .

Martinez L, Cords O, Liu Q, et al. Infant BCG vaccination and risk of pulmonary and extrapulmonary tuberculosis throughout the life course: a systematic review and individual participant data meta-analysis .  Lancet Glob Health . 2022;10(9):e1307-e1316. doi:10.1016/S2214-109X(22)00283-2

Family Health Service. Bacille Calmette-Guerin (BCG) Vaccine .

Schildknecht KR, Pratt RH, Feng PI, Price SF, Self JL. Tuberculosis — United States, 2022 . MMWR Morb Mortal Wkly Rep 2023;72:297–303. doi:10.15585/mmwr.mm7212a1

Trunz BB, Fine P, Dye C. Effect of BCG vaccination on childhood tuberculous meningitis and miliary tuberculosis worldwide: a meta-analysis and assessment of cost-effectiveness .  The Lancet . 2006;367(9517):1173-1180. doi:10.1016/S0140-6736(06)68507-3

National Health Service. Side effects of the BCG vaccine .

Mohamed L, Madsen AMR, Schaltz-Buchholzer F, Ostenfeld A, Netea MG, Benn CS, Kofoed PE. Reactivation of BCG vaccination scars after vaccination with mRNA-Covid-vaccines: Two case reports . BMC Infect Dis . 2021;21(1):1264. doi:10.1186/s12879-021-06949-0

By Laura Dorwart Dr. Dorwart has a Ph.D. from UC San Diego and is a health journalist interested in mental health, pregnancy, and disability rights.

• About the Handbook

Tuberculosis

Information about tuberculosis (TB) disease, vaccines and recommendations for vaccination from the Australian Immunisation Handbook.

Recently added

This page was added on  05 June 2018 .

Updates made

This page was updated on 05 August 2022 .  View history of updates

Vaccination for certain groups of people is funded by states and territories .

Tuberculosis is caused by the bacterium Mycobacterium tuberculosis . Most people who become infected with M. tuberculosis have latent tuberculosis infection , which means they are not ill and not infectious. People with tuberculosis disease, in contrast, are ill and usually infectious.

BCG (bacille Calmette–Guérin) vaccine is recommended for:

• Aboriginal and Torres Strait Islander children aged <5 years in some parts of Australia
• Healthcare workers with a high risk of exposure to tuberculosis
• Young children who will be travelling to settings with high tuberculosis incidence
• Some children born to parents from countries with high tuberculosis incidence
• Young children who are a household contact of a person with leprosy

BCG vaccine is given as a single dose by intradermal injection .

A tuberculin skin test pre-vaccination is recommended for some people prior to BCG vaccination, based on a risk assessment of the likely exposure to tuberculosis in the past.

The World Health Organization considers tuberculosis a global emergency. BCG vaccine is recommended for those at highest risk of severe outcomes of tuberculosis.

Recommendations

Aboriginal and torres strait islander people.

Aboriginal and Torres Strait Islander people in some states and territories experience a significant burden of tuberculosis (see Tuberculosis in Australia ). BCG vaccine is recommended for young children living in these regions. 1 Consult state and territory guidelines for more details on BCG vaccination programs for Aboriginal and Torres Strait Islander children.

Tuberculin skin test screening before vaccination is only required in some circumstances. See Tuberculin skin testing before vaccination for details.

See also Vaccination for Aboriginal and Torres Strait Islander people .

Occupational groups

The efficacy of BCG vaccination in adults is more limited compared with in children. See Vaccine Information.

In the workplace, tuberculosis prevention and control should focus on:

• infection control measures
• employment-based screening
• therapy for latent tuberculosis infection

Healthcare workers working overseas in high tuberculosis incidence settings, particularly those with limited infection prevention and control measures, have an increased risk of acquiring tuberculosis. Assess the need for BCG vaccination in these workers.

Consider BCG vaccination for TST negative health care workers in any setting who are at high risk of exposure to drug resistant tuberculosis. This is because drug resistant infections are difficult to treat.

Some other occupational groups have a risk of tuberculosis exposure but are not recommended to receive BCG vaccine because the evidence of benefit of BCG vaccination is limited and infection prevention can be undertaken. This includes healthcare workers at tuberculosis clinics or refugee health clinics, embalmers and people involved in autopsies.

Children aged <5 years travelling to countries with high tuberculosis incidence (>40 cases per 100,000 population per year) are at increased risk of acquiring tuberculosis and developing severe disease. 2 BCG vaccine is most effective at preventing severe tuberculosis (miliary tuberculosis and tuberculous meningitis) in children. See Epidemiology and Vaccine information .

Children should ideally receive the vaccine at least 3 months before departure to a high risk destination. Consider discussing future travel plans with parents and carers of young infants at the earliest possible age.

The risk assessment should take account of the following:

• the child’s age
• how long they are in the high-risk area — the longer the exposure the higher the risk of infection
• the proximity of contact to others — staying with friends or family members in the community increases the risk of infection, particularly if they have a history of recent tuberculosis
• the tuberculosis incidence at the destination

See the World Health Organization’s country-specific incidence data . 3

If additional information is needed to support the risk assessment, seek expert input. Discuss with state or territory tuberculosis services, a paediatric infectious diseases specialist or travel vaccine centres.

BCG vaccine is not as effective in older children and adults. It is not recommended for people in these age groups who are travelling to a country with high tuberculosis incidence, except in some healthcare workers.

Other groups

Children aged <5 years born to parents from countries with high tuberculosis incidence who are now living in Australia are not recommended to receive BCG vaccine, because of the low incidence of tuberculosis in Australia and the uncertainty of the benefit of vaccination compared with the risk of vaccine adverse events.

Tuberculosis is uncommon in children born in Australia. However, children born in Australia to parents from countries with a high tuberculosis incidence (>40 cases per 100,000 population per year) may have a higher risk of tuberculosis exposure from parents and travelling family, in their early life. 4 BCG vaccination may be recommended in some cases, based on an individual risk assessment.

Children born outside of Australia may also be at high risk of disease, but have often previously received a BCG vaccine shortly after birth in their country of birth. See Epidemiology .

Tuberculin skin test (TST) screening before vaccination is only required in some circumstances. See Tuberculin skin testing before vaccination for details.

BCG vaccine provides some protection against infection with Mycobacterium leprae , the organism that causes leprosy. 5 Children aged <5 years with family or household contacts who have leprosy may be recommended to receive BCG vaccine, based on an individual risk assessment.

Tuberculin skin testing before vaccination

The need for TST should be determined by an individual risk assessment that considers whether the person:

• was born in a tuberculosis-endemic country (>40 cases per 100,000 population per year)
• has lived or travelled to a tuberculosis-endemic country or region (>40 cases per 100,000 population per year)
• had exposure to a close contact with tuberculosis or who is under investigation for tuberculosis

If an immunocompetent person who was required to have a TST is confirmed to be negative (induration of <5mm), they can receive BCG vaccine. A person with a TST of 5mm or greater or who has an accelerated BCG reaction (see below), should be considered for further investigation of latent or active tuberculosis.

The TST uses tuberculin, a purified protein derivative. This causes a hypersensitivity reaction in people who have previously been infected with Mycobacterium tuberculosis . ‘False positive’ hypersensitivity reactions can also occur in:

• people infected with other (non-tuberculous) mycobacteria

• people who have previously received BCG vaccine. Vaccination interferes with the interpretation of tuberculin skin test (TST) results

Interferon-gamma release assays (IGRAs) are a type of blood test that can detect M. tuberculosis infection (similar to the TST), but the TST is still the preferred method of screening for past tuberculosis exposure before BCG vaccination. Although TST and IGRA essentially provide the same information, there is uncertainty about whether hypersensitivity detected by IGRA is also associated with an accelerated local BCG reaction (as is the case with a positive TST). 6

Both measles virus and measles-containing live attenuated vaccines  7,8 inhibit the response to tuberculin. TST-positive people may become TST-negative for 4–6 weeks after measles infection or vaccination. This should be taken into account when considering the timing of a TST in people who have had a measles-containing vaccine.

You can give a tuberculin skin test on the same day or visit with a COVID-19 vaccine. There is no specific time interval restriction between a tuberculin skin test and receiving a COVID-19 vaccine. Inhibition of response to a tuberculin skin test is not expected following administration of COVID-19 vaccines.

People with cellular immune compromise may also have a false negative TST, and BCG vaccination is generally contraindicated in this group since it is an attenuated live vaccine. See Contraindications and precautions .

Health professionals must correctly administer and interpret the TST. Consult state or territory tuberculosis guidelines for advice.

Vaccines, dosage and administration

Tuberculosis vaccines available in australia.

The Therapeutic Goods Administration website provides product information for each vaccine .

See also Vaccine information  for more details.

Note: The only BCG vaccine registered for use in Australia ( BCG vaccine (Sanofi-Aventis Australia)) has not been available for some time.

Other BCG vaccines are available in Australia under a special prescribing arrangement (e.g. BCG Vaccine SSI [Statens Serum Institut, Denmark]). These vaccines can be used in the same manner as the registered unavailable vaccine. Contact state and territory public health authorities for more information on obtaining BCG vaccines. See also Public health management .

Please note that different vaccines may use different strains of M. tuberculosis which may differ slightly in antigenic properties. See Vaccine information .

Dose and route

BCG vaccine is a single dose given by intradermal injection . The standard dose is:

• In newborns and infants <12 months of age, the dose is 0.05 mL.
• In children ≥12 months of age and adults, the dose is 0.1 mL.

If BCG is inadvertently given subcutaneously, there is no need to repeat vaccination as the vaccine will still have a protective effect. The person should be informed that they may be more likely to experience an injection site reaction, or regional lymph node involvement, but overall BCG is a very safe vaccine.

Only healthcare workers who are trained in intradermal vaccination procedures should administer BCG vaccine. See Administration of vaccines .

BCG revaccination is generally not recommended, because of a lack of evidence for increased efficacy . 9

BCG vaccine may be available from state and territory tuberculosis services , and may be available through some travel medicine clinics.

BCG vaccination procedures

BCG vaccination steps are:

1. Wear protective eyewear

The following people should wear protective eyewear:

• the person giving the vaccine
• the person receiving the vaccine
• the parent or carer holding a small child who is receiving the vaccine

Eye splashes can ulcerate. If eyes are splashed, wash the eyes with saline or water immediately. Any irritation to the eye as a result of a should be followed up in the subsequent weeks, with an assessment by a medial practitioner and/or a specialist ophthalmologist.

2. Identify the correct injection site

Inject BCG vaccine into the skin over the region where the deltoid muscle inserts into the humerus. This is just above the midpoint of the upper arm. This site is recommended to minimise the risk of keloid formation.

By convention, use the left upper arm, if possible. This can assist people who may later look for evidence of BCG vaccination.

3. Inject the vaccine intradermally

See Administration of vaccines for information on the intradermal vaccination technique.

Response to BCG vaccination

After BCG vaccination, a small, red papule forms and ulcerates within 2–3 weeks of vaccination. The ulcer heals with minimal scarring over several weeks. Local lymph nodes may be swollen and tender.

More serious injection site reactions are less common. See Adverse events .

People who have latent or previous tuberculosis infection and receive BCG vaccine are likely to have an accelerated response. An accelerated cutaneous reaction to BCG is not more severe than typical BCG reactions and have no long-term detrimental effect - it simply occurs more rapidly. This is characterised by:

• induration within 24–48 hours
• pustule formation within 5–7 days
• healing within 10–15 days

Clinical trials have not shown a consistent relationship between the size of tuberculin reactions after BCG vaccination and the level of protection.

Performing a TST to demonstrate immunity after BCG vaccination is not recommended. 10,11

Co-administration with other vaccines

People can receive BCG vaccine at the same time as, or at any time after, other inactivated vaccines.

People can receive BCG vaccine and another live parenteral vaccine (such as MMR [measles-mumps-rubella], varicella or yellow fever) either on the same day or at least 4 weeks apart.

For three months following a BCG vaccine, do not give any other vaccine in the same arm.

People can receive BCG vaccine at any time in relation to oral live vaccines. These include rotavirus vaccine and oral poliovirus vaccine (in infants who have received it overseas).

Contraindications and precautions

Contraindications.

BCG vaccine is contraindicated in people who have had anaphylaxis after any component of a tuberculosis vaccine.

BCG is an attenuated live vaccine that is contraindicated in the following groups:

• people with known or suspected HIV infection ,12 even if they are asymptomatic or have normal immune function. This is because of the risk of disseminated BCG infection 13,14
• people treated with high doses of corticosteroids or other immunosuppressive therapy . These therapies include monoclonal antibodies against tumour necrosis factor (TNF)-alpha, such as infliximab, etanercept and adalimumab. See Vaccination for people who are immunocompromised
• people with congenital cellular immunodeficiencies, including specific deficiencies of the interferon-gamma pathway
• people with active malignancies involving bone marrow or lymphoid systems, any person with cancer receiving immunosuppressive therapy , or people who completed chemotherapy within the previous 3 months. See People with cancer in Vaccination for people who are immunocompromised
• people with any serious underlying illness, including severe malnutrition
• pregnant women. BCG vaccine has not been shown to harm the fetus, but receiving live vaccines in pregnancy is not recommended
• people who have previously had tuberculosis or a positive (≥5 mm) TST

Precautions

Defer BCG vaccination in the following groups:

• neonates who are medically unstable, until the neonate is in good medical condition and ready for discharge from hospital
• infants born to mothers who are suspected or known to be HIV-positive, until HIV infection of the infant can be confidently excluded
• people with active skin disease such as eczema, dermatitis or psoriasis at or near the site of vaccination
• people being treated for latent tuberculosis infection , because the therapy is likely to inactivate the BCG vaccine
• people with significant febrile illness, until 1 month after recovery
• infants aged <6 months born to mothers who were treated with bDMARDs (biologic disease-modifying anti-rheumatic drugs) in the 3rd trimester of pregnancy. These medicines include TNF-alpha-blocking monoclonal antibodies. These infants often have detectable TNF-alpha-blocking antibodies for several months.15-17 See also Use of immunosuppressive therapy during pregnancy in Vaccination for women who are planning pregnancy, pregnant or breastfeeding

Vaccination before or after administration of immunoglobulin or blood products

People can receive BCG vaccine at any time before or after receiving immunoglobulin or any antibody-containing blood product. These preparations and BCG vaccines have minimal interaction. 18 See also Vaccination for people who have recently received normal human immunoglobulin and other blood products .

Adverse events

The normal reaction to BCG vaccination is described in Vaccines, dosage and administration . With the proper procedure less than 5% of vaccinated people experience adverse events; ~2.5% may develop a local injection site ‘cold abscess’ and ~1% regional lymphadenitis with/without ‘cold abscess’ formation. 19

Other adverse events include: 20

• local suppurative complications. This does not require treatment with anti-tuberculosis medicine unless there is perceived risk of disseminated BCG disease (see below), but BCG is inherently resistant to pyrazinamide and optimal treatment requires careful consideration; 21 it is best to seek specialist advice from state or territory tuberculosis services .
• keloid formation. This risk is minimised if the injection is no higher than the level of insertion of the deltoid muscle into the humerus
• disseminated BCG disease, but the risk is extremely low (1–4 cases per million vaccinated people) and it is only observed in people with immune compromise. Treatment with anti-tuberculosis medicines may be warranted, but BCG is inherently resistant to pyrazinamide and optimal treatment requires careful consideration; 21 it is best to seek specialist advice from state or territory tuberculosis services.

Nature of the disease

Tuberculosis is caused by Mycobacterium tuberculosis and other organisms of the M. tuberculosis complex (M. TB complex) 22 . M. tuberculosis is the cause of almost all tuberculosis in Australia 23

Pathogenesis

Infection usually occurs when a person inhales the tuberculosis bacteria , which reach the lungs. M. bovis can be ingested from unpasteurised milk, consumed in countries where M. bovis remains prevalent (not Australia)

If the person’s immune system can contain the bacteria , the person will be infected but not develop active disease. This is called latent tuberculosis infection .

If the bacteria overcome the immune system, which may occur after many years of immune control, the person develops active disease and may become infectious if pulmonary disease occurs. 24

Transmission

M. tuberculosis is usually transmitted by the airborne route. Factors affecting transmission include: 22

• duration of exposure
• frequency of exposure
• proximity to the infected person (high-density or communal living situations may increase the risk of transmission)

Persons with extrapulmonary TB with no lung or larygneal involvement are not infectious.

Clinical features

Tuberculosis most commonly presents as lung disease, which accounts for 60% of notified tuberculosis cases in Australia. 25 Common symptoms of pulmonary tuberculosis are:

• weight loss
• coughing up blood (mainly in adults with late-stage pulmonary disease)

Extrapulmonary tuberculosis can occur in any part of the body. Tuberculosis lymphadenitis is the most common extrapulmonary manifestation.

