The Immunological Basis for Immunization Series

Transcription

1 The Immunological Basis for Immunization Series Module 5: Tuberculosis Immunization, Vaccines and Biologicals

2 The Immunological Basis for Immunization Series Module 5: Tuberculosis Immunization, Vaccines and Biologicals

3 WHO Library Cataloguing-in-Publication Data The immunological basis for immunization series: module 5: tuberculosis. (Immunological basis for immunization series ; module 5) 1.Tuberculosis - prevention and control. 2.Tuberculosis -microbiology. 3.Tuberculosis - immunology. 4.BCG vaccine - therapeutic use. 5.BCG Vaccine - adverse effects. 6.Vaccination. I.World Health Organization. II.Series. ISBN (NLM classification: WF 200) World Health Organization 2011 All rights reserved. Publications of the World Health Organization are available on the WHO web site ( or can be purchased from WHO Press, World Health Organization, 20 Avenue Appia, 1211 Geneva 27, Switzerland (tel.: ; fax: ; bookorders@who.int). Requests for permission to reproduce or translate WHO publications whether for sale or for noncommercial distribution should be addressed to WHO Press through the WHO web site ( The designations employed and the presentation of the material in this publication do not imply the expression of any opinion whatsoever on the part of the World Health Organization concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. Dotted lines on maps represent approximate border lines for which there may not yet be full agreement. The mention of specific companies or of certain manufacturers products does not imply that they are endorsed or recommended by the World Health Organization in preference to others of a similar nature that are not mentioned. Errors and omissions excepted, the names of proprietary products are distinguished by initial capital letters. All reasonable precautions have been taken by the World Health Organization to verify the information contained in this publication. However, the published material is being distributed without warranty of any kind, either expressed or implied. The responsibility for the interpretation and use of the material lies with the reader. In no event shall the World Health Organization be liable for damages arising from its use.

4 The Department of Immunization, Vaccines and Biologicals thanks the donors whose unspecified financial support has made the production of this document possible. This module was produced for Immunization, Vaccines and Biologicals, WHO, by: Lewellys F. Barker Aeras Global TB Vaccine Foundation, Rockville MD, USA Gregory Hussey University of Cape Town, Cape Town, South Africa Printed in October 2011 Copies of this publication as well as additional materials on immunization, vaccines and biological may be requested from: World Health Organization Department of Immunization, Vaccines and Biologicals CH-1211 Geneva 27, Switzerland Fax: vaccines@who.int World Health Organization 2011 All rights reserved. Publications of the World Health Organization can be obtained from WHO Press, World Health Organization, 20 Avenue Appia, 1211 Geneva 27, Switzerland (tel: ; fax: ; bookorders@who.int). Requests for permission to reproduce or translate WHO publications whether for sale or for noncommercial distribution should be addressed to WHO Press, at the above address (fax: ; permissions@who.int). The designations employed and the presentation of the material in this publication do not imply the expression of any opinion whatsoever on the part of the World Health Organization concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. Dotted lines on maps represent approximate border lines for which there may not yet be full agreement. The mention of specific companies or of certain manufacturers products does not imply that they are endorsed or recommended by the World Health Organization in preference to others of a similar nature that are not mentioned. Errors and omissions excepted, the names of proprietary products are distinguished by initial capital letters. All reasonable precautions have been taken by the World Health Organization to verify the information contained in this publication. However, the published material is being distributed without warranty of any kind, either expressed or implied. The responsibility for the interpretation and use of the material lies with the reader. In no event shall the World Health Organization be liable for damages arising from its use. The named authors alone are responsible for the views expressed in this publication. Printed by the WHO Document Production Services, Geneva, Switzerland ii

5 Contents Abbreviations and acronyms...v Preface... vii 1. TB disease Bacteriology The host response to infection Characteristics of BCG vaccines Response to vaccination with BCG vaccines Route of administration of BCG vaccines BCG vaccination scars Delayed type hypersensitivity reactions and cell-mediated response Adverse events Protective efficacy and duration of immunity Future prospects...18 References...20 iii