Disseminated disease (miliary tuberculosis) and meningeal tuberculosis are more common in very young children. 26 These are among the most serious manifestations of tuberculosis disease. 20

Most people infected with M. tuberculosis remain asymptomatic. There is a 10% lifetime risk of developing clinical illness. Clinical disease can develop many years after the original infection . The risk varies depending on age and immune status.

Groups more prone to rapidly progressive disease include: 22

• young children (infants and children <5 years of age)
• elderly people
• people who are immunocompromised as a result of medical treatment, disease or adverse socioenvironmental circumstances.

Epidemiology

Tuberculosis in australia.

In Australia, tuberculosis is an uncommon disease, with annual incidence remaining below 7 per 100,000 population since 1980s. 27

Most tuberculosis cases in Australia (more than 85%) occur in people who were born overseas, especially in countries with a high incidence of tuberculosis. 3,28 See latest WHO Global Tuberculosis Report for up-to-date information . 29

The rate of multidrug-resistant (MDR) tuberculosis in Australia remains low (approximately 2% of bacteriologically confirmed cases with drug susceptibility testing available). 30

Tuberculosis in animals ( Mycobacterium bovis ) has been eradicated in Australia by screening and culling programs. 31

Tuberculosis in Aboriginal and Torres Strait Islander people

In most states and territories, rates of tuberculosis among Aboriginal and Torres Strait Islander people overall are comparable to rates among Australian-born non-Indigenous people. However, notifications of tuberculosis among Aboriginal and Torres Strait Islander people in some states and territories are disproportionately higher than for Aboriginal and Torres Strait Islander people and non-Indigenous people in other states.

Regions with higher rates of tuberculosis include: 25

• the Northern Territory
• Far North Queensland

See Vaccination for Aboriginal and Torres Strait Islander people .

Screening for tuberculosis

Screening programs in Australia focus on:

• contacts of notified cases
• people at increased risk of tuberculosis infection , including refugees and healthcare workers

Tuberculosis in other countries

The World Health Organization declared tuberculosis a global emergency in 1993, and recent reports have reaffirmed the threat to human health. 32 In 2019, there were about 7.1 million incident cases of tuberculosis globally. 29

Vaccine information

When reconstituted, BCG vaccine is a suspension of a live attenuated strain of M. bovis . Worldwide, many BCG vaccines are available, but they are all derived from the original strain selected by Calmette and Guerin, which was first tested in humans in 1921. 33

Sanofi-Aventis Australia markets the only BCG vaccine registered for use in Australia, although this vaccine is currently unavailable. See Vaccines, dosage and administration . Contact your state or territory health authority to access a BCG vaccine.

Efficacy of BCG vaccine

In children.

BCG vaccination in young children provides: 34

• ~25% protection against tuberculosis infection
• ~70% protection against active tuberculosis
• >70% protection against severe forms of tuberculosis disease in young children, including miliary tuberculosis and tuberculosis meningitis. 35-39

The efficacy of BCG vaccine against pulmonary disease in adults is less consistent, and has ranged from no protection to 80% in controlled trials. 35   The reason for the wide variation is not clear, but it has been attributed to differences in:

• study quality
• BCG strains
• host factors, such as age at vaccination and nutritional status
• the prevalence of infection with environmental mycobacteria

Duration of protection

The duration of protection after BCG vaccination has been difficult to measure because the time between infection and disease can be decades. Benefit from infant vaccination has been found in studies with follow-up of up to 40 years, but protection is thought to decline over 10–20 years. 20 Immune memory responses may remain for 10–50 years. 41-43

Other uses of BCG vaccine

BCG vaccination offers some protection against Mycobacterium leprae , which causes leprosy. 5

BCG is also used as treatment for bladder cancer, but this is a different preparation that is instilled directly into the bladder.

Transporting, storing and handling vaccines

The currently available vaccine, BCG Vaccine SSI, is presented in a multidose vial. For more information on use of multidose vials, see Administration of vaccines .

Transport according to National Vaccine Storage Guidelines: Strive for 5. 44 Store at +2°C to +8°C. Do not freeze. Protect from light.

BCG vaccine must be reconstituted . Add the entire contents of the diluent container to the vial and shake until the powder completely dissolves. Reconstituted vaccine is very unstable. Use within 4–6 hours.

Public health management

Tuberculosis is a notifiable disease in all states and territories in Australia. The Communicable Diseases Network Australia national guidelines for the public health management of tuberculosis 40 have details on the management of tuberculosis cases and their contacts.

State and territory public health authorities can provide further advice about:

• public health management of tuberculosis
• using alternative vaccine products in special circumstances, such as during shortages of the registered vaccine
• National Tuberculosis Advisory Committee. The BCG vaccine: information and recommendations for use in Australia – National Tuberculosis Advisory Committee update October 2012. Communicable Diseases Intelligence 2013;37:E65-72.
• Toms C, Stapledon R, Coulter C, Douglas P, National Tuberculosis Advisory Committee. Tuberculosis notifications in Australia, 2014. Communicable Diseases Intelligence 2017;41:E247-63.
• World Health Organization. World health statistics - Tuberculosis profile. 2022. (Accessed 25 April 2022). https://worldhealthorg.shinyapps.io/tb_profiles/?_inputs_&entity_type=%22country%22&lan=%22EN%22&iso2=%22AF%22
• Smith BB, Hazelton BJ, Heywood AE, et al. Disseminated tuberculosis and tuberculous meningitis in Australian-born children; case reports and review of current epidemiology and management. Journal of Paediatrics and Child Health 2013;49:E246-50.
• Zodpey SP, Bansod BS, Shrikhande SN, Maldhure BR, Kulkarni SW. Protective effect of Bacillus Calmette Guerin ( BCG ) against leprosy: a population-based case-control study in Nagpur, India. Leprosy Review 1999;70:287-94.
• National Tuberculosis Advisory Committee. Position statement on interferon-γ release assays in the detection of latent tuberculosis infection . Communicable Diseases Intelligence 2012;36:125-31.
• McLean HQ, Fiebelkorn AP, Temte JL, Wallace GS. Prevention of measles, rubella, congenital rubella syndrome, and mumps, 2013: summary recommendations of the Advisory Committee on Immunization Practices (ACIP). [erratum appears in MMWR Morb Mortal Wkly Rep. 2015 Mar 13;64(9):259]. MMWR. Recommendations and Reports 2013;62(RR-4):1-34.
• Starr S, Berkovich S. Effects of measles, gamma-globulin-modified measles and vaccine measles on the tuberculin test. New England Journal of Medicine 1964;270:386-91.
• World Health Organization. SAGE Evidence to recommendations framework. 2017. (Accessed 28 March 2022). https://www.who.int/immunization/sage/meetings/2017/october/2_EvidencetoRecommendationFramework_BCG.pdf
• Menzies D. What does tuberculin reactivity after bacille Calmette-Guérin vaccination tell us? Clinical Infectious Diseases 2000;31 Suppl 3:S71-4.
• Hart PD, Sutherland I, Thomas J. The immunity conferred by effective BCG and vole bacillus vaccines in relation to individual variations in induced tuberculin sensitivity and to technical variations in the vaccines. Tubercle 1967;48:201-10.
• Hesseling AC, Marais BJ, Gie RP, et al. The risk of disseminated Bacille Calmette-Guerin ( BCG ) disease in HIV-infected children. Vaccine 2007;25:14-8.
• Hesseling AC, Cotton MF, Fordham von Reyn C, et al. Consensus statement on the revised World Health Organization recommendations for BCG vaccination in HIV-infected infants. International Journal of Tuberculosis and Lung Disease 2008;12:1376-9.
• Mansoor N, Scriba TJ, de Kock M, et al. HIV-1 infection in infants severely impairs the immune response induced by Bacille Calmette-Guérin vaccine. Journal of Infectious Diseases 2009;199:982-90.
• Cheent K, Nolan J, Shariq S, et al. Case report: Fatal case of disseminated BCG infection in an infant born to a mother taking infliximab for Crohn's disease. Journal of Crohn's and Colitis 2010;4:603-5.
• Mahadevan U, Terdiman JP, Church J, et al. Infliximab levels in infants born to women with inflammatory bowel disease. Gastroenterology 2007;132 Suppl 2:A144.
• Mahadevan U, Miller JK, Wolfe DC. Adalimumab levels detected in cord blood and infants exposed in utero. Gastroenterology 2011;140 Suppl 1:S61-2.
• Connelly Smith K, Orme IM , Starke JR. Tuberculosis vaccines. In: Plotkin SA, Orenstein WA, Offit PA, eds. Vaccines. 6th ed. Philadelphia, PA: Elsevier Saunders; 2013.
• Turnbull FM, McIntyre PB, Achat HM, et al. National study of adverse reactions after vaccination with bacille Calmette-Guérin. Clinical Infectious Diseases 2002;34:447-53.
• World Health Organization. BCG vaccines: WHO position paper – February 2018. Weekly Epidemiological Record 2018;93:73-96.
• Hesseling AC, Rabie H, Marais BJ, et al. Bacille Calmette-Guérin vaccine-induced disease in HIV-infected and HIV-uninfected children. Clinical Infectious Diseases 2006;42:548-58.
• Fitzgerald DW, Sterling TR, Haas DW. Mycobacterium tuberculosis. In: Bennett JE , Dolin R, Blaser MJ, eds. Mandell, Douglas, and Bennett's principles and practice of infectious diseases. 8th ed. Philadelphia, PA: Elsevier Saunders; 2015.
• Lumb R, Bastian IB, Jelfs PJ, et al. Tuberculosis in Australia: bacteriologically-confirmed cases and drug resistance, 2011. A report of the Australian Mycobacterium Reference Laboratory Network. Communicable Diseases Intelligence 2014;38:E369-75.
• National Center for HIV/ AIDS , Viral Hepatitis, STD, and TB Prevention, Division of Tuberculosis Elimination. Introduction to the Core Curriculum on Tuberculosis: what the clinician should know. 6th ed. Atlanta, GA: Centers for Disease Control and Prevention; 2013. https://www.cdc.gov/tb/education/corecurr/pdf/corecurr_all.pdf
• Barry C, Konstantinos A, National Tuberculosis Advisory Committee. Tuberculosis notifications in Australia, 2007. Communicable Diseases Intelligence 2009;33:304-15.
• Perez-Velez CM, Marais BJ. Tuberculosis in children. New England Journal of Medicine 2012;367:348-61.
• Bareja C, Waring J, Stapledon R, Toms C, Douglas P. Tuberculosis notifications in Australia, 2011. Communicable diseases intelligence quarterly report 2014;38:E356-68.
• NSW Health. List of countries with a tuberculosis incidence of 40 cases per 100,000 persons or greater. 2021. (Accessed 28 March 2022). https://www.health.nsw.gov.au/Infectious/tuberculosis/Pages/high-incidence-countries.aspx
• World Health Organization. Tuberculosis data: global tuberculosis report. 2021. (Accessed 28 March 2022). https://www.who.int/teams/global-tuberculosis-programme/data
• World Health Organization. BCG vaccines: WHO position paper – February 2018. 2018. (Accessed 28 March 2022). https://apps.who.int/iris/bitstream/handle/10665/260307/WER9308-73-96.pdf?sequence=1&isAllowed=y
• Ingram PR, Bremner P, Inglis TJ, Murray RJ, Cousins DV. Zoonotic tuberculosis: on the decline. Communicable Diseases Intelligence 2010;34:339-41.
• World Health Organization (WHO). The End TB Strategy: global strategy and targets for tuberculosis prevention, care and control after 2015. Geneva: WHO; 2014. http://www.who.int/tb/strategy/End_TB_Strategy.pdf
• Wittes RC. Immunology of bacille Calmette-Guérin and related topics. Clinical Infectious Diseases 2000;31 Suppl 3:S59-63.
• Roy A, Eisenhut M, Harris RJ, et al. Effect of BCG vaccination against Mycobacterium tuberculosis infection in children: systematic review and meta-analysis. BMJ 2014;349:g4643.
• Bourdin Trunz B, Fine PE, Dye C. Effect of BCG vaccination on childhood tuberculous meningitis and miliary tuberculosis worldwide: a meta-analysis and assessment of cost-effectiveness. The Lancet 2006;367:1173-80.
• Colditz GA, Berkey CS, Mosteller F, et al. The efficacy of bacillus Calmette-Guérin vaccination of newborns and infants in the prevention of tuberculosis: meta-analyses of the published literature. Pediatrics 1995;96:29-35.
• Rodrigues LC, Diwan VK, Wheeler JG. Protective effect of BCG against tuberculous meningitis and miliary tuberculosis: a meta-analysis. International Journal of Epidemiology 1993;22:1154-8.
• Colditz GA, Brewer TF, Berkey CS, et al. Efficacy of BCG vaccine in the prevention of tuberculosis: meta-analysis of the published literature. JAMA 1994;271:698-702.
• Brewer TF. Preventing tuberculosis with bacillus Calmette-Guérin vaccine: a meta-analysis of the literature. Clinical Infectious Diseases 2000;31 Suppl 3:S64-7.
• Barreto ML, Pereira SM, Ferreira AA. BCG vaccine: efficacy and indications for vaccination and revaccination. Jornal de Pediatria 2006;82(3 Suppl):S45-54.
• Aronson NE, Santosham M, Comstock GW, et al. Long-term efficacy of BCG vaccine in American Indians and Alaska Natives: a 60-year follow-up study. JAMA 2004;291:2086-91.
• Sterne JA, Rodrigues LC, Guedes IN. Does the efficacy of BCG decline with time since vaccination? International Journal of Tuberculosis and Lung Disease 1998;2:200-7.
• Weir RE, Gorak-Stolinska P, Floyd S, et al. Persistence of the immune response induced by BCG vaccination. BMC Infectious Diseases 2008;8:9.
• National vaccine storage guidelines: Strive for 5. 2nd ed. Canberra: Australian Government Department of Health and Ageing; 2013. https://beta.health.gov.au/resources/publications/national-vaccine-storage-guidelines-strive-for-5-2nd-edition
• Communicable Diseases Network Australia ( CDNA ). Tuberculosis ( TB ): CDNA national guidelines for the public health management of TB . Canberra: Australian Government Department of Health; 2015. http://www.health.gov.au/cdnasongs

Page history

Recommendations for skin testing before BCG vaccination have changed. A tuberculin skin test before BCG vaccination is now only recommended in limited circumstances, based on a risk assessment.

Updates to all sections of the Tuberculosis chapter have been made including Recommendations, Vaccines, dosage and administration, Contraindications and precautions, Adverse events, Nature of the disease, Clinical features, Epidemiology, Vaccine information, Transporting, storing and handling vaccines, Public health management and Variations from product information.

Changes to 4.20.10 Precautions

4.20.10 Precautions

Addition of text to clarify when BCG vaccination should be deferred in people with skin conditions.

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• Hum Vaccin Immunother
• v.17(12); 2021

BCG vaccination strategies against tuberculosis: updates and perspectives

a College of Veterinary Medicine, China Agricultural University, Beijing, China

b Key Laboratory of Animal Epidemiology and Zoonosis, Ministry of Agriculture, National Animal Transmissible Spongiform Encephalopathy Laboratory, China Agricultural University, Beijing, China

Xiangmei Zhou

c Center for Infectious Disease Research, School of Medicine, Tsinghua University, Beijing, China

Bacillus Calmette-Guérin (BCG) is the only licensed vaccine against tuberculosis (TB). However, BCG has variable efficacy and cannot completely prevent TB infection and transmission. Therefore, the worldwide prevalence of TB calls for urgent development of a more effective TB vaccine. In the absence of other approved vaccines, it is also necessary to improve the efficacy of BCG itself. Intravenous (IV) BCG administration and BCG revaccination strategies have recently shown promising results for clinical usage. Therefore, it is necessary for us to revisit the BCG vaccination strategies and summarize the current research updates related to BCG vaccination. This literature review provides an updated overview and perspectives of the immunization strategies against TB using BCG, which may inspire the following research on TB vaccine development.

1. Introduction

BCG, an attenuated strain of Mycobacterium bovis ( M.bovis ), remains the only approved vaccine against TB for clinical use since 1921. 1 Since 1974, BCG vaccination has been included in the World Health Organization (WHO) Expanded Programme on Immunization (EPI), which was dedicated for infant vaccination worldwide. Different countries have subsequently formulated more favorable BCG vaccination policies according to their own conditions. 1 Countries with high TB incidences continue universal BCG vaccination strategies, while most countries with low to moderate incidence rates consider selective vaccination strategies to target high-risk groups. 2–4 In 2020, 154 countries reported that BCG vaccination is a standard part of childhood immunization programs, of which 53 reported more than 95% coverage. 5 However, previous studies showed that BCG can be only modestly protective, and even completely ineffective against TB in human populations. 6 , 7 The latest WHO report on global TB is still shocking, with an estimated 9.9 million people infected and more than 1.43 million deaths due to the disease in 2020. 5 TB mortality has been more severely impacted by the COVID-19 pandemic in 2020. 5 TB is the leading cause of infectious death worldwide at present, which calls for the development of effective vaccination strategies. 8

The initial development of TB vaccines was mainly focused on devising a vaccine more effective than BCG. Although TB vaccines development have made some progress in the past few years, vaccine evaluation is an extremely long-term, high-risk, and expensive program. 9 On the other hand, BCG has a beneficial heterologous effect, which may prevent diseases other than TB, and modulate immune responses to other vaccines in children. The BCG replacement strategy must take its substantial nonspecific effects into consideration. 10–12 Moreover, strategies of improving existing vaccines by modifying immunization schedules or routes are more cost-effective ways than developing totally new vaccines. Therefore, novel BCG vaccination strategies are being developed. These have shown promising results against Mycobacterium tuberculosis (Mtb) infection.

BCG, which has been used for 100 years as an effective strategy for TB control has protected millions of people from TB. 8 By improving BCG immunization strategies, new and remarkable immune effects have been demonstrated. This has rekindled the hope of BCG to be more effective against TB. 13–17 Under the raging of the global Corona Virus Disease 2019 (COVID-19) pandemic in 2020, BCG has also shown its potential to be protective against COVID-19, which has reignited the research interest in it. 18 , 19 In a retrospective study, among health care workers in a multisite Los Angeles health care organization, BCG vaccination was associated with a reduction in the seroprevalence of anti-SARS-CoV-2 IgG, as well as a decrease in the number of participants who self-reported clinical symptoms associated with COVID-19. 20 Therefore, it is extremely necessary to summarize and update the immunization strategies based on BCG vaccination against TB to provide guidance and inspiration for future research.

2. Why does intradermal BCG vaccination have limited protection against TB?

It is known that BCG is administered via the intradermal (ID) route shortly after birth in TB endemic areas. 6 Although this inoculation method can be easily performed and induce a strong systemic immunity, it can only Mtb provide partial protection in humans and animals models. 21 In addition, this method can produce positive results for the tuberculin skin test (TST), however, it has been shown that the positive conversion rate of TST is irrelevant to the efficacy of BCG immunity. 22 An in-depth discussion on the defects of ID BCG immunization may provide indicative information for the improvement of BCG immune strategy ( Figure 1 ).

The adaptive immune response to ID BCG vaccination. ID BCG vaccination can arouse a strong adaptive immune response in human body, but these immune responses are not enough to resist Mtb infection for the long term. There are three main reasons: (a). Cellular immunity plays a crucial role in fighting against Mtb infection. Although the T helper 1 (Th1) immune response induced by ID BCG vaccination is relatively robust, it would be inhibited by the Th2 and Treg immune response. In addition, the immune response of Th17 and CD8+ induced by ID BCG vaccination is weak. (b). Recently more and more evidences indicate that humoral immune responses play important roles in protection against Mtb, but the level of antibodies induced by ID BCG immunization is very low, or even almost undetectable. (c). For the memory immune responses, although ID BCG vaccination can induce a large number of effector memory T (TEM) cells, the number of central memory T (TCM) cells and resident memory T (TRM) cells account for small population. Such a composition of memory cells would result in vaccine-induced protection not being sustained for long and make it difficult to respond quickly to the presence of pathogens. The red“ – ” represents a weak immune response, the red“-” represents an adverse immune response, and the red “↓” represents a decrease in the intensity of the immune response.

The performance in inducing T cells immune responses of ID BCG immunization could be an important factor for BCG protection against TB ( Figure 1(a )). First of all, CD4+ and CD8 + T cells cannot be induced efficiently. 23–26 The airway luminal T (ALT) cells are important for the host against Mtb infection, however ID BCG vaccination can only induce a small population of ALT cells and these cells are deficient for at least 10 days after Mtb infection in a TB mouse model. 23 , 24 In a Guinea pig infection model, ID BCG vaccination in the early stage can produce abundant antigen (Ag)-specific lipopeptide-reactive CD4 + T cells in peripheral blood mononuclear cells (PBMCs), but lack functional diversity to prevent granuloma formation. 25 As for CD8 + T cells, BCG can cause significant activation of Ag-specific CD8 + T cells, but its delivery of Ag to the sites of T cell activation is inefficient. 26 Secondly, ID BCG vaccination is not good at inducing T helper 1 (Th1) and Th17 cells. Although ID BCG immunization can induce a robust Th1 immune response, it does not provide sufficient protection and is also negatively regulated by the Th2 and regulatory T cell (Treg) responses. 27 , 28 The same conclusion was reached in a study of neonatal BCG immunization. 29 Human cord blood mononuclear cells selectively produced Th2-type cytokines IL-10 and IL-5 in response to BCG stimulation, and the level of IL-10 was higher than that of unvaccinated infants aged 10 weeks. 29 Furthermore, infants who received BCG at the age of 10 weeks had a stronger lymphoproliferative and Th1 immune response than newborns who received BCG. 29 Th17 cells can trigger the expression of CXCR3 chemokine ligand 9 (CXCL9), CXCL10, and CXCL11 which recruit CD4 + T cells producing interferon (IFN)-γ, and ultimately restrict Mtb growth. 30 Furthermore, interleukin (IL)-17 plays an important role in preventing Mtb infection by inducing CXCL13 to drive neutrophil recruitment to the infection site for pathogens’ control. 31 , 32 However, ID BCG vaccination cannot induce enough Th17 immune responses. 33 Thirdly, as Mtb infection progresses, BCG-induced CD4 + T cells and subsequently CD8 + T cells functionally fade away, gradually resulting in the immune system paralysis. 34 , 35 CD4+ and CD8 + T cells exhaustion after infection is related to mitochondrial dysfunction and the expression of T cell immunoglobulin and mucin domain-containing protein 3 (TIM3), programmed cell death protein 1 (PD1), and other inhibitory receptors. 36 , 37 Therefore, it is necessary to block or offset these complex signals about T cell exhaustion and maintain a reserve of specific self-renewing T cells that can mediate long-term containment in order to improve BCG efficacy.

Although Mtb is an intracellular pathogen, B cells and antibodies also have important roles in resisting Mtb infection. 38–41 Total immunoglobulin (Ig) isolated from Mtb-exposed healthcare workers in a TB-specialized hospital can offer protection against Mtb infection in the aerosol infection mouse model. 39 Monoclonal antibodies against Mtb phosphate transporter subunit PstS1 isolated from active tuberculosis infection (ATBI) patients could reduce bacterial lung burden by 50% in Mtb infected Balb/c mice. 40 Antibodies may mediate protection against Mtb by Mtb neutralization, phagocytosis enhancement, inflammasome activation, and cytotoxic natural killer (NK) cell activities. 41 Some studies showed that ID BCG immunization not only could produce Mtb-specific antibodies but also antibody levels would increase slightly but significantly with the increase of dose and immunization times, while some studies indicated the opposite results. 42 These results suggest that BCG-mediated humoral immunity is heterogeneous, which may be because of different BCG strains, the health state of the immunized subjects, the number of subjects, and the diagnosis methods. 42 However, it is undeniable that the antibody levels induced by ID BCG vaccination are indeed very low ( Figure 1(b )). Further analysis of the antigenic targets for specific antibodies produced by BCG vaccination revealed that BCG only significantly induced specific antibody against lipoarabinomannan (LAM). 43 , 44 Although the antibody against LAM had been clarified to limit the growth of Mtb, 45 , 46 the antibody produced by ID BCG immunization is obviously not enough to control the invasion of Mtb, probably because of insufficient antibody levels. It was also reported that the inhibitory activity of anti-Mtb antibodies was directly associated with their isotypes. 47 IgA antibodies that target Mtb surface antigens could mediate the blocking effect of Mtb uptake independently of Fc alpha receptors expression, while IgG antibody promoted host cell infection. 47 However, the level of IgA induced by ID BCG vaccination is not adequate to make it effective. 42 Therefore, humoral immunity should be taken into account to improve the immune effects of BCG when develop new BCG vaccine strategies.

For memory immune responses, insufficient induction of the central memory T (TCM) cells and tissue-resident memory T (TRM) cells by ID BCG immunization is another important reason for its immune failure ( Figure 1(c )). The long-term memory response mainly depends on the magnitude of TCM cells, not the T effector memory (TEM) cells. 48 ID BCG immunization induces much fewer TCM cells than TEM cells in the lung. 49 Moreover, TCM cells in the host are gradually depleted due to the long-term exposure to environmental mycobacteria, and this leads to the loss of IL-2 producing CD4 + T cells and the increase of KLRG1+ terminally differentiated T cells. 50 TRM cells, also called the local specialists in immune defense, have the ability to detect infected cells and can respond quickly before host recruitment of circulating memory T cells when exposed to Mtb. 51 CD8+ TRM cells can restrict the entry of Mtb into lung tissue by killing infected macrophages, and trigger protective innate and adaptive immune responses by secreting IFN-γ, TNF-α, and IL-2. When these cytokines are blocked, this protective immune responses disappear completely. 14 , 52 Although ID BCG vaccination can also induce TB-specific lung TRM cells, the frequency of TRM cells is relatively low. 14 Moreover, TRM cells in the lungs are not stable, causing a gradual protection loss. 53 In mouse models, ID BCG vaccination could induce antigen-specific CD4+ TRM cells in lung parenchyma for at least 12 months, but this duration time is still short for vaccination protection. 54 Therefore, the improvement of BCG vaccination strategy should be designed to induce both TRM cells and circulating memory T cells especially TCM cells to obtain a high level of protection against Mtb infection.

3. BCG alternative vaccination routes

The immunogenicity and immuno-protection level of BCG may be improved to some extent by changing the administration route. 21 In recent years, the research of BCG mucosal delivery and intravenous injection ( Figure 2 ) has produced satisfactory results, revealed the importance of immune approaches on the immune response, and also provided a paradigm shift in TB vaccine research. 14–16 , 55

The immune mechanisms of BCG delivered by mucosal and intravenous vaccination. (a). BCG is first taken up by M cells of mucosal epithelium and transported to mucosa-associated lymphoid tissue (MALT). After BCG is processed by dendritic cells, effector T and B lymphocytes are generated, and then differentiated into memory cells. The effector T and B lymphocytes play their protective functions in the effective sites after lymphatic circulation and blood circulation. Except for that tissue-resident memory T (TRM) cells remain constrained within local tissue, central memory T (TCM) cells and effector memory T (TEM) cells migrate to the corresponding lymphatic organs or non-lymphoid tissues. When the body is attacked by Mtb, TRM cells respond quickly, and then the circulating memory cells perform their effector functions. At the same time, memory B cells also rapidly differentiate and secrete IgA (sIgA). (b). Darrah groups 16 showed IV BCG made that 9 out of 10 macaques were highly protected and even 6 showed no signs of infection. The possible protective mechanism of IV BCG vaccination: increased markedly antigen-responsive T cells, higher significantly antibody response, and well-trained immunity. Red shows the presence of bacteria and pulmonary tuberculosis disease, and Orange indicates reduced bacterial burdens and disease, whereas brown remarks no detectable infection.

3.1. Oral immunization

BCG was developed by Calmette and Guerin in 1921 and initially administered orally. 56 And the oral BCG vaccination was used in neonates until 1976 in Brazil, and many data support safety of BCG oral immunization. 13 , 56 Combined with the poor ID BCG immunization, there is a renewed interest in oral BCG. Since Mtb enters the host through infectious aerosols, the mucosa is often the first site to contact with Mtb, and mucosal immunity can trigger a specific protective immune response in the mucosa-associated lymphoid tissue (MALT), which is extremely important for prevention of Mtb infection. 57 The specific immune cells in MALTs are then transported throughout the body generating a systemic immune response ( Figure 2(a )). 58 Besides, B and T cells acquire mucosal homing properties only in the draining lymph nodes from specialized dendritic cells that migrate from the mucosal tissue to these lymph nodes, thus rapidly responding to Mtb. 58 Hence, mucosal delivery can rapidly induce both local and systemic immune responses. 58 Moreover, mucosal immunity can produce specific secretory antibodies in the mucosa to mediate the protective effects against Mtb. 47 , 59 Oral immunization is not only easier to operate but also safer than other mucosal immunization strategies. 60 In this delivery method, BCG can effectively penetrate through the tonsils and intestinal epithelium in newborns and induce specific immunity in the MALTs. 55

Oral BCG can also be used as a booster vaccine. In healthy adults, a combined ID and oral BCG vaccination approach could induce the optimal combination of mucosal and systemic immune responses associated with resistance to TB infection and disease progression. 13 Furthermore, no major safety hazards had been found in the combined ID and oral BCG vaccination approach. 13 ID BCG vaccination-induced systemic Th1 response more powerfully, whereas oral BCG induced a stronger mucosal secretory IgA (sIgA) response and a higher frequency of mucosal cytotoxic T lymphocytes. 60 Therefore, combination of the two vaccination strategies can be considered in clinical practice to enhance the effectiveness of BCG. However, fewer vaccines are using oral immunization methods in clinical practice currently, because this route of administration passes through the body’s first-pass effect, which reduces the drugs’ bioavailability and makes functional burden to the livers and kidneys. 61 In the early days of BCG administration, it was required to take BCG repeatedly to achieve the desired protective effect. 56 However, excessively high doses tend to induce mucosal tolerance, which would avoid triggering an immune response. 62 Hence, it is necessary to use potent adjuvants to make BCG more immunogenic and stable. Nowadays, new materials are often used as transport carriers to wrap BCG, which contribute to mucosal uptake and enhance the BCG protective immunogenicity. 63 , 64

Another promising application of oral BCG is among wild animals living in the nature. 65 , 66 BCG oral vaccination can protect European badgers from virulent M. bovis both experimentally and in the field. 65 , 66 In addition to the general drawbacks of oral vaccines, it is also necessary to consider how to ensure that animals voluntarily consume enough BCG to provide a protective effect. Furthermore, it is required to consider whether BCG could be mixed in the feed avoiding any damage to the vaccine and also preventing environmental pollution.

3.2. Intratracheal and intranasal vaccination

Intratracheal (IT) and intranasal (IN) vaccination are also representative routes of mucosal immunity to deliver BCG. This method of immunization does not require large doses compared to oral administration, and vaccine delivery via aerosol spray is more convenient and attractive due to the upgrading of delivery equipment. 67 Furthermore, Mtb usually enters the host through the respiratory tract, suggesting that IT and IN BCG vaccination are highly effective for the induction of protective immunity. 68 These vaccination methods generate a large number of effector T cells and TRM cells in the lung airway, which are the main components of the BCG efficacy. 14 The airway-resident CD8 + T cells exhibit typical TRM characteristics, in addition to expressing IFN-γ and TNF-α, two cytokines that are not only primary mediators of protective immunity against TB but also recruit CD4 + T cells and B cells to the Mtb infected site to enhance local immunity. 14 In contrast, the airway-resident CD4 + T cells contain a mixture of T-bet + effectors and Foxp3 + -expressing regulatory T cells. 14 Furthermore, CD4 + T cells induced by BCG in this manner also exhibit a specific cellular phenotype compared to those induced by intradermal delivery of BCG. 69 Ag-specific CD4 + T cells expressing a PD-1+ KLRG1- phenotype are present in lung parenchyma and bronchoalveolar lavage fluid (BALF), and these cells can enhance the local immune effect at the infection site by improving the homing effect. Such phenotype determines that these cells can be purified from the lung parenchyma rather than the pulmonary vasculature. 69 CD4 + T cells from the lung parenchyma have greater control over Mtb infection because of the homing effect than the ones from the pulmonary vascular system. 70 , 71 Besides these immune cells, the human alveolar lining fluid contains hydrolytic enzymes which can help BCG improve Mtb control in the mouse infection model. 72

In TB animal models, rhesus monkeys, which share the greatest anatomical and physiological similarities with humans, are the most important “gateways” into human performance testing. 73 The IT BCG vaccination route also shows excellent immune protection effects in rhesus monkeys. Dijkman et al . 15 showed the differences between IT and ID BCG vaccination in rhesus macaques by repeatedly infecting the test population with very low doses of Mtb (1 CFU Mtb) to simulate human natural infection. Surprisingly, infection of rhesus macaques immunized by endobronchial instillation was significantly delayed, or even completely absent. In contrast, all unvaccinated animals and animals that received BCG through the skin got infected and subsequently developed TB. 15 The Th1/Th17 response and the expression of IL-10 in lung cells may be significantly associated with the enhanced immune protection of BCG. 15 IL-17-mediated specific mucosal immune responses triggered by BCG mucosal immunity also offer robust protection against Mtb infection. 74 On the contrary, IL-10 is related to Mtb ability to evade immune responses and mediate long-term lung infections. 75 However, a role for IL-10 in protective immunity cannot be excluded as well. A balance between pro- and anti-inflammatory cytokines was associated with clearance of Mtb at the granulomas level. 76 The balance between activities of IL-17 and IL-10 induced by IT BCG immunization constitute the host defense mechanism in overcoming chronic infection established by Mtb. 15 Interestingly, there is no correlation between sIgA and immune protection in this study, possibly due to the limited number of experimental animals. 15

Mimicking the natural infection route of Mtb has been suggested as a possible means to improve the protective efficacy of the vaccine. 69 In conclusion, both oral BCG and IT or IN BCG are effective. Moreover, treatment of BCG with petroleum either removes inflammatory lipids on the surface of BCG while maintaining the vitality of bacteria, thereby reducing the inflammation caused by lung inoculation with BCG. 77 The technical aspects of BCG mucosal immunization need further research including oral or aerosol delivery, immune dosage, and immune adjuvant mechanism, etc. Overall, it is believed that BCG mucosal immunization has a bright application prospect.