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7 Abbreviations and acronyms AIDS BCG CFU CI DOTS DTH ELISPOT EPI GACVS HIV HLA IGRA IFN-γ IL-2 LTBI acquired immunodeficiency syndrome bacille Calmette-Guérin (vaccine) colony-forming units confidence interval WHO-recommended directly observed treatment tuberculosis control strategy delayed type hypersensitivity enzyme-linked immunospot assay Expanded Programme on Immunization Global Advisory Committee on Vaccine Safety (WHO) human immunodeficiency virus human leukocyte antigen interferon gamma release assay interferon-gamma interleukin-2 latent tuberculosis infection M. Mycobacterium MDR-TB MRC Mtb NRAMP PBMC PPD multi drug-resistant tuberculosis Medical Research Council (United Kingdom) Mycobacterium tuberculosis natural resistance associated monocyte protein peripheral blood mononuclear cells purified protein derivative RD1 deletion region 1 SCID severe combined immunodeficiency v

8 TB tuberculosis TH1/2 T helper cells type 1/2 TNF-α ToC TST WHO USA XDR-TB tumour necrosis factor-alpha test-of-concept (trial) tuberculin skin test World Health Organization United States of America extensively drug-resistant tuberculosis vi

9 Preface This module is part of the series The Immunological Basis for Immunization, which was initially developed in 1993 as a set of eight modules focusing on the vaccines included in the Expanded Programme on Immunization (EPI) 1. In addition to a general immunology module, each of the seven other modules covered one of the vaccines recommended as part of the EPI programme diphtheria, measles, pertussis, polio, tetanus, tuberculosis and yellow fever. The modules have become some of the most widely used documents in the field of immunization. With the development of the Global Immunization Vision and Strategy (GIVS) ( ) ( EN.pdf) and the expansion of immunization programmes in general, as well as the large accumulation of new knowledge since 1993, the decision was taken to update and extend this series. The main purpose of the modules which are published as separate disease/vaccinespecific modules is to give immunization managers and vaccination professionals a brief and easily-understood overview of the scientific basis of vaccination, and also of the immunological basis for the World Health Organization (WHO) recommendations on vaccine use that, since 1998, have been published in the Vaccine Position Papers ( WHO would like to thank all the people who were involved in the development of the initial Immunological Basis for Immunization series, as well as those involved in its updating, and the development of new modules. 1 This programme was established in 1974 with the main aim of providing immunization for children in developing countries. vii

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11 1. TB disease Tuberculosis (TB) is a bacterial disease which has been prevalent since ancient times. TB is currently one of the most important health problems in developing countries where it imposes a tremendous burden of suffering and economic loss. Mycobacte rium tuberculosis (Mtb) is responsible for almost nine million new TB cases, and 1.7 million deaths per year, mostly in developing countries, although annually there are over new cases in industrialized countries. In fact, as infection with human immunodeficiency virus (HIV), drug resistance and travel between developing and developed countries have become increasingly common, tuberculosis has become a serious global-health problem. Substantial progress has been made worldwide in slowing the TB pandemic, especially through the WHO-recommended directly observed treatment short course strategy (DOTS). However, serious obstacles to DOTS success include low case-detection rates, the emergence of multi-drug resistant (MDR-TB) and extensively drug-resistant (XDR-TB) TB strains, the severe and widespread risk of HIV/TB coinfection, and the logistic shortcomings of modern tools to diagnose, treat and prevent the disease. In sub-saharan Africa the rise in TB disease incidence has been strongly associated with HIV infection; in some countries more than half of individuals with TB have concurrent HIV infection, and HIV-infected persons have a ten-fold greater risk of developing overt clinical TB disease upon infection with Mtb than non-hiv-infected persons. TB is also the leading cause of death among HIV-infected persons, accounting for approximately 23% of AIDS deaths worldwide. In 2008 WHO estimated that there were nearly half a million new cases of multi-drug-resistant TB (MDR-TB) a year, which is about 5% of over nine million new TB cases of all types (1). WHO also found that extensively drug-resistant tuberculosis (XDR-TB), a virtually untreatable form of the respiratory disease, has been recorded in 45 countries, and that there is also a link between HIV infection and MDR-TB. Surveys in several countries found nearly twice the level of MDR-TB among TB patients living with HIV compared with patients without HIV. TB is a poverty-related disease; it has long been recognized that war, malnutrition, population displacement and crowded living and working conditions favour the spread of TB among human populations, whereas improvements in social conditions and hygiene lead to its decline. By far the most important source of human infec tion is an already infected person who spreads the infectious bacilli via respiratory droplets when they cough. TB is highly contagious; left untreated, each patient with active pulmonary TB can infect between 10 and 15 people every year. Transmission is common in families, schools, hospitals and prisons. Another factor that contributes to the spread of TB is the global movement of people; as many as 50% of the world s refugees may be infected with TB. In many industrialized countries half or more of new TB cases occur in foreign-born people or migrant populations from endemic areas. 1