3.3. Intravenous vaccination

Intravenously (IV) BCG immunization can effectively prevent Mtb infection. 16 , 21 , 78 , 79 As early as the 1970s, IV BCG in rhesus monkeys had been shown to provide more protection compared with other conventional BCG inoculation methods. 78 Of 7 IV BCG-immunized rhesus monkeys to mimic natural infection with Mtb, 4 had no gross lesion and the other 3 had the only mild disease. 79 A study published in 2016, further confirmed that IV immunization could induce the highest IFN spot-forming units and multifunctional CD4 + T cell frequency to reduce disease pathology caused by TB. 21 The latest research has shown that IV administration of BCG could achieve unprecedented levels of protection to resist Mtb infections and diseases in non-human primates (NHP) ( Figure 2(b )). 16 In the Mtb challenge experiment after BCG vaccination 6 months, 9 out of 10 macaques given BCG intravenously were highly protected, and of which 6 showed no signs of infection. 16 Compared to aerosol and intradermal delivery, IV BCG immunization resulted in large and sustained recruitment of T cells into the airway and parenchyma. Moreover, IV injection-induced more intense antigen-specific CD4+ and CD8 + T cell responses in BALF and PBMCs, which helped rapid elimination of Mtb. 16 In addition, the antibody response aroused by IV BCG vaccination in BALF and plasma was also significantly higher than other routes. IV BCG vaccination in mice models could induce trained immunity to enhance innate immunity, thereby generating better protection against Mtb infection by means of producing epigenetically modified macrophages. 80 However, neither BCG was detected in bone marrow after one month of IV BCG vaccination, nor was there increase in the innate activation of PBMCs against non-Mtb antigens in NHP models. 16 Nevertheless, it was undeniable that trained immunity played a role in this process. The immune correlation of high protection of IV BCG vaccination still need to be further studied.

It is hard to imagine that BCG as a 100 years old vaccine has remarkable high protection level against TB after changing vaccination route. 16 Importantly, the limited set of clinical safety parameters measured suggested that IV BCG might be well tolerated in NHP, which indicates that this immunization strategy may have good prospects in human applications. 16 It is known that IV injection is currently used for drug therapy and is rarely used for vaccination because of difficulties to implement it in mass vaccination. However, IV immunization has shown excellent immune effects in prevention of many diseases. In a recent study, it was reported that IV vaccination induced a higher proportion of TCF1+ PD-1+ CD8 + T cells and produced a higher anti-tumor response as compared to subcutaneous immunization. 81 Another study of the malaria preventive vaccine PfSPZ showed that IV immunization had produced superior immunogenicity and protective effects in humans compared with subcutaneous and ID administration. 82 Similarly, a series of clinical trials have begun in Africa, Europe, the United States and other regions, in anticipation of applying this immunization method to a small number of high-risk groups. 82 However, the PfSPZ is a non-replicating sporeworm vaccine. 82 A recent study showed that intravenous administration of COVID-19 mRNA vaccine might cause acute myopericarditis in mouse model. 83 As for BCG, although such delivery had previously been used in humans to treat cancer, 84 , 85 further in-depth research is still required to study the safety and effectiveness of injecting pathogenic bacteria with replication ability into human blood.

4. Prime-boost vaccination strategy to enhance BCG efficacy

Except for the immunization routes, various booster vaccines are developed to “repair” the immunogenicity and enhance immune memory persistence of BCG. 86–96 The current BCG booster vaccine research strategy is mainly based on the several dominant antigens of Mtb with the help of live virus expression vectors or adjuvants. 33 WHO has made this approach to improving BCG a priority for the research and development of a new TB vaccine. 97 A total of 9 BCG booster candidate vaccines are currently under active evaluation in clinical trials ( Figure 3 ) and only “best-in-class” candidates to late-stage clinical trials. A really excellent BCG booster vaccine can prevent not only the primary Mtb infection but also the progression of the disease in those latently infected individuals. In the recently completed final analysis of the clinical phase IIb trial (ClinicalTrials.gov Identifier: {"type":"clinical-trial","attrs":{"text":"NCT01755598","term_id":"NCT01755598"}} NCT01755598 ), M72/AS01 E could provide 49.7% protection against active pulmonary TB for latent Mtb-infected adults for at least 3 years, excluding differences in age or gender for vaccine efficacy, which was a milestone in the development of a new tuberculosis vaccine. 93 , 98 Notably, the vaccine had a clinically acceptable safety profile and immunogenicity in HIV-infected people, no matter in TB endemic areas or in low-risk areas, regardless of their antiretroviral therapy status. 99 , 100 Although TB is highly prevalent among HIV-positive people, WHO does not recommend BCG vaccination in infants infected with HIV. 101 Hence, there is an urgent need for an effective TB vaccine that can be safely vaccinated to HIV-infected people. M72/AS01 E is expected to fill the gap.

BCG booster vaccine candidates in clinical development. There are currently 9 BCG booster candidates in clinical development, including viral vector vaccines, protein subunit vaccines, live attenuated vaccines, and whole cell vaccines 69–77. The stage of clinical development of vaccine candidates is inferred from data available at ClinicalTrials.gov. Abbreviation: TLR = toll-like receptor.

The easiest and most convenient way to apply the prime-boost strategy is a second BCG vaccination. Previous large randomized clinical trials had shown that BCG revaccination do not contribute to TB prevention. 102 , 103 In 2018, the WHO also announced the same conclusion and did not recommend BCG revaccination. 104 However, recent clinical trials make us rethink about this strategy. BCG revaccination was safe in QuantiFERON-TB Gold In-tube assay (QFT)-negative adolescents and can significantly improve BCG-specific CD4 + T cell response. But not the specific CD8 + T cell response. 17 , 105 Remarkably, BCG revaccination did not prevent the initial conversion of QFT in a context of TB high-transmission, but reached 45.4% efficacy against persistent QFT conversion, while the efficacy of clinical TB vaccine candidate H4:IC31 (Ag85B-TB10.4 fusion proteins in IC31 adjuvant) was only 30.5%. 17 The sustained QFT conversion might reflect sustained Mtb infection and progression to disease. This study reflected BCG revaccination could help prevent sustained Mtb infection, which was of great public health significance.

The immune effect of BCG will be affected by the infection status. This is one of the reasons for the variable immune effect of BCG. 106 Therefore, it is very important to see whether BCG revaccination will be affected by the Mtb infection status, since approximately just under a quarter of the global population are latent tuberculosis infection (LTBI) patients in 2014. 107 The above clinical trials of BCG revaccination were carried out in QFT-negative adolescents. 17 , 105 Similarly, BCG revaccination in healthy adults infected with Mtb, whether or not they were treated with isoniazid before vaccination, had the same robust immunogenicity. 108 , 109 BCG revaccination could transiently promote BCG-specific CD4+, CD8+ and γδ T cell responses, it could particularly boost highly specific natural killer T (NKT) cell and NK cell responses persistently (at least for 1 year) to improve trained immunity, 109 , 110 which might indicate that BCG revaccination could also produce an additional immune effect. In addition, BCG vaccination could significantly enhance Mtb-specific Th17 responses, especially regulatory IL-10+ Th17 responses. 108 The protectiveness of BCG revaccination against Mtb infection in LTBI patients still needs further research.

5. Perspectives

Mtb is an extremely “robust” and “tricky” intracellular pathogen that has highly efficient mechanisms for immune evasion and can coexist with infected hosts for a lifetime. 57 TB vaccines should have the ability to modulate moderately the complex regulatory signals induced by Mtb, create a delicate balance between inflammation and regulatory immune responses, and maintain strong memory immune responses for a long time. The BCG immunization strategy must be continuously improved to ensure the efficacy of the Mtb control strategy worldwide.

Either changing the vaccination route or relying on the prime-boost immune strategy, is a good way to improve the immune effect of BCG. However, there are significant challenges in conducting the process of clinical trials, one of the biggest obstacles in this process is the lack of accurate and reliable immune markers. It is not feasible to overemphasize the Th1 immune response before the classical and reliable immune markers are determined, which may ignore the truly effective immune response and enable Mtb to perform immune evasion. This might suggest that the sample size should be as large as possible and the scope of immunization evaluation should be as wide as possible when conducting TB vaccine research.

Another major challenge is the often glaring difference between the immune assessment of clinical trials and those based on animal models for TB vaccines. To minimize discrepancies, animal models that reflect human infection, such as NHPs, should be selected when evaluating TB vaccines in animal models. Secondly, the number of experimental animals can be increased as much as possible to reduce the randomness of experimental results and the differences caused by the heterogeneity of experimental animals. Besides, TB vaccines need to be evaluated in the context of ongoing chronic infections to reflect people’s lifelong exposure to pathogens and their antigens in many cases. 15 It has been reported that the combination of Monte-Carlo methods and compartmental models can reduce the uncertainty in impact evaluations to a certain extent, improve the evaluation of vaccine candidates and help the decision-making processes of funding agencies. 111

An obvious limitation in the development of BCG booster vaccines is that the type of vaccine function is extremely limited. For most TB vaccine candidates entering preclinical trials and clinical trials, their functional profiles are extremely limited. In most cases, they can be differentiated mostly by the magnitude of antigen-specific T-cell responses. 33 So the studies should focus more on finding promising protective antigens that are not confined only in inducing cellular immunity. The role of antibodies in TB has been initially elucidated and should be taken into account in the design of TB vaccines. 39–41 Additionally, adjuvants are usually required for vaccines to exert enough protective immune responses against pathogens, which can increase the vaccine efficacy significantly. 112 Therefore, new adjuvants technologies should be studied in parallel with vaccines research and development. Moreover, TB vaccine design cannot be limited to its small field and should learn from the experiences of other successful vaccines such as Hib and meningococcal conjugate vaccines. 113 Over last century, BCG vaccine has saved countless lives around world. With the rapid development of science and technologies, we believe that BCG vaccination strategies development will be a crucial and important research direction and will exert its positive roles in public health.

Funding Statement

This research was funded by National Natural Science Foundation of China to Hao Li (No. 32070937), 2015 Talent Development Program of China Agricultural University to Hao Li (No. 00109029) and National Natural Science Foundation of China to Xiangmei Zhou (No.31873005).

Disclosure statement

No potential conflict of interest was reported by the author(s).

Author contributions

Hao Li: Conceptualization, Writing-Review & Editing, Supervision, Project administration and Funding acquisition. Xiangmei Zhou: Writing-Review & Editing, Supervision, Project administration and Funding acquisition. Mengjin Qu: Writing- Original draft preparation.

Travel vaccination advice

If you're planning to travel outside the UK, you may need to be vaccinated against some of the serious diseases found in other parts of the world.

Vaccinations are available to protect you against infections such as yellow fever , typhoid and hepatitis A .

In the UK, the  NHS routine immunisation (vaccination) schedule protects you against a number of diseases, but does not cover all of the infectious diseases found overseas.

When should I start thinking about the vaccines I need?

If possible, see the GP or a private travel clinic at least 6 to 8 weeks before you're due to travel.

Some vaccines need to be given well in advance to allow your body to develop immunity.

And some vaccines involve a number of doses spread over several weeks or months.

You may be more at risk of some diseases, for example, if you're:

• travelling in rural areas
• backpacking
• staying in hostels or camping
• on a long trip rather than a package holiday

If you have a pre-existing health problem, this may make you more at risk of infection or complications from a travel-related illness.

Which travel vaccines do I need?

You can find out which vaccinations are necessary or recommended for the areas you'll be visiting on these websites:

• Travel Health Pro
• NHS Fit for Travel

Some countries require proof of vaccination (for example, for polio or yellow fever vaccination), which must be documented on an International Certificate of Vaccination or Prophylaxis (ICVP) before you enter or when you leave a country.

Saudi Arabia requires proof of vaccination against certain types of meningitis for visitors arriving for the Hajj and Umrah pilgrimages.

Even if an ICVP is not required, it's still a good idea to take a record of the vaccinations you have had with you.

Find out more about the vaccines available for travellers abroad

Where do I get my travel vaccines?

First, phone or visit the GP practice or practice nurse to find out whether your existing UK vaccinations are up-to-date.

If you have any records of your vaccinations, let the GP know what you have had previously.

The GP or practice nurse can give you general advice about travel vaccinations and travel health, such as protecting yourself from malaria.

They can give you any missing doses of your UK vaccines if you need them.

Not all travel vaccinations are available free on the NHS, even if they're recommended for travel to a certain area.

If the GP practice can give you the travel vaccines you need but they are not available on the NHS, ask for:

• written information on what vaccines are needed
• the cost of each dose or course
• any other charges you may have to pay, such as for some certificates of vaccination

You can also get travel vaccines from:

• private travel vaccination clinics
• pharmacies offering travel healthcare services

Which travel vaccines are free?

The following travel vaccines are available free on the NHS from your GP surgery:

• polio (given as a combined diphtheria/tetanus/polio jab )
• hepatitis A

These vaccines are free because they protect against diseases thought to represent the greatest risk to public health if they were brought into the country.

Which travel vaccines will I have to pay for?

You'll have to pay for travel vaccinations against:

• hepatitis B
• Japanese encephalitis
• tick-borne encephalitis
• tuberculosis (TB)
• yellow fever

Yellow fever vaccines are only available from designated centres .

The cost of travel vaccines that are not available on the NHS will vary, depending on the vaccine and number of doses you need.

It's worth considering this when budgeting for your trip.

Other things to consider

There are other things to consider when planning your travel vaccinations, including:

• your age and health – you may be more vulnerable to infection than others; some vaccines cannot be given to people with certain medical conditions
• working as an aid worker – you may come into contact with more diseases in a refugee camp or helping after a natural disaster
• working in a medical setting – a doctor, nurse or another healthcare worker may require additional vaccinations
• contact with animals – you may be more at risk of getting diseases spread by animals, such as rabies

If you're only travelling to countries in northern and central Europe, North America or Australia, you're unlikely to need any vaccinations.

But it's important to check that you're up-to-date with routine vaccinations available on the NHS.

Pregnancy and breastfeeding

Speak to a GP before having any vaccinations if:

• you're pregnant
• you think you might be pregnant
• you're breastfeeding

In many cases, it's unlikely a vaccine given while you're pregnant or breastfeeding will cause problems for the baby.

But the GP will be able to give you further advice about this.

People with immune deficiencies

For some people travelling overseas, vaccination against certain diseases may not be advised.

This may be the case if:

• you have a condition that affects your body's immune system, such as HIV or AIDS
• you're receiving treatment that affects your immune system, such as chemotherapy
• you have recently had a bone marrow or organ transplant

A GP can give you further advice about this.

Non-travel vaccines

As well as getting any travel vaccinations you need, it's also a good opportunity to make sure your other vaccinations are up-to-date and have booster vaccines if necessary.

Although many routine NHS vaccinations are given during childhood, you can have some of them (such as the MMR vaccine ) as an adult if you missed getting vaccinated as a child.

There are also some extra NHS vaccinations for people at higher risk of certain illnesses, such as the flu vaccine , the hepatitis B vaccine and the BCG vaccine for tuberculosis (TB) .

Your GP can advise you about any NHS vaccinations you might need.

Find out about NHS vaccinations and when to have them

Page last reviewed: 16 March 2023 Next review due: 16 March 2026

• People/Staff

Researchers discover a potential vaccine to prevent tuberculosis in people of all ages

19 June 2024 - Wits University

In a critical global public health development, a candidate vaccine for tuberculosis (TB) has been created using a gene-editing approach.

TB remains the leading cause of death by infectious disease globally, with South Africa having one of the highest incidence rates in the world.

While the BCG vaccine used to prevent TB is widely available for infants, no vaccine has shown lasting protection. The BCG is also the only existing effective vaccine.

“South Africa committed to the Sustainable Development Goal of ending the TB epidemic by 2030. While we are doing relatively well as a country – TB deaths have come down since 2015 – we need to do a lot better to reach the milestones,” says Professor Bavesh Kana .

Kana, the Head of the School of Pathology and former director of the Centre of Excellence for Biomedical TB Research at Wits University, contributed to the groundbreaking study .

Researchers sought to modify the BCG vaccine to make it more effective at controlling the growth of M. tuberculosis .

Mice injected with the edited BCG vaccine had less M. tuberculosis growth in their lungs than mice that received the original vaccine.

“We can now offer a new candidate vaccine in the fight against this deadly disease," says Kana. "The work also demonstrates that gene editing is a powerful way to develop vaccines. This is particularly important for researchers working on vaccine development."

About the tuberculosis vaccine

The BCG vaccine is given to children around the time of birth and is effective at preventing TB disease.

However, BCG does not protect teenagers and adults and has not been effective at eradicating TB.

This has spurred the need to develop novel TB vaccine candidates to replace or boost BCG.

“We also see that the BCG can evade the immune system and that this reduces its efficacy as a vaccine,” says Kana.

He noted that the importance of vaccines cannot be overstated.

When humans get sick, their body's defence system spots particular signs, called PAMPs (p athogen-associated molecular patterns) , on the outside of bacteria, viruses, or other harmful germs.

This helps the body tell the difference between invaders and its own cells and then starts fighting the infection.

Vaccines work by looking like germs, so that they can start the first defence without making a person sick.

Kana has lamented the funding gap in developing tools to eliminate TB – a disease which dates back over 9000 years.

“Until recently, our diagnostic approaches were a century old. With some novel vaccine candidates in the pipeline, we can finally begin to adequately address this devastating illness.”

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United States Government’s Response to the Clade I Mpox Outbreak in the Democratic Republic of the Congo and Other Countries in the Region

This week, the Africa Centres for Disease Control and Prevention (Africa CDC) declared the mpox clade I outbreak a Public Health Emergency of Continental Security, and the World Health Organization (WHO) declared this outbreak a Public Health Emergency of International Concern. The United States government supports those declarations. The United States will continue to work closely with African governments, Africa CDC and WHO to ensure an effective response to the current outbreak and to protect the health and lives of people of the region.

In 2022, the world experienced a global outbreak of clade II mpox, which led to more than 95,000 cases across 115 non-endemic countries. Clade I mpox tends to cause a higher number of severe infections and have a higher mortality rate than clade II mpox. The evidence for clade I mpox clinical outcomes is based primarily on data from endemic countries, particularly DRC. We expect it would cause lower morbidity and mortality in the United States than in the DRC.

DRC is currently experiencing the largest number of annual suspect cases ever recorded and the disease has now been identified in several neighboring countries where mpox (clade I or clade II) has not been found in the past.

U.S. Government Partnership on the Mpox Response

The United States Government has been closely monitoring the spread of clade I mpox in the DRC and neighboring countries since 2023, and we have been working closely with the Government of DRC, as well as regional and global health partners to reduce the impact of this outbreak and safeguard public health. U.S. government support for the mpox response also builds on our robust, longstanding health partnerships with DRC and throughout Africa, which have helped combat infectious diseases such as HIV, tuberculosis, and malaria for over 20 years. In fiscal year 2023, the United States allocated more than $2.65 billion in bilateral health funding in Central and Eastern Africa. This response also builds on a longstanding partnership on global health security between DRC and the United States.

In addition to ongoing health support, in the last few months the United States has provided an additional $17 million USD to support clade I mpox preparedness and response efforts in Central and Eastern Africa. The funding has enabled stronger surveillance, risk communication, and community engagement, as well as needed laboratory supplies and diagnostics, clinical services, and vaccine planning.

Vaccination will be a critical element of the response to this outbreak. To support this effort, the United States is donating 50,000 doses of the Food and Drug Administration (FDA)-approved JYNNEOS vaccine to DRC. The United States is working with other countries, WHO, and international partners to encourage donations that support vaccine efforts and address challenges to vaccine delivery.

Mpox Preparedness for the United States

The risk to the general public in the United States from clade I mpox circulating in the DRC is very low, and there are no known cases in the United States at this time. Due to efforts over the last nine months, the United States is well prepared to rapidly detect, contain, and manage clade I cases should they be identified domestically. The United States has a robust surveillance system in place, including through clinical testing and wastewater analysis. We continue to encourage those at high risk to get vaccinated with the JYNNEOS mpox vaccine, which has been demonstrated to be safe and highly effective at preventing severe disease from mpox. Those who have already had clade II mpox or are fully vaccinated against mpox are expected to be protected against severe illness from clade I mpox.

CDC has issued an updated Health Alert Network advisory urging clinicians to consider clade I mpox in people who have been in DRC or neighboring countries in the previous 21 days; clinicians are also asked to submit specimens for clade-specific testing for these patients if they have symptoms consistent with mpox. Given the geographic spread of clade I mpox, the U.S. CDC issued an updated Travel Health Notice on Aug. 7, 2024, recommending travelers to DRC and neighboring countries practice enhanced precautions.

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Open Access

Peer-reviewed

Methods and Resources

Methods and Resources report novel methods, substantial improvements to current methodologies, or informational datasets.

See Journal Information »

A noninvasive BCG skin challenge model for assessing tuberculosis vaccine efficacy

Roles Data curation, Formal analysis, Investigation, Methodology, Writing – original draft, Writing – review & editing

Affiliation Department of Infectious Disease, Imperial College London, London, United Kingdom

Roles Conceptualization, Data curation, Formal analysis, Investigation, Methodology

Roles Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Writing – review & editing

Roles Data curation, Formal analysis, Investigation, Methodology, Resources, Software

Roles Investigation, Methodology

Affiliation Department of Life Sciences, Centre for Bacterial Resistance Biology, Imperial College London, London, United Kingdom

Roles Conceptualization, Formal analysis, Investigation, Methodology, Resources, Software, Writing – review & editing

Affiliation Stanford Photonics Research Center, Stanford University, Stanford, California, United States of America

Roles Data curation, Formal analysis, Methodology, Software, Writing – review & editing

Affiliation Department of Pharmaceutical Biosciences, Uppsala University, Uppsala, Sweden

Roles Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Software, Supervision, Writing – review & editing

Roles Conceptualization, Funding acquisition, Project administration, Supervision, Writing – original draft, Writing – review & editing

* E-mail: [email protected]

• Nitya Krishnan, 
• Miles Priestman, 
• Iria Uhía, 
• Natalie Charitakis, 
• Izabella T. Glegola-Madejska, 
• Thomas M. Baer, 
• Albin Tranberg, 
• Alan Faraj, 
• Ulrika SH Simonsson, 
• Brian D. Robertson

• Published: August 19, 2024
• https://doi.org/10.1371/journal.pbio.3002766
• Peer Review
• Reader Comments

This is an uncorrected proof.

We report here on the characterisation in mice of a noninvasive bacille Calmette-Guérin (BCG) skin challenge model for assessing tuberculosis (TB) vaccine efficacy. Controlled human infection models (CHIMs) are valuable tools for assessing the relevant biological activity of vaccine candidates, with the potential to accelerate TB vaccine development into the clinic. TB infection poses significant constraints on the design of a CHIM using the causative agent Mycobacterium tuberculosis (Mtb). A safer alternative is a challenge model using the attenuated vaccine agent Mycobacterium bovis BCG as a surrogate for Mtb, and intradermal (skin) challenge as an alternative to pulmonary infection. We have developed a unique noninvasive imaging system based on fluorescent reporters (FluorBCG) to quantitatively measure bacterial load over time, thereby determining a relevant biological vaccine effect. We assessed the utility of this model to measure the effectiveness of 2 TB vaccines: the currently licenced BCG and a novel subunit vaccine candidate. To assess the efficacy of the skin challenge model, a nonlinear mixed effect model was built describing the decline of fluorescence over time. The model-based analysis identified that BCG vaccination reduced the fluorescence readout of both fluorophores compared to unvaccinated mice ( p < 0.001). However, vaccination with the novel subunit candidate did not alter the fluorescence decline compared to unvaccinated mice ( p > 0.05). BCG-vaccinated mice that showed the reduced fluorescent readout also had a reduced bacterial burden in the lungs when challenged with Mtb. This supports the fluorescence activity in the skin as a reflection of vaccine induced functional pulmonary immune responses. This novel noninvasive approach allows for repeated measurements from the challenge site, providing a dynamic readout of vaccine induced responses over time. This BCG skin challenge model represents an important contribution to the ongoing development of controlled challenge models for TB.

Citation: Krishnan N, Priestman M, Uhía I, Charitakis N, Glegola-Madejska IT, Baer TM, et al. (2024) A noninvasive BCG skin challenge model for assessing tuberculosis vaccine efficacy. PLoS Biol 22(8): e3002766. https://doi.org/10.1371/journal.pbio.3002766

Academic Editor: Matthew K. Waldor, Brigham and Women’s Hospital, UNITED STATES OF AMERICA

Received: October 24, 2023; Accepted: July 25, 2024; Published: August 19, 2024

Copyright: © 2024 Krishnan et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: Relevant data are within the paper and its Supporting Information files. Flow cytometry FCS files are available from https://doi.org/10.5281/zenodo.12794251 . Python scripts are available from https://doi.org/10.5281/zenodo.12781429 .

Funding: BDR received funding from Aeras grant “Human Challenge Model for TB”. BDR and TMB received funding from the Bill and Melinda Gates Foundation grant OPP1180610. The sponsors played no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Abbreviations: BCG, bacille Calmette-Guérin; CFU, colony-forming unit; CHIM, controlled human infection model; FOCEI, first-order conditional estimation method with interaction; IAV, inter-animal variability; ID, intradermally; Mtb, Mycobacterium tuberculosis ; OFV, objective function value; PsN, Perl-speaks-NONMEM; RFU, relative fluorescence unit; SCM, stepwise-covariate modelling; TB, tuberculosis; VPC, visual predictive check; YFP, yellow fluorescent protein

Introduction

Despite advances in modern medicine and better living conditions for many people, tuberculosis (TB) remains a major killer of the poor and disadvantaged around the world. Notwithstanding major efforts in the diagnosis, treatment, and prevention of TB—which have saved an estimated 54 million lives between 2000 and 2017—these have had little impact on transmission, and there are still nearly 10 million newly diagnosed cases and around 1.5 million deaths every year [ 1 ]. The current bacille Calmette-Guérin (BCG) vaccine is routinely given to neonates in endemic countries and high-risk populations and protects against the disseminated forms of TB disease to which children are susceptible. However, BCG works poorly in adolescents and adults who are the significant drivers of TB in high incidence communities [ 2 , 3 ], meaning that large-scale vaccination programmes have had negligible impact on transmission. Consequently, we need new vaccines with efficacy in all ages and all populations to successfully control TB and reach the WHO End-TB targets of a 95% decrease in deaths and a 90% decrease in incidence by 2035 (both relative to 2015) [ 4 ].

We have learnt much about the pathogenesis and immunology of TB using the mouse model. However, TB vaccine discovery has been hampered by animal models that poorly reflect the stages of infection and disease found in humans. Candidate vaccines that show good efficacy in a range of animal models do not always perform as well in human trials, where a lack of biomarkers for vaccine efficacy make incident TB the primary outcome, which requires several years of follow-up post-vaccination [ 5 , 6 ]. As the number of vaccine candidates completing preclinical studies increases [ 7 – 10 ], there is a need to prioritise those that proceed to expensive large-scale clinical trials without relying solely on the current suite of animal model data.

Human infection/challenge models have a long history in experimental medicine [ 11 ]. The malaria sporozoite challenge model has a safe history of use to assess new drugs and vaccines with a relatively short antimalarial treatment at the conclusion of the study [ 12 , 13 ]. A human challenge model for TB creates several challenges, including pulmonary infection, lengthy 6-month drug treatment, and the potential for latent or asymptomatic infection. The establishment of a pulmonary human infection model using BCG has demonstrated its feasibility for advancing the understanding of pulmonary immunobiology during TB infection [ 14 ]. A strategy to avoid issues associated with Mycobacterium tuberculosis (Mtb) as a challenge agent is to use Mycobacterium bovis BCG, which has a long history of safe use and has been used as a surrogate [ 14 – 18 ]. Our study utilises BCG as the basis for a fluorescent reporter strain (FluorBCG) that can be introduced intradermally, with the fluorescent signal measured noninvasively through the skin using a sensitive and cost-effective imaging system. This system reproducibly measures an accelerated loss of fluorescent signal over time in vaccinated compared to nonvaccinated animals. In this report, we describe the development and characterisation of the system and demonstrate its ability to detect relevant vaccine effects in the mouse model of TB. This skin-based challenge model has the potential to be used as an early indicator of vaccine efficacy and provide useful data to inform and advance vaccine development. This feasibility study is a step towards developing a human challenge model for TB.

Materials and methods

Animal studies.

All animal procedures were performed under the Animal Scientific Procedures Act (1986) under the licence issued by the UK Home Office (PPL 70/7160). Six- to 8-week-old female BALB/c mice (Charles River, United Kingdom) were maintained in Biosafety Containment Level 3 facilities (BSL3) according to institutional protocols.

Bacterial strains and growth conditions

Experiments with Mtb H37Rv (a kind gift from Dr Christophe Guilhot, Institut de Pharmacologie et de Biologie Structurale, France) were carried out in BSL3 facilities according to institutional protocols. BCG-Pasteur and BCG Pasteur Δ panCD strains were a kind gift from Dr Nathalie Cadieux, Aeras Global TB Vaccine Foundation. Mycobacterial strains were cultured in Difco Middlebrook 7H9 liquid medium (Becton Dickinson, UK) supplemented with 0.2% glycerol (Sigma, UK), 0.05% Tween 80 (Sigma, UK), and 10% oleic acid-albumin-dextrose-catalase (OADC, USBiological, UK). For growth on solid medium, Middlebrook 7H10 plates were supplemented with 0.5% glycerol and 10% OADC. BCG Pasteur Δ panCD was grown in medium supplemented with 24 μg/ml calcium pantothenate (Sigma-Aldrich, UK).

Construction of the fluorescent mycobacterial strain

We constructed a recombinant fluorescent mycobacterium, BCG Pasteur Δ panCD [Psmyc Turbo635asv-YFP] (FluorBCG), expressing dual fluorophores (one unstable to increase detection of growth/death). The pCB22 backbone plasmid (6572 bp; Dr Nathalie Cadieux, Aeras Global TB Vaccine Foundation) is a shuttle vector containing E . coli and mycobacterial origins of replication, a hygromycin resistance cassette, and the panCD operon driven by the constitutive BCG_3667c promoter; the plasmid complements the panCD auxotrophy in the host BCG Δ panCD strain ensuring the plasmid is stably maintained without antibiotic selection. Turbo635 has been previously used for in vivo imaging of mycobacteria [ 19 ], and Turbo635asv has a C-terminal degradation tag added to promote protein turnover [ 20 ]. The monomeric superfolder (msf) yellow fluorescent protein (YFP) is a derivative of GFP [ 21 ]. An artificial operon was constructed with both genes driven by the Psmyc mycobacterial strong promoter [ 22 ] and cloned into the XbaI-PacI sites in the pCB22 MCS downstream of the panCD operon ( S1 Fig ). The fluorescent BCG reporter strain was obtained by electroporation of the plasmid into BCG Pasteur Δ panCD as previously described [ 23 ].

A dual fluorescent reporter strain of Mtb was also constructed by transforming the same Psmyc Turbo635asv-YFP reporter plasmid into an H37Rv panCD mutant kindly provided by Prof Bill Jacobs, Albert Einstein College of Medicine to create Fluor-Mtb.

Immunisation and challenge

Mice were vaccinated subcutaneously with 1 × 10 4 BCG Pasteur and rested for 4 weeks before challenge. In Mtb challenge groups, each mouse was infected with 1 × 10 3 colony-forming units (CFUs) of strain H37Rv in 35 μl via the intranasal route. For the fluorescent strain challenge, either 5 × 10 6 CFU of FluorBCG or 5 × 10 6 CFU of Fluor-Mtb was injected intradermally (ID) with a 30G insulin needle (Easy Touch, United States of America) into the skin on the dorsal side of the mouse ear. Ears were imaged immediately post-challenge to provide a reading for baseline fluorescence; this was the day 0 time point. Imaging of the ears was repeated at regular intervals until day 28. Four weeks post-challenge with Mtb, the bacterial burden was determined in the lungs and spleen. Lungs and spleen were removed aseptically, homogenised in PBS containing 0.05% Tween 80, and serial dilutions of the organ homogenates were plated on Middlebrook 7H10 agar plates. The number of CFU was enumerated 21 days later. A summary of experiments and treatments is provided in S1 Table .