12 Pri mary infections can occur at any age, but children in areas of high incidence and high population density are most often affected. Even after resolution, the disease can be reactivated and spread again. Agents that depress the immune system, such as corticosteroid treatment or HIV infection, facilitate reactivation. Primary infection may be asymptomatic and often resolves spontaneously without progression to active disease. In 90% of infected persons the bacterium is contained by the host immune response as a latent TB infection (LTBI). In approximately 5% of infections, progression to disease occurs in the first 2 3 years after infection and an additional 5% of infections progress later to active TB disease from LTBI, including in old age. TB may progress by local spread in the lungs to cause pleurisy or bron chopneumonia. If the infection spreads through the bloodstream, it can affect many organs, including the meninges, the bones or the internal organs. TB disease can be accompanied by tuberculous lymphadenopa thy or, in the absence of other features, this manifestation can occur. 2 The Immunological Basis for Immunization Series - Module 5: Tuberculosis - Update 2011

13 2. Bacteriology Mycobacte rium tuberculosis (Mtb), the main agent of human TB disease, was discovered in 1882 by Robert Koch. All members of the Mycobacte rium genus are slender, non-motile rod, Gram-positive bacteria with the property of acid-fast (Ziehl-Nielsen) staining due to their complex mycolic acid-rich cell wall structure. The genus includes four members of the M. tuberculosis complex: M. tuberculosis and M. africanum, which are primary human pathogens, M. bovis, the agent of TB in cattle and other animals which can also cause disease in humans, and M. ulcerans, which causes Buruli ulcer disease. Other mycobacteria include M. leprae, the agent which causes leprosy, and a large number of nontuberculous environmental mycobacteria such as those belonging to the M. avium-intracellulare complex, of which M. avium is an opportunistic pathogen in AIDS patients. M. bovis, from which bacille Calmette-Guérin (BCG) vaccine was derived, causes disease in a wide range of domestic animals, including cattle, bison, buffalo and pigs, and also in feral hosts such as badgers, New Zealand brush-tail possums, kudus, antelopes, deer, lion and baboons. Infection of humans with M. bovis can occur by inhalation of aerosols (especially affecting farmers, abattoir workers and veterinarians), or through consumption of contaminated milk and milk products. M. tuberculosis has a long generation time (18 24 hours) and growth on solid media such as Lowenstein-Jensen medium is detectable only after 2 6 weeks. Using fluid medium and automated detection systems, growth can be detected in 1 2 weeks. Under field conditions, the diagnosis of pulmonary TB is usually based on microscopy by demonstrating acid-fast bacilli in sputum; isolation of the organism is required for a definitive species diagnosis and for drug sensitivity testing. Modern molecular techniques based on nucleic acid amplification and genetic probes can provide rapid species information on clinical material or laboratory isolates, and are now widely used in modern laboratories to supplement traditional microbiological diagnostic methods. Since the late 1980s, an increase in mycobacterial drug resistance has been reported from many countries. The success of mycobacteria as pathogens comes mostly from their ability to survive in the environment and to persist in infected hosts for long periods of time. No Mtb toxins are known, and the virulence of different strains of Mtb rests largely within its ability to grow and persist within host cells. The waxy cell wall protects the bacteria and promotes their intracellular persistence by inhibiting phagosome-lysosome fusion, which in turn keeps them away from terminal endocytic processes. Mycobacteria induce the formation of granulomas that wall-off sites of infection and limit its spread, but also restrict macrophage killing of the bacteria resulting in persistence of latent infection. 3

14 Starting in 1998, the complete genome of Mtb (H37Rv strain) and subsequently other mycobacterial genomes, including BCG, have been sequenced (2 6). Comparative genomics have uncovered interesting polymorphisms among species of the M. tuberculosis complex, as well as among and within BCG strains. Research revealed a number of genes that are expressed during intracellular infection, that encode products required for survival and progressive infection or dormancy within the host (7 10). Mycobacterial mechanisms to direct or block host-cell immune responses have also been uncovered (11,12). 4 The Immunological Basis for Immunization Series - Module 5: Tuberculosis - Update 2011