ChAdOx1.PPE15 strain and immunisations

ChAdOx1.PPE15 was a kind gift from Dr Elena Stylianou and Prof Helen McShane, University of Oxford [ 24 ]. Female BALB/c mice were immunised intranasally with 1 × 10 8 infectious units of the virus in a final volume of 35 μl. A summary of experiments and treatments is provided in S1 Table .

Portable imager

The portable imager instrument was designed to provide quantitative images of the fluorophore emission from intradermal injections of FluorBCG in the mouse ears. FluorBCG is excited by green (Cree XPE2, Green) and amber (Cree XPE2, Amber) LEDs, which are chosen to have emission maxima near the peak excitation of the YFP and Turbo635 fluorophores, respectively. The LEDs broad band emissions are narrowed using dichroic filters centred at 505 nm (YFP) and 590 nm (Turbo-365) with passbands of 20 nm. The Nikon D5300 camera exposure settings are typically ISO 400 and shutter speed 1/30 second. The images are captured in RAW format and then are converted to 48-bit TIFF images for quantitation. The images are analysed using custom software that integrates the fluorescent intensities over a user-specified region, typically at 4 mm circular region centred on the intradermal injection location.

Construction details of the portable imager

The imager consists of a Nikon 5300 SLR colour camera with a 24.2-megapixel sensor with a 3.89-μm pixel pitch, and a 14-bit resolution depth. The image is formed on the camera sensor using an image relay system consisting of a 75-mm focal length, 25-mm diameter objective lens and a 100-mm focal length, 50-mm diameter tube lens. The field of view of the imaging system is approximately 18 mm by 12 mm. The mouse ear is located at the focus of the objective lens and the camera sensor is located at the focal plane of the tube lens. The mouse is located in a sealed chamber with a transparent cover. The mouse ears are gently held flat in the chamber and are positioned to be at the image plane of the camera. The filtered LED broad band emission of YFP (505 nm) and Turbo635 (590 nm) is partially collimated by a 25-mm focal length, 25-mm diameter lens located approximately 25 mm from the LED source. Typical LED intensities at the image plane are 20 mW/cm 2 at 505 nm and 25 mW/cm 2 at 590 nm. The fluorescent emission from the bacteria passes through a dichroic filter placed between the objective lens and the tube lens with passbands centred at 542 nm and 639 nm, with passband widths of 27 nm and 42 nm, respectively.

Python pipeline

A semiautomated, user-friendly python pipeline was created for data analysis. The pipeline requires python 3.7.0 to be installed and packages: numpy 1.20.1, matplotlib 3.0.2, pandas 0.23.4, scipy 1.6.2, and xlsxwriter 1.3.8. Inputs for the pipeline are the text files generated from the custom-built imager containing the average intensity for YFP and Turbo635 fluorescence. The imager software has established analytical routines for measuring the average fluorescence over a defined area and storing this information as text files rather than images, allowing for a less memory intensive pipeline input. It is possible to collect background measurements of fluorescence and subtract these values from the measurements of the injected area to decrease background noise. While different visualisations can be generated using the pipeline, the initial processing steps are consistent. First, as images of each ear are taken separately, these fluorescence intensity values are averaged for each mouse and the standard deviation for all mice at each time point is calculated. The pipeline can visualise any number of mice and time points but assumes there is a maximum of 2 fluorescence colours. In addition to plotting the average fluorescence intensity value for all mice at each time point, the pipeline can also plot the normalised average values, where each time point after the first recorded fluorescence value is calculated as a percentage of the initial value. The code generated for the python pipeline is available here: https://doi.org/10.5281/zenodo.12781429 .

Isolation of cells from murine ear

Mouse ears were removed postmortem using dissecting scissors and cut into small pieces to facilitate tissue digestion. The tissue pieces were incubated in a digestion cocktail of 300 μg/ml Liberase TM (Roche, UK) and 50 U/ml of DNaseI (Sigma-Aldrich, UK) at 37°C with slow agitation for 90 min [ 25 ]. The digested skin fragments were passed through a nylon 100 μm cell strainer (Falcon, Thermo Fisher Scientific, UK) to obtain single-cell suspensions. Cell viability was >90% as determined by Trypan Blue (Sigma-Aldrich, UK) exclusion.

Flow cytometry and cell sorting

Red blood cells were lysed using RBC lysing buffer (Sigma-Aldrich, UK). Cells were subsequently washed, enumerated, and the cell concentration adjusted to 1 × 10 7 cells/ml in PBS (Thermo Fisher Scientific, UK). The single-cell suspension was incubated with TruStain FcX anti-mouse CD16/CD32 antibody (BioLegend, UK) for 10 min at 4°C. Following Fc block, cells were stained using horizon fixable viability stain 510 (Becton Dickinson, UK) for 15 min at room temperature, washed, and then resuspended in staining buffer (PBS +1 mM EDTA (Invitrogen, UK) +0.1% BSA (SigmaAldrich, UK)). Cells were then surface stained with fluorochrome-conjugated antibodies summarised in Table 1 . Following antibody staining, cells were washed, resuspended in PBS, filtered through a Falcon tube with an integrated cell strainer (Fisher Scientific, UK) before being analysed and sorted using the FACSAria III (Becton Dickinson, UK) cell sorter housed in a biosafety cabinet. Flow cytometry data were acquired using the FACSDiva software. The gating strategy used to identify the immune subsets in the ear are presented as supporting data ( S5 Fig ). Post-acquisition analysis was performed using FlowJo 10.7.1 (Becton Dickinson).

• PPT PowerPoint slide
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All antibodies were purchased from BioLegend, UK.

https://doi.org/10.1371/journal.pbio.3002766.t001

Statistical analysis

To assess the efficacy of the skin challenge, 2 nonlinear mixed effect models were built to, empirically, describe the decline of fluorescence over time. The model was based on data collected from 5 experiments with a total of 100 mice ( S1 Table ), including 45 vaccinated with BCG, 45 unvaccinated, and 10 vaccinated with ChAdOx1.PPE15. Eighty mice received the FluorBCG reporter, and 20 Fluor-Mtb. There were 1,030 observations in total. Among these, 455 observations were from unvaccinated mice, 455 observations were from BCG-vaccinated mice, and 120 observations were from ChAdOx1.PPE15-vaccinated mice. Forty-five mice underwent pulmonary challenge with Mtb. This subset included 20 mice vaccinated with BCG, 20 unvaccinated mice, and 5 mice vaccinated with ChAdOx1.PPE15. The median and range of log10_fluoroscence from the YFP reporter was 8.754 (min: 6.326, max: 9.476). Similarly, for the Turbo635 reporter, the median and range was 7.575 (min: 6.847, max: 8.836). Twenty animals received the Fluor-Mtb reporter, of which 10 were vaccinated with BCG, and 10 unvaccinated. A total of 220 observations were collected from the Fluor-Mtb reporter animals.

Separate models were developed for each fluorophore using log transformed data. Different structural models were evaluated (Eqs 1 and 2 ) to find a base structural model to empirically describe the 2 endpoints.

An additive residual error model was used. Thereafter, all combinations of inter-animal variability (IAV) were explored on the different parameters of the base model. The individual parameters were assumed to be log-normally distributed and covariance between different IAV were explored. Furthermore, Box-cox transformation of the IAV was evaluated on all parameters.

The final base model was taken forward to study the effects of available covariates (vaccination status, pulmonary challenge status, reporter, and baseline fluorescence) using stepwise-covariate modelling (SCM) [ 26 ]. The SCM was configured to include a covariate on a parameter if it would result in an objective function value (OFV) drop corresponding to a statistical significance change of p ≤ 0.05 during the forward step. For the backward step, it was configured to only remove covariates if the removal corresponded to a statistically significant increase of the OFV using a stricter criterion of p ≤ 0.01. Only the reporter covariate was allowed to be implemented on the first intercept parameter, while all covariates were allowed to be implemented on the remaining parameters.

Parameter estimation was performed using nonlinear mixed effects modelling in NONMEM (version 7.5.1; ICON plc, North America, Gaithersburg) [ 27 ]; the first-order conditional estimation method with interaction (FOCEI) was used [ 28 ]. Perl-speaks-NONMEM (PsN) (version 5.3.1, Department of Pharmacy, Uppsala University, Sweden) was utilised to run models, generate visual predictive checks (VPCs), and SCM runs [ 29 ].

Model evaluation was performed by comparing the OFV of nested hierarchical models, where a decrease in OFV of 3.84 can be considered statistically significant at a 5% level for one degree of freedom change using a χ 2 distribution. In addition, stratified VPCs, goodness of fit plots, precision of model parameters, shrinkage, scientific plausibility, and model stability were considered in the model selection and evaluation procedure. Graphical and numerical analysis of the data was performed in R (version 4.2.3, R Foundation for Statistical Computing, Vienna, Austria). All model evaluation plots were generated in R package Xpose4 (version 4.7.2; Department of Pharmacy, Uppsala University, Sweden). Documentation and comparison between models were performed using Pirana (Version 23.1.1, build 1, Certara, Princeton, USA).

The final model for each fluorophore was used to simulate the typical predictions (no IAV or residual error) over 30 days to visualise the covariate effects found on the measured endpoints fluorescence over time.

Fluorophore selection

There are 2 issues to consider when imaging fluorescent reporters in vertebrates. Firstly, the ability of light to penetrate tissue is a function of its wavelength, with red light above 600 nm showing less light absorption by haemoglobin [ 30 ]. Secondly, fluorescent proteins are stable, so signals take time to decay after the cell stops producing the fluorophore. To address these issues, we decided to construct a dual reporter strain, FluorBCG, including an unstable version of a red fluorophore that would ensure tissue penetration and be degraded faster as the bacteria stop growing and die, plus a stable fluorophore protein as an additional marker of bacteria should degradation of the unstable fluorophore take it below detectable levels. We surveyed the available fluorophores for those reported to be bright and stable in mycobacteria. We have previously demonstrated the utility of the red fluorophore Turbo635 for imaging in murine systems [ 19 ] and made an unstable derivative by the addition of the tripeptide ASV to the C-terminus [ 20 ]. We selected a YFP reported as bright and stable in mycobacteria [ 21 ]. A YFP is a monomeric superfolder derivative of GFP that folds faster and more efficiently with superior solubility and brightness [ 31 ]. Fig 1A shows each fluorophore was detected by the custom imager, but Turbo635ASV signals were noticeably dimmer. Quantification showed 10-fold less Turbo635ASV RFUs compared to YFP ( S2 Fig ).

( A ) Experimental schema ( B ) and ( D ) raw YFP fluorescence ( C ) and ( E ) normalised YFP fluorescence from both control and BCG vaccinated mice post-ID skin challenge with fluorescent BCG (FluorBCG). Representative data from 2 experiments are shown. Data represent mean fluorescence ± SD from n = 5 mice (average of 2 ears per mouse). The data underlying this figure can be found in S1 Data . BCG, bacille Calmette-Guérin; ID, intradermal; YFP, yellow fluorescent protein.

https://doi.org/10.1371/journal.pbio.3002766.g001

Utilising fluorescence output as a measure of vaccine efficacy in a murine skin challenge model

We chose the skin of the mouse ear as a suitable model to establish proof-of-concept for noninvasive monitoring of vaccine responses using a fluorescence-based readout. The mouse ear is thin with little fur or pigmentation, making it ideal for imaging and avoiding the autofluorescence associated with fur. A dose escalation study was carried out to determine the bacterial dose that would provide the optimum signal to noise ratio for imaging. Female BALB/c mice were dosed with 3 different concentrations of fluorescent BCG (FluorBCG): low dose of 5 × 10 4 CFU, mid-dose of 5 × 10 5 CFU, and high dose of 5 × 10 6 CFU. The high dose inoculum provided the optimum signal to noise ratio for YFP and was well tolerated in mice with no adverse effects ( S2 Fig ). Female BALB/c mice were then vaccinated with BCG and 4 weeks later challenged ID in each ear with 5 × 10 6 CFU of FluorBCG. Control mice were unvaccinated. Fluorescence readings were taken at the indicated time points until 28 days post-challenge ( Fig 1A ). A time-course experiment captures the YFP fluorescence dynamics observed in the control and BCG-vaccinated groups ( Fig 1B and 1D ). Data were normalised to day 0 to allow for operator-dependent variations in the initial intradermal inoculation in the ear ( Fig 1C and 1E ). The initial YFP readout (days 0 to 2) was similar in both vaccinated and control groups. From day 6 to day 21, there is a marked difference in groups, with a lower YFP readout in the vaccinated compared to control mice. After day 21, the YFP output declines in both vaccinated and control groups ( Fig 1B–1E ). In the control group, the increase in YFP fluorescence on day 5 and day 6 could represent a short growth period. In contrast, in the BCG-vaccinated group, the fluorescence readout declines throughout the measurement period. There are some variations in signal, but these are mirrored in both vaccinated and control groups suggesting a measurement anomaly on those days. The YFP fluorescence kinetics in the vaccinated and control groups are paralleled in the Turbo635 output, with the only major difference being a 10-fold decrease in relative fluorescence units (RFUs) ( S3 Fig ).

Fluorescence output related to viable BCG burden in the ears

Fluorescent proteins have relatively long half-lives, which could result in false-positive signals from residual fluorescent proteins when bacteria are no longer viable. To investigate BCG replication kinetics with fluorescence output, a time-course experiment was performed measuring the YFP readout ( Fig 2A and 2B ) and quantifying bacterial load ( Fig 2C and 2D ) over 21 days. Both vaccinated and control mice had similar fluorescence readout on days 0 and 2; however, BCG-vaccinated mice displayed lower YFP output between days 7 and 21 ( Fig 2A ). The trend was mirrored in the readout from the Turbo635 channel ( S4 Fig ). The effect of BCG immunisation is more evident post-normalisation, with the fluorescence readout being lower in the vaccinated mice in comparison to the controls ( Fig 2B ).

Mice were imaged and fluorescence from ( A ) YFP channels were quantified and normalised to day 0 ( B ) following intradermal skin challenge with fluorescent BCG. Data represent mean fluorescence ± SD from n = 5 mice. The bacterial load was quantified in the ears ( C ) and in the lymph nodes ( D ). The ears and proximal auricular draining lymph nodes from n = 5 mice were pooled, and CFUs were quantified at various time points. Data from one of 2 experiments are shown ( A and B ) and average of 2 experiments ( C and D ). Error bars represent mean ± SD. The data underlying this figure can be found in S1 Data . BCG, bacille Calmette-Guérin; CFU, colony-forming unit; YFP, yellow fluorescent protein.

https://doi.org/10.1371/journal.pbio.3002766.g002

To link bacterial viability with fluorescence output, bacterial numbers were enumerated in the ear at days 2, 7, and 21 post-challenge ( Fig 2C ). There is a decrease in the CFUs after day 2, with a further decrease in the vaccinated animals between days 7 and 21, which parallels the onset of adaptive immunity.

It is well documented that BCG is trafficked by host cells from the site of injection to the draining lymph nodes [ 32 – 37 ].To confirm movement of FluorBCG from the ear, bacterial load was quantified in the proximal auricular draining lymph nodes at day 21 post-challenge and showed that the BCG-vaccinated mice had lower bacteria load than the control group, although this was not statistically significant ( Fig 2D ).

Antigen presenting cells dominate the local immune environment in the mouse ear following BCG vaccination

To study the cellular milieu in mouse ears after FluorBCG challenge, a panel of defined markers was used to characterise the different cellular subsets by flow cytometry. To obtain single-cell suspensions, mouse ears were enzymatically digested and passed through a cell strainer to obtain viable cells. The gating strategy used for flow cytometry was adapted from Yu and colleagues‘ study ( S5 Fig ) [ 38 ]. The ear skin consists of several types of innate immune cells including neutrophils, macrophages, dendritic cells, T cells, and mast cells. The local environment of the mouse ear at day 7 following FluorBCG challenge of control animals is dominated by neutrophils, Langerhans cells, and macrophages ( Fig 3A ). In response to intradermal injection of FluorBCG in control animals, neutrophils in the ear increased to a maximum of 5.79% on day 7, decreasing to 1.74% by day 14 ( Fig 3A and 3B ). In vaccinated mice challenged with FluorBCG, the frequency of neutrophils at day 7 (3.13%) is lower in comparison to control animals (5.79%), dropping further by day 14 ( Fig 3A and 3B ). The marker Langerin (CD207) identifies the main subsets of dendritic cells that reside in the mouse ear. Langerhans cells are CD207 + , and, although conventionally thought of as belonging to the dendritic cell cohort, recent evidence has redefined these cells to be more like tissue-resident macrophages that have acquired dendritic cell-like functions. Langerhans cells possess both in situ functions and migratory functions that promote antigen presentation and T cell priming [ 39 ]. A high frequency of Langerhans cells is present in both control and vaccinated mice with an increase overall in vaccinated mice between day 7and day 14 ( Fig 3A and 3B ). Localisation of Langerhans cells in the mouse ear corroborates the known prevalence of this subset in the skin epidermis [ 39 ]. Another important subset of cells in the skin are the dermal dendritic cells, which are subdivided into CD207 − CD103 +/− and CD207 + CD103 + . Both these subsets of dendritic cells are up-regulated in response to BCG vaccination, with the vaccinated mice exhibiting a large influx of CD207 + dermal dendritic cells ( Fig 3A and 3B ) in comparison to the control mice. In response to BCG vaccination, there is an influx of macrophages that persist in the skin until day 14 ( Fig 3A and 3B ).

Mice were immunised with BCG and post-4-week rest, mice were challenged ID in the ear with FluorBCG and cellular infiltrates at the site of challenge were measured at day 7 ( A ) and day 14 ( B ). Gates in contour plots contains single-cell populations—neutrophils (CD45 + Ly6G + ), dendritic cells subdivided into Langerhans cells* (CD45 + MHC-II + CD207 + CD103 − ), dermal DCs (CD45 + MHC-II + CD207 − CD103 + ) and dermal langerin + DCs (CD45 + MHC-II + CD207 + CD103 + ); monocytes (CD45 + CD11b + CD64 int ) and macrophages (CD45 + MHC-II + F4/80 + CD64 + ). *Langerhans cells, although classified under the dendritic cells group here, are more like tissue-resident macrophages, which acquire a phenotype-like dendritic cells. The data underlying this figure can be found at https://doi.org/10.5281/zenodo.12794251 . BCG, bacille Calmette-Guérin; DC, dendritic cell; ID, intradermally.

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These results suggest that the response to FluorBCG intradermal challenge leads to an inflammatory influx dominated by migratory dendritic cells, neutrophils, and macrophages.

Next, we investigated if viable FluorBCG can be recovered from selected subsets of immune cells from the ear. Neutrophils, macrophages, and dendritic cells were gated for the presence of YFP + BCG cells, single-cell sorted using a FACS Aria sorter, and plated to determine CFUs. Neutrophils (CD45 + Ly6G + ) accounted for a minor population of cells in the ear ( Fig 4A and 4B ) but had a high BCG burden, which was comparable to the macrophage and dendritic cells present in the ear at day 7 ( Fig 4E ) with a reduced bacterial load on day 14 ( Fig 4F ). The reduction in viable bacterial numbers in neutrophils noted at day 14 ( Fig 4F ) does not match with the expression of YFP + bacteria in neutrophils ( Fig 4D ); however, the frequency of YFP+ neutrophils does match with frequency of YFP+ macrophages and dendritic cell subsets. In contrast, macrophages were the dominant population in the mouse ear ( Fig 4A and 4B ), but only 12% of this subset carried YFP + BCG ( Fig 4C and 4D ). Overall, reduction of the viable bacterial load was observed by day 14 in neutrophils, macrophages, and dendritic cells ( Fig 4F ).

Bar graphs ( A , B ) depicting immune cell profiles of neutrophils (expressed as a frequency of CD45+ cells), macrophages, and dendritic cells (expressed as a frequency of the myeloid cell population). Immune cellular subsets were further gated on YFP+ cells to isolate populations that phagocytosed fluorescent BCG ( C , D ). These gated populations were sorted and plated to quantify viable BCG ( E , F ). Flow cytometry data are an average of 2 independent experiments with n = 5 mice per group. Error bars represent mean ± SD. The data underlying this figure can be found in S1 Data . BCG, bacille Calmette-Guérin; CFU, colony-forming unit; YFP, yellow fluorescent protein.

https://doi.org/10.1371/journal.pbio.3002766.g004

Fluorescence readout from the skin relates to protective immunity in the lung

The ideal route of challenge in humans would be the pulmonary route as it mimics the natural route of infection with Mtb but detecting challenge bacteria in vivo in the lung poses obstacles. Exposure via the skin is a more feasible approach, and detection of the bacteria can be achieved by noninvasive methods. It has been reported that the efficacy of BCG vaccination against intradermal BCG skin challenge has comparable outcomes to an aerosol Mtb challenge, highlighting the viability of using a BCG-based skin challenge as an alternative to a pulmonary challenge [ 34 ]. To test whether the reduction in skin fluorescence in response to BCG vaccination matched an immune response in the lungs, mice were BCG vaccinated, challenged with FluorBCG in the ear, and given a pulmonary Mtb challenge by the intranasal route. In response to BCG vaccination, there was a reduced YFP fluorescence readout overall from the skin of vaccinated mice ( Fig 5A and 5B ) and Turbo635 ( S6 Fig ). In the lungs and spleen, BCG vaccination significantly reduced the bacterial burden in comparison to the control group ( Fig 5C and 5D ). The group of control mice that received a fluorescent BCG challenge (Unvaccinated_FluorBCG_H37Rv) in the ear had a statistically lower bacterial load in the lungs and spleen in comparison to the control mice (Unvaccinated_H37Rv) that did not receive a fluorescent BCG inoculation ( Fig 5C and 5D ), suggesting the single-dose BCG challenge in the ear affords a degree of protection. The results show that the reduced fluorescence readout from FluorBCG in the skin provides a sensitive and reproducible measure of a relevant biological effect that is shown to reflect traditional measures of TB vaccine efficacy in the lung.

BALB/c mice were immunised with a single dose of BCG and after a 4-week rest period, mice were either challenged ID in the ear with 5 × 10 6 CFU of FluorBCG and IN with 1 × 10 3 H37Rv or only challenged IN with 1 × 10 3 H37Rv (H37Rv). Fluorescence intensities from the site of ID challenge in the ear are represented as raw outputs from the YFP channel ( A ) and fluorescence values normalised to day 0, YFP ( B ). Lungs and spleen were harvested 4 weeks post-challenge and processed to quantify the bacterial burden ( C and D ). Representative data from duplicate experiments are displayed. Data represent mean ± SD from n = 5 mice. The data underlying this figure can be found in S1 Data . BCG, bacille Calmette-Guérin; CFU, colony-forming unit; ID, intradermally; IN, intranasally; YFP, yellow fluorescent protein.

https://doi.org/10.1371/journal.pbio.3002766.g005

Evaluating the skin challenge model using a novel TB subunit vaccine candidate

The skin challenge model was utilised to assess the efficacy of a novel vaccine candidate, ChAdOx1.PPE15. This subunit vaccine candidate comprises a replication-deficient, recombinant chimpanzee adenovirus vector (ChAdOx1), which expresses the mycobacterial antigen PPE15 [ 24 ]. Subunit vaccines are primarily designed to boost protective immune responses conferred by the BCG vaccine, but it has been shown that administering ChAdOx1.PPE15 as a single intranasal dose followed by a Mtb challenge resulted in a significant reduction of bacteria load in the lungs compared to controls [ 16 , 24 ]. We immunised BALB/c mice with a single intranasal dose of ChAdOx1.PPE15 and 4 weeks later challenged them with intranasal Mtb and intradermal FluorBCG in the ear. The fluorescence output from immunised mice was stable until day 16 after which there was a decline in the YFP readout. A diminished fluorescence output from the ChAdOx1.PPE15 immunised group in comparison to the control group was noted ( Fig 6A ). However, the YFP ( Fig 6B ) and Turbo635 ( S7 Fig ) normalisation data did not indicate a vaccine effect in mice immunised with only ChAdOx1.PPE15 ( Fig 6B ). Normalisation of data to day 0 is critical as it removes any confounding variables such as differences in initial dosing. Mice immunised with ChAdOx1.PPE15 also failed to demonstrate a protective effect in the lungs and spleen ( Fig 6C and 6D ), which corroborates the fluorescence data ( Fig 6A and 6B ), and mirrors the poor responses reported [ 24 ]. A reduction in bacterial load in both lungs and spleen were primarily noted in the groups that received a dose of FluorBCG in the ears ( Fig 6C and 6D ). Comparison of ChAdOx1.PPE15 vaccinated with BCG vaccinated groups showed no overall difference in terms of RFU ( S10 Fig ). BCG performed better than ChAdOx1.PPE15 at controlling Mtb H37Rv CFU in spleen but not lungs ( S11 Fig , panels A and B); ChAdOx1.PPE15 showed better control of lung CFU but not spleen CFU in the groups that also received intradermal challenge with FluorBCG ( S11 Fig , panels C and D).

BALB/c mice were immunised with a single intranasal dose of ChAdOx1.PPE15, and, after a 4-week rest period, mice were either challenged ID in the ear with 5 × 10 6 CFU of FluorBCG and challenged IN with 1 × 10 3 H37Rv (H37Rv). Fluorescence intensities from the site of ID challenge in the ear are represented as raw outputs from the YFP channel ( A ) and fluorescence values normalised to day 0, YFP ( B ). Lungs and spleen were harvested 4 weeks post-challenge and processed to quantify the bacterial burden ( C and D ). Data represent mean ±SD from n = 5 mice. The data underlying this figure can be found in S1 Data . CFU, colony-forming unit; ID, intradermally; IN, intranasally; YFP, yellow fluorescent protein.

https://doi.org/10.1371/journal.pbio.3002766.g006

Statistical analysis of aggregated experimental data

We built nonlinear mixed-effects models of the aggregated data collected from 5 independent experiments. Based on the graphical and numerical analysis, the baseline fluorescence of YFP in 1 mouse was excluded as it was approximately 100 times lower compared to the median value.

The final model for YFP fluorescence consisted of a biexponential structural model that provided a better fit to the data than a monoexponential model. In addition, a biexponential model was deemed more suitable to describe the initial rapid decline of fluorescence for some groups of mice, which was confirmed by comparing the ability of the mono- and biexponentials models to describe the data through stratified VPCs. IAV on the second intercept and the slope were statistically significant; no covariance between IAV parameters were supported by the data. Box-cox transformation on individual error (eta) distributions were not supported. An additive residual error model was found to be adequate. Covariates in the SCM procedure were implemented as percentage change of typical parameter values. Statistically significant covariates were vaccination (1.52% decrease, p < 0.001), baseline fluorescence (10 th-quantile = 2.2% decrease and 90 th-quantile = 1.36% increase, p < 0.001), Mtb pulmonary challenge (1.31% increase, p < 0.001), and the reporter (3.43% decrease, p < 0.001) on the second intercept, reporter on first intercept (24.2%, increase, p < 0.001), and reporter on the second SLOPE (52.4% decrease, p < 0.001). However, the covariate effect of vaccination on the second intercept was not statistically significantly different from unvaccinated for the ChAdOx1.PPE15 vaccine, and, as such, the group of mice treated with ChAdOx1.PPE15 was treated as unvaccinated.

The structural base model for Turbo635 fluorescence also consisted of a biexponential model. IAV on the first and second intercept were statistically significant, and no covariance between IAV parameters was found to be significant. Box-cox transformation on ETA distributions were not supported, and an additive residual error model was found to be adequate. Statistically significant covariates were vaccination (1.47% decrease, p < 0.001), baseline fluorescence (10 th-quantile = 2.3% decrease and 90 th-quantile = 3.11% increase, p < 0.001), and reporter (4.93% decrease, p < 0.001) on the second intercept, reporter (12.3% increase, p < 0.001) on the first intercept, reporter (45.2% decrease, p < 0.001) and baseline fluorescence (10 th-quantile = 22.3% decrease and 90 th-quantile = 30.3% increase, p < 0.001) on the second slope. Similar to the YFP model, the ChAdOx1.PPE15-vaccinated groups was not statistically significantly different from unvaccinated mice.

The final models were considered suitable to describe the data in all different experimental groups based on stratified VPCs ( S8 and S9 Figs). In Figs 7 and 8 , typical predictions can be found for all the possible covariate combinations for both the models. The final parameter estimates for the 2 models are provided in Tables 2 and 3 .

Log10 RFU based on Turbo635 versus time for different combinations of covariate effects. The typical predictions were performed using the final model for Turbo635 (one prediction of the dependent variable per time point, using a fine time grid, over 28 days). This is done for each possible covariate effect. The plots can be recreated by using the parameter values in Table 2 and the covariate relationships described for Turbo635 in the results section.

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Log10 RFU based on YFP versus time for different combinations of covariate effects. The typical predictions were performed using the final model for YFP (one prediction of the dependent variable per time point, using a fine time grid, over 28 days). This is done for each possible covariate effect. The plots can be recreated by using the parameter values in Table 3 and the covariate relationships described for YFP in the results section.

https://doi.org/10.1371/journal.pbio.3002766.g008

https://doi.org/10.1371/journal.pbio.3002766.t002

https://doi.org/10.1371/journal.pbio.3002766.t003

We report on a proof-of-concept noninvasive, skin-based challenge model for detection and quantification of relevant biological activity reflecting traditional measures of TB vaccine efficacy in the mouse (viable bacterial loads in the lung). Although the utility of the BCG skin challenge model has previously been demonstrated in humans [ 16 ], to our knowledge, this is the first reported noninvasive, skin-based challenge study to demonstrate the feasibility of using a fluorescence-based readout as a measure of vaccine efficacy. We also show that the fluorescence output from the skin after challenge with fluorescent BCG serves as a reliable indicator of vaccine-induced immunity in the lung. The fluorescence-based quantitative measurement of protective efficacy described in this study solves several problems associated with vaccine assessment in humans. It uses a proven safe challenge organism, and it allows for repeated noninvasive measurement at the challenge site, providing high-quality temporal data on vaccine responses in vivo. It uses a low-cost imaging platform that can be used in the field, and the technology and approach have potential applications in other areas.

The maximal difference in fluorescence signal between the control and vaccinated groups were observed between day 4 and day 21, providing a window in which new vaccine candidates can be assessed for their efficacy using this noninvasive approach. This defined interval for determining vaccine efficacy will cover the period of collective input from both the innate and adaptive immune system, thus providing a more comprehensive output of vaccine protective immunity. This time interval is consistent with the optimum period for detecting live BCG in the skin [ 34 , 40 ]. Although in this first-generation approach, there was some variability in the fluorescence readout, making it harder to achieve statistical significance at specific time points. However, a trend for reduced fluorescence output is noted in the groups of mice vaccinated with BCG compared to control. The relatively long half-life of fluorescent proteins does not seem to impact their utility in this system. The addition of the tripeptide ASV degradation tag to Turbo635 did not change the fluorescent dynamics in this system over time compared to YFP but instead reduced the overall fluorescent signal. Whether Turbo635-ASV is advantageous in other systems remains to be tested and there is potential for fluorescence optimisation.

We used the dorsal surface of the mouse ear as the site for intradermal injection and imaging. Use of the mouse ear as a surrogate for intradermal vaccination site has several advantages: (1) autofluorescence is low, so background fluorescence levels are negligible; (2) ease of accessibility; and (3) repeated imaging of the same area requires no advance preparation, such as hair removal. Using a microfine, 30G insulin needle, we could precisely deliver the dose needed while minimising disruption of the local microenvironment.

Live BCG was detectable in the skin till day 21, although BCG counts were not quantified at later time points in this study. In comparison, Minassian and colleagues reported that BCG could be detected until 12 weeks in the mouse ear by culture. However, a significant decline in BCG CFUs was observed after 4 weeks post-immunisation [ 34 ]. Furthermore, in a clinical study, live BCG could be cultured from skin punch biopsy specimens up to 4 weeks [ 16 ] and 2 months [ 41 ] after the BCG challenge. We observed a slight increase in YFP output from unvaccinated mice between day 2 and day 7; however, this increase in fluorescence readout did not correspond to an increase in CFU counts between day 2 and day 7. Hence, we conclude there was no replication of BCG at the immunisation site between day 2 and day 7. However, a delicate equilibrium may exist between bacterial replication, migration, and bacterial death in the ears, a critical point that needs to be addressed in future experiments. Chambers and colleagues [ 35 ] demonstrated that early accumulation of inflammatory cells at the immunisation site reflected the clearance of live BCG from the footpad of mice to the draining lymph nodes. In contrast, dead BCG persisted at the immunisation site for extended periods [ 35 ]. We have shown that bacteria migrate out of the ears to the draining lymph nodes with no significant difference in CFU counts between vaccinated and control mice at day 21 post-FluorBCG challenge in the ear. A decline in CFUs was observed by day 7 in both control and BCG-vaccinated mice. By day 21, a reduction in bacterial load was noticed in mice immunised with BCG, which matches the onset of the adaptive immune response and likely to involve more efficient priming of T cells in BCG-vaccinated mice [ 36 ].