15 3. The host response to infection Primary infection by tubercle bacilli induces both innate and adaptive immune and nonimmune inflammatory reac tions. Several different types of immune responses include: interferon gamma (IFN-γ) producing Th1 CD4+ T-cells and major histocompatibility complex Class I restricted CD8+ T cell-mediated immune responses; increased macrophage activity; delayed hypersensitivity, granulomatous inflammation and circulating antibodies. These responses may be protective, or contribute to pathology depending on the specificity and function of the activated T-cells. Typically a strong T cell-mediated immune response kills infected macrophages and also induces extensive necrosis in host tissue. The production of anti-inflammatory cytokines in response to Mtb antigens may downregulate the immune response and limit T helper cells type 2 (TH-2) driven tissue injury. Cell-mediated immunity, not production of humoral antibodies, is considered the major kind of immunological defence in tuberculosis. The granulomatous reaction is an immune re sponse that limits the dissemination of the organisms. The first exposure of the host to the tubercle bacillus may be completely asymptomatic, with a small Ghon focus of inflammation seen in the lung. A Ghon complex is a usually calcified healed granuloma in the lung and the draining lymph nodes. As an intracellular pathogen, Mtb has developed mechanisms to survive inside host mononuclear phagocytic cells and thus evade the host immune response. TB bacilli usually multiply first in pulmonary alveolar macrophages and draining lymph nodes. Killing of infected macrophages progressively creates a primary tubercle. Delayed cutaneous hypersensitivity develops and, together with other cellular host immune reactions, leads to caseous necrosis of the primary complex. CD4+ T-cells accumulate in large numbers in early granulomatous lesions, where CD8+ T-cells later join them (13,14). The bacilli can spread to many parts of the body including spleen, liver, meninges, bones, kidneys and lymph nodes, where they are sometimes a source of overt extrapulmonary TB disease or, more commonly, remain dormant (15). When the infection is brought under control, Mtb may remain in a dormant state in host tissue for life. Reactivation of dormant bacilli is often associated with immunodeficiency, for instance in patients with HIV/AIDS (16). CD4+ T-cells producing interferon gamma (IFN-γ) and tumour necrosis factor-alpha (TNF-α) play a major role in containment of TB infection, but they do not appear to be the whole story. The TH1 type cytokines contribute to walling of Mtb inside granulomas and the controlling of the evolution of disease. In addition, CD8+ T-cells and T-cells expressing γδ T-cell receptors with specificity for small phosphorylated ligands, and T- cells for glycolipids are stimulated (17). As long as the host remains immunocompetent, granulomas can persist for years and can keep Mtb in a dormant state. Individuals with deficient CD4+ T-cells or deficient IFN-γ signalling, suffer from rapid evolution of TB disease (18), and individuals with latent TB infection treated with anti-tnf-α antibodies are at increased risk of reactivation of TB disease (19,20). 5

16 A positive tuberculin skin test, erythema and induration 24 to 48 hours after injection of purified protein derivative (PPD) antigens, has long been used as evidence of active or latent TB infection, and as a sign of an immune response to BCG vaccine. However, it has not been possible to establish a clear relationship between this delayedtype hypersensitivity reaction and protective immunity, and a positive test does not necessarily indicate immunity to reinfection. Newer tests use IFN-γ release following in vitro stimulation of peripheral blood mononuclear cells (PBMCs) to detect current or past infection with Mtb; appropriate selection of antigens that are missing in BCG for PBMC stimulation make these interferon gamma release assays (IGRAs) more specific than the skin test with PPD (21,22). A number of antigens found in Mtb (e.g. Ag85A and B, ESAT-6, CFP10, MPT64 and others) have been identified, that may play a major role in protective cellular immunity and are being evaluated in new vaccines for TB (see below). 6 The Immunological Basis for Immunization Series - Module 5: Tuberculosis - Update 2011

17 4. Characteristics of BCG vaccines By culturing an M. bovis strain isolated from a cow by Nocard in 1904 for a period of 13 years, and a total of 231 passages, on potato slices cooked in beef bile supplemented with glycerol, physician Calmette and Guérin, a veterinarian, at the Institut Pasteur in Lille, France, created a live, attenuated vaccine, bacillus Calmette-Guérin or BCG (23). The first human vaccination by Weil-Halle in Paris in 1921 was given to an infant by the oral route with several successive doses (24). Additional methods of administration employed subsequently include intradermal, and percutaneous by multiple puncture or scarification. Since 1974, BCG vaccination of infants has been included in the WHO Expanded Programme on Immunization (EPI) resulting in more than three billion cumulative vaccinations worldwide and approximately 100 million vaccinations per year. BCG has never been cloned and there are now multiple different BCG seed strains (sub-strains) used in BCG manufacture. There have been many (WHO estimated 40 or more in 1999) manufacturers of BCG around the world. The most widely-used strains in international immunization programmes include: Danish 1331 strain; Moscow strain, and Tokyo 172 strain. The use of other strains is largely limited to their country of origin, e.g. Moreau strain (Brazil) or Tice strain (USA). In 1966, the WHO Expert Committee on Biological Standardization established the first requirements for BCG vaccine (WHO Expert Committee on Biological Standardization, 1966). These requirements were subsequently revised (WHO Expert Committee on Biological Standardization, 1987 and 1988). They outline procedures for production of BCG vaccines to ensure potency, safety and efficacy, and describe certain tests which should be done on the vaccine seeds and on the final vaccine itself. The WHO requirements were designed to reduce the variability among BCG strains seen in clinical and animal trials, by requiring each manufacturer to correlate laboratory test results with clinical efficacy data. Recent genomic analysis of BCG strains by Behr, Cole and others (25,26,27) has documented multiple molecular changes between the introduction of Pasteur BCG in 1921 and lyophilization of the BCG Pasteur 1173 strain in With continuous passage in vitro prior to lyophilization of seed strains, all BCG strains presumably continued to evolve, and possibly became over-attenuated (28). As recently shown by genome sequencing, the original Pasteur BCG strain probably lost the deletion region 1 (RD1) of M. bovis in the course of multiple passages used for the original selection process. Loss of the RD1 region appears to have occurred in , so that strains prior to that time contained mpt64 and those subsequent to 1931 did not. Multiple other deletions probably contribute to phenotypic differences between BCG strains, but their specific relevance to the protective efficacy and reactogenicity/safety of different BCG strains is not entirely clear. Although there are clear reactogenicity 7

18 differences between strains (Adverse Events, see below), Brewer & Colditz (29) did not find evidence based on limited data that strain differences are a significant factor contributing to variable BCG vaccine efficacy against tuberculosis. A WHO consultation on the characteristics of BCG strains in 2003 (30) discussed the extensive evidence for molecular variation in BCG sub-strains, which was also reflected in laboratory studies of their phenotypic and immunological properties. Although there is good evidence to support the notion that the induced immune response and protection afforded against tuberculosis differs between BCG vaccine strains, currently there are insufficient data to favour or recommend one particular strain (31). Clinical and epidemiological studies also indicated differences in vaccine performance in clinical trials, but the presence of many confounding variables made it difficult to determine the influence of sub-strain. Nevertheless, the consultation reached a consensus that for future use of BCG vaccines, both as conventional preparations for routine vaccination, or as part of a prime-boost strategy and as genetically modified strains, better characterization was needed, and that the current WHO requirements for BCG vaccines were outdated and should be reviewed. Application of molecular characterization methods to the modern production and quality control of BCG is being undertaken, as well as efforts to determine the significance of known genetic variations and genomic decay in relation to the quality and performance of current BCG vaccines. 8 The Immunological Basis for Immunization Series - Module 5: Tuberculosis - Update 2011

19 5. Response to vaccination with BCG vaccines Clinical trials in the past have shown that BCG vaccine may induce some protec tion against tuberculosis. Vaccination with BCG spreads from the inoculation site via the lymphatic system to local lymph nodes, and produces an immune response with similar characteristics to that produced by natural infection with Mtb. As in the case of natural tuberculosis infection, the resistance is cell-mediated and is largely attributable to activation and destruction of macrophages by sensitized T-cells. BCG-induced immunity develops about six weeks after vaccination. Experimental studies indicate that the mechanism of protection by BCG vaccination consists in reduc tion of the haematogenous spread of bacilli from the site of primary infection (32,33) mediated by memory, and T-lymphocytes induced by the first exposure to BCG and effector T-lymphocytes induced by Mtb infection in BCG vaccinated persons. There is no evidence that BCG reduces the risk of becoming infected with tuberculo sis bacilli, but the fact that it prevents disseminated childhood tuberculosis is taken as an indication that it impairs the haematogenous spread of the bacillus. This inhibition of the haemato genous spread of bacilli thus reduces the risk of disseminated primary disease and possibly also of disseminated disease due to reactivation. 5.1 Route of administration of BCG vaccines WHO recommends BCG vaccination by the intradermal route, generally by injection in the deltoid insertion region of the upper arm with a 25 or 26 gauge needle. The infant dose is generally 0.05 ml. Some countries, especially Japan, employ percutaneous administration with special multipuncture devices. Percutaneous administration methods are generally simpler to perform than intradermal, but are less consistent with regard to the amount of vaccine delivered. Fewer adverse events were reported following percutaneous administration of BCG compared with intradermal administration (34,35). However, more positive delayed type hypersensitivity (DTH) skin reactions and greater in vitro cellular responses to BCG were demonstrated when the vaccine was given to healthy adults by intradermal rather than by percutaneous administration (36). Similarly, greater lymphoproliferative and TH1-type cytokine responses were found in newborns in response to intradermal BCG compared with percutaneous BCG (37). A literature review comparing intradermal and percutaneous routes reached the same conclusion, adding that the lower dosage inoculated by the percutaneous route may account for the lower immune responses (38). However, in a large randomized prospective trial that compared administration of BCG vaccine by either the intradermal or percutaneous route in over newborns, there was no difference in TB events or adverse events, i.e. the two different routes gave equivalent results (39). 9

20 BCG can also be delivered via the oral routes. Moreover, there is limited past experience with injection by jet-injector and by aerosol (40 43). The original route recommended by Calmette and used by Weil-Halle in 1921 was oral administration given three times at 2-day intervals shortly after birth in doses ranging from 2 to 10 mg approximately colony-forming units (CFU) (44). Many infants immunized by the oral route did not react to tuberculin skin testing or, not until after many months had passed. Increased reactivity to skin testing could be accomplished by increasing the oral BCG dose to mg (45). Factors favouring intradermal (or percutaneous) vaccination (46) include: oral vaccination requires much larger doses of BCG for skin test conversion ( mg compared with 0.1 mg for intradermal) and hence was more expensive; it is more difficult to control the effective dose with oral administration, as some bacilli are inactivated in the stomach and some pass through the intestinal tract; intradermal administration is much more efficient at inducing tuberculin conversion; there were reports of cervical lymphadenopathy attributed to oral administration. 5.2 BCG vaccination scars Following intradermal injection of BCG vaccine into humans, a papule with induration appears within two to three weeks. The papule ulcerates at six to eight weeks, followed by a scar at the end of three months. The presence of such a scar in the appropriate place (commonly the right arm just below the insertion of the deltoid) has been used as evidence for prior BCG vaccination. With multiple puncture percutaneous inoculation of BCG, there are many small papules which disappear more quickly, and often without scarring. Although the size of the scar follows a simple dose-response, various other factors have been shown to influence the size and shape of the scar, including: the technique of vaccine administration (intradermal administration is more likely to leave a uniform scar, while improper, i.e. subcutaneous, administration may not; the characteristics of the recipient (keloid formation may be associated with race), and the strain of BCG used. A number of studies have reported on the agreement between a documented history of BCG vaccine and the presence of a BCG scar at one or two years after vaccination. In studies in Brazil and Peru, BCG scar was shown to be a highly sensitive indicator of vaccination in children vaccinated during the first month and before three months of age, and examined respectively up to three years of age and in early adolescence (47,48). However, Malawian infants who were vaccinated when they were less than one month old or 1 4 months old, 24% and 16% respectively, did not have recognizable scars four years later (49). From the latter results it was concluded that, when the vaccine had been administered in infancy as is recommended by WHO and widely practiced, use of presence or absence of BCG scar years later was not a highly sensitive and reliable indicator of prior vaccination status. There is also no evidence of a correlation between increased BCG scar size and protection against either tuberculosis or leprosy (50). 10 The Immunological Basis for Immunization Series - Module 5: Tuberculosis - Update 2011

21 5.3 Delayed type hypersensitivity reactions and cell-mediated response Delayed hypersensitivity tuberculin reaction, tuberculin skin test (TST) to screen people for prior exposure to tuberculosis, is measured by injection intradermally of purified protein derivative (PPD), 0.1 ml containing 5 TU, on the volar surface of the forearm (Mantoux test). The results are read 48 to 72 hours later as the area of induration, with at least 5 mm in diameter as the threshold for indication of a positive reaction. An alternative skin puncture method (Heaf test) uses a multiple needle device to inject PPD spread on the forearm; results are read 5 7 days later and defined Grades 2 to 4 are interpreted as positive reactions (Table 1). Table 1: Heaf testing and Mantoux equivalents Heaf grade Reaction Mantoux equivalent (mm induration) Negative No induration papules <5 2 Confluent papules form indurated ring Central filling to form disc >15 4 Disc > 10mm with or without blistering >15 Tuberculin positivity appears following BCG vaccination and decreases with time at a rate depending on the age of vaccination. When BCG is administered to newborns, a high proportion have reactions after a few years <5 mm in size, so that under these circumstances a large reaction to PPD (>10 15 mm) remains a useful