Two empirical models were successfully developed to describe both fluorophores over time with good predictive performance for both FluorBCG and Fluor-Mtb. For Turbo635, the impact of covariate effects on the fluorescence decay was investigated. The vaccinated population with the FluorBCG reporter exhibits a decline compared to the unvaccinated group ( Fig 7A ), in addition to reaching a lower fluorescence level. For the Fluor-Mtb reporter, both the unvaccinated and vaccinated groups demonstrate a decline compared to the FluorBCG reporter, with the vaccinated group reaching a lower fluorescence level ( Fig 7C ) and displaying a decline compared to the unvaccinated group. For the identified covariate effects using YFP data, it was observed that the vaccinated population with the FluorBCG reporter exhibits a decline compared to the unvaccinated group and reaching a lower fluorescence level ( Fig 8C ). Additionally, the effect of pulmonary infection with Mtb leads to an earlier stabilisation of fluorescence levels and a decline for both unvaccinated and vaccinated ( Fig 8D and 8B ). In the case of the Fluor-Mtb reporter, both the vaccinated and unvaccinated groups show a decline compared to the FluorBCG reporter, with the vaccinated group ( Fig 8G and 8H ) reaching an even lower fluorescence level and displaying a decline compared to the unvaccinated group ( Fig 8E and 8F ). Similar to BCG, the effect of pulmonary infection results leads to an earlier stabilisation of fluorescence levels and a decline for both unvaccinated and vaccinated.

In the typical prediction plots, a rapid decline for the Fluor-Mtb-reporter was also noticed, and from Tables 2 and 3 , there is a difference in parameter values between the 2 reporters (FluorBCG and Fluor-Mtb). This could be attributed to the Fluor-Mtb reporter being more antigenically complete, thereby facilitating a quicker initial immunological response. However, the reasons behind the reduction in the second intercept value for the YFP reporter due to pulmonary challenge are uncertain. To the best of our knowledge, there are no physiological explanations for this phenomenon compared to the Turbo635 reporter. It is possible that there are minor outlier values within the experiments involving pulmonary infection or that the inclusion of the Mtb-challenge (MTBCH) factor accounts for variances stemming from factors other than the actual pulmonary infection in the YFP model. The effect of the baseline fluorescence was statistically significant for both the second slope and the second intercept in the case of Turbo635, while only for the second intercept in the case of YFP. This discrepancy may possibly be attributed to a higher variance in the readouts from the Turb635 fluorophore. The empirical models described the data well. However, the evaluations of model performance were performed on the same data that were used for model building. The typical prediction plots (Figs 7 and 8 ) are specific to the mouse model, which has been shown to be a useful screening tool for early-stage TB vaccines, and, although these specific data have yet to be confirmed in human studies, current evidence is supportive. The models analysed all data over time from all experiments simultaneously using a mixed effects approach, which is suitable for longitudinal datasets. An alternative would be to select a datapoint of interest and perform group-wise statistical comparisons. However, such an approach is not as informative and does not utilise the full set of generated experimental data.

Our study demonstrates the presence of migratory immune cells, particularly an accumulation of dendritic cells at the site of FluorBCG administration in both control and BCG-vaccinated mice. The skin milieu is complex with multiple migratory immune cell subsets [ 42 , 43 ], so we utilised additional cell surface markers to study the different populations of prominent dendritic cell populations in the ear. In this study, the marked decrease in the fluorescence readout in BCG-vaccinated mice from day 7 likely corresponds to an increase in the presence of migratory immune cells such as dermal langerin+ dendritic cells and, to a lesser extent, the Langerhans cells in the ear. BCG vaccination influences the early appearance of migratory dendritic cells [ 35 ]. Migratory immune cells are likely to phagocytose and carry BCG to the draining lymph nodes, although the migration of free bacteria to the lymph nodes cannot be discounted. The dermal langerin+ dendritic cells are known to migrate to lymph nodes in response to infection and inflammation and presents antigens to T cells [ 44 ]. These cells are also early responders and are continually replenished from the blood [ 45 ]. The presence of a large pool of FluorBCG at the administration site is likely to indicate continuous migration and replenishment of dendritic cells, or persistence in the ear. We observed that the population of dermal langerin+ dendritic cells are maintained in the ear until day 14. Neutrophils were observed in both control and vaccinated mouse skin, with the neutrophil population being smaller in the BCG-vaccinated group. Neutrophils are present in the skin under steady-state conditions, and they are part of the early responders to appear in the skin following sterile injury [ 46 ]. Neutrophils can be detected as early as 4 hours following intradermal BCG vaccination [ 33 ]. Neutrophils have also been shown to migrate to the draining lymph nodes in response to specific microbial stimuli [ 47 ]. Although neutrophils were not the dominant population of cells in the ear, they can phagocytose BCG, as the bacterial load in neutrophils was comparable to the bacterial load enumerated from dendritic cells and macrophages on day 7. Abaide and colleagues also reported on the colocalisation of neutrophils with BCG in the ear and demonstrated that neutrophils could phagocytose BCG present in the skin dermis [ 33 ].

By day 14, there was a smaller population of neutrophils present in the ear, with the bacterial load in neutrophils lower than the CFUs in macrophages and dendritic cells. However, the frequency of YFP+ neutrophils present in the skin was similar to YFP+ macrophages and YFP+ dendritic cells. The fluorescence readout is indicative that BCG is present in the neutrophils. However, the lower bacterial load by culture from these cells suggests that at day 14, the neutrophils are predominantly harbouring nonviable BCG. The role of neutrophils in the direct clearance of mycobacteria is not clear [ 33 , 48 – 50 ]; however, they are known to function cooperatively with dendritic cells and macrophages to orchestrate the killing of mycobacteria [ 51 ]. Moorlag and colleagues demonstrated that BCG vaccination could induce trained immunity in neutrophils, which contributed to a more efficient response to C . albicans infection by the up-regulation of reactive oxygen species and enhanced expression of degranulation markers [ 52 ]. Further work needs to be carried out to confirm if neutrophils play a direct role in mycobacterial killing due to increased neutrophil antimicrobial factors or an indirect role by establishing an early inflammatory response following BCG vaccination in our skin challenge model.

The skin-based challenge model described in this study has a unique advantage for detecting and measuring the protective efficacy by a noninvasive method. Furthermore, repeated sampling of the injection site with minimal discomfort could be carried out, eliminating the need for invasive skin sampling procedures. This study demonstrates that the skin-based fluorescence output is a measurable indicator of the vaccine protective response in the lung. A reduction in the fluorescence output from BCG-vaccinated group compared to the control mirrors a significant decrease in bacterial load in both the lungs and spleen of BCG-vaccinated mice. Control mice that received FluorBCG ID in the ear also demonstrate a vaccine effect that was expected and agrees with previously published data [ 36 ]. Additionally, a BCG boosting effect was observed in response to the intradermal administration of FluorBCG in vaccinated mice, which resulted in further reduction of CFU counts in the lungs and spleen. In our study, we have confirmed and extended on the results demonstrating that protection against a skin-based challenge is associated with a protective vaccine response in the lung [ 37 ].

This skin challenge model study is a feasibility study that will ultimately aid the development of a human challenge model for assessment of TB vaccines. Such a model needs to be tested for its utility of measuring responses from vaccines other than BCG. We used a subunit vaccine ChAdOx1.PPE15 to test the skin challenge model with a vaccine other than BCG. In our study, ChAdOx1.PPE15 was not effective in conferring a protective immune response, which was confirmed by both the fluorescence readout from the skin and CFU data from the lungs and spleen of BALB/c mice. This observation agrees with data published by Stylianou and colleagues, demonstrating that in CB6F1 mice, ChAdOx1.PPE15 was only effective in boosting BCG vaccination, and administration of the ChAdOx1.PPE15 vaccine on its own demonstrated a moderate vaccine efficacy in CB6F1 mice but not in BALB/c mice [ 24 ]. Significant protective immune responses were observed in the groups that were administered FluorBCG. BCG is a vaccine that induces strong protective immune responses in mice, so any effects of ChAdOx1.PPE15 will likely to be masked unless it can significantly boost the BCG immune response.

Conclusions

Several animal models exist for TB, but none encompasses the complexity of the spectrum of clinical disease, representing a significant hurdle in translational TB research. For TB, where prognostic animal models are lacking, human challenge trials will be beneficial to identify correlates of immune protection and test vaccine efficacy. TB, a chronic disease caused by a virulent bacterium, does not immediately fit the criteria as a disease for which a human challenge trial can be designed easily. Using alternative approaches to test the efficacy of vaccines is therefore warranted. This skin-based challenge model is one such approach to stratify and select vaccine candidates based on their immune response to accelerate the most relevant candidates along the clinical trial pipeline.

In summary, in this vaccination-challenge model, serially tracking fluorescently labelled bacteria in the skin using a portable imager is a novel noninvasive strategy for measuring vaccine efficacy. This novel detection method has unique advantages for clinical implementation, and this feasibility study establishes the first steps towards developing a safe, tractable, relevant human challenge model for TB.

Supporting information

S1 fig. pcb22-turbo635-asv-yfp plasmid map..

https://doi.org/10.1371/journal.pbio.3002766.s001

S2 Fig. Optimisation of BCG dose.

BALB/c mice were infected ID with 3 concentrations of FluorBCG: FluorBCG_High of 5 × 10 6 CFU, FluorBCG_Mid of 5 × 10 5 CFU, and FluorBCG_Low of 5 × 10 4 CFU. ( A ) Raw YFP fluorescence. ( B ) Raw Turbo-635 fluorescence from unvaccinated mice. Data represent mean fluorescence ± SD from n = 3 mice (mean of 2 ears per mouse). The data underlying this figure can be found in S1 Data . BCG, bacille Calmette-Guérin; CFU, colony-forming unit; ID, intradermally; YFP, yellow fluorescent protein.

https://doi.org/10.1371/journal.pbio.3002766.s002

S3 Fig. Measuring the Turbo-635 signal from the skin in response to a fluorescent BCG challenge.

( A , C ) Raw Turbo-635 fluorescence. ( B , D ) Normalised Turbo-635 fluorescence from both control and vaccinated mice post-ID skin challenge with fluorescent BCG. Representative data from one of 2 experiments are shown. Data represent mean fluorescence ± SD from n = 5 mice (average of 2 ears per mouse). The data underlying this figure can be found in S1 Data .

https://doi.org/10.1371/journal.pbio.3002766.s003

S4 Fig. Turbo-635 fluorescence output from BCG-vaccinated mice.

Mice were imaged and fluorescence from ( A ) Turbo-635 channels were quantified and normalised to day 0 ( B ) following intradermal skin challenge with fluorescent BCG. Data from one of 2 experiments are shown representing mean fluorescence ± SD from n = 5 mice. The data underlying this figure can be found in S1 Data .

https://doi.org/10.1371/journal.pbio.3002766.s004

S5 Fig. Gating strategy for flow cytometry.

Total cells were isolated from murine ear, and single cells were identified using the FSC-A and FSC-H plot. The viability of the skin cell suspension was determined using the SSC-A and viability plot. Viable cells were gated to identify the CD45 + cells and then neutrophils (CD45 + Ly6G + ), dendritic cells subdivided into Langerhans cells* (CD45 + MHC-II + CD207 + CD103 − ), dermal DCs (CD45 + MHC-II + CD207 − CD103 + ) and dermal langerin + DCs (CD45 + MHC-II + CD207 + CD103 + ); monocytes (CD45 + CD11b + CD64 int ) and macrophages (CD45 + MHC-II + F4/80 + CD64 + ).

https://doi.org/10.1371/journal.pbio.3002766.s005

S6 Fig. Fluorescence output from the ear and bacterial burden in the lungs.

BALB/c mice were immunised with a single dose of BCG, and, after a 4-week rest period, mice were challenged ID in the ear with 5 × 10 6 CFU of FluorBCG and IN with 1 × 10 3 H37Rv (FluorBCG+H37Rv). Fluorescence intensities from the site of ID challenge in the ear are represented as raw outputs from the Turbo-635 channel ( A ) and fluorescence values normalised to day 0 ( B ). Representative data from duplicate experiments are displayed. Data represent mean ± SD from n = 5 mice. The data underlying this figure can be found in S1 Data . BCG, bacille Calmette-Guérin; CFU, colony-forming unit; ID, intradermally; IN, intranasally.

https://doi.org/10.1371/journal.pbio.3002766.s006

S7 Fig. Testing the skin challenge model with a TB subunit vaccine.

BALB/c mice were immunised with a single dose of ChAdOx1.PPE15, and, after a 4-week rest period, mice were challenged ID in the ear with 5 × 10 6 CFU of FluorBCG and IN with 1 × 10 3 H37Rv (FluorBCG+H37Rv). Fluorescence intensities from the site of ID challenge in the ear are represented as raw outputs from the Turbo-635 channel ( A ) and fluorescence values normalised to day 0 ( B ). Representative data from duplicate experiments are displayed. Data represent mean ± SD from n = 5 mice. The data underlying this figure can be found in S1 Data . BCG, bacille Calmette-Guérin; CFU, colony-forming unit; ID, intradermally; IN, intranasally.

https://doi.org/10.1371/journal.pbio.3002766.s007

S8 Fig. VPC based on the final model for TURBO635 data stratified by all existing covariate effects based on 2,000 simulations.

The solid line is the median of the observed data, the shaded area is the 95% confidence interval for the median, and the open blue circles are the observations. The VPC was generated in NONMEM using the PsN VPC command. It can be regenerated by utilising the software specified in the statistical analysis method section and the S2 Data .

https://doi.org/10.1371/journal.pbio.3002766.s008

S9 Fig. VPC based on the final model for YFP data stratified by all existing covariate effects based on 2,000 simulations.

The solid line is the median of the observed data, the shaded area is the 95% confidence interval for the median, and the open blue circles are the observations. The VPC was generated in NONMEM using the PsN VPC command. It can be regenerated by utilising the software specified in the statistical analysis method section and the S3 Data .

https://doi.org/10.1371/journal.pbio.3002766.s009

S10 Fig. Measuring the skin fluorescence signal from BCG- and ChAdOx.PPE15-vaccinated mice in response to a fluorescent BCG challenge.

BALB/c mice were either immunised with a single dose of BCG or a single intranasal dose of ChAdOx1.PPE15, and, after a 4-week rest period, mice were challenged ID in the ear with 5 × 10 6 CFU of FluorBCG. ( A , C ) Raw YFP fluorescence. ( E , G ) Raw Turbo-635 fluorescence. ( B , D ) Normalised YFP fluorescence from both BCG- and ChAdOx.PPE15-vaccinated mice post-ID skin challenge with fluorescent BCG ( F , H ) Normalised Turbo-635 fluorescence from both BCG- and ChAdOx.PPE15-vaccinated mice post-ID skin challenge with fluorescent BCG. Data represent the mean fluorescence ± SD from n = 5 mice (an average of 2 ears per mouse). The data underlying this figure can be found in S1 Data . BCG, bacille Calmette-Guérin; CFU, colony-forming unit; ID, intradermally; YFP, yellow fluorescent protein.

https://doi.org/10.1371/journal.pbio.3002766.s010

S11 Fig. Comparing the skin challenge model using a novel vaccine candidate and BCG.

BALB/c mice were either immunised with a single dose of BCG or a single intranasal dose of ChAdOx1.PPE15, and, after a 4-week rest period, mice were either challenged ID in the ear with 5 × 10 6 CFU of FluorBCG and challenged IN with 1 × 10 3 H37Rv (H37Rv) or only challenged with 1 × 10 3 H37Rv (H37Rv). Lungs ( A , C ) and spleen ( B , D ) were harvested 4 weeks post-challenge and processed to quantify the bacterial burden. Data represent mean ± SD from n = 5 mice. * p > 0.05, ** p > 0.01. The data underlying this figure can be found in S1 Data . BCG, bacille Calmette-Guérin; CFU, colony-forming unit; ID, intradermally; IN, intranasally.

https://doi.org/10.1371/journal.pbio.3002766.s011

S1 Table. Summary of experiment names and treatments.

Each experiment had 5 mice per group and was measured for both ears; mean ears are tabulated. VC-2, VC-4, and VC-5 had 12 RFU readings taken over 28 days. Flow1 and Flow2 had 5 RFU readings taken over 21 days and were also used for flow cytometry and bacterial load measurements in ears and lymph nodes.

https://doi.org/10.1371/journal.pbio.3002766.s012

S1 Data. Numerical data for plots in all figures.

https://doi.org/10.1371/journal.pbio.3002766.s013

S2 Data. Details of software and statistical analysis methods to recreate S8 Fig .

https://doi.org/10.1371/journal.pbio.3002766.s014

S3 Data. Details of software and statistical analysis methods to recreate S9 Fig .

https://doi.org/10.1371/journal.pbio.3002766.s015

Acknowledgments

Thanks to the TB-Human Challenge Team for their input and discussions. Special thanks to Barry Walker for his constant support for the project.

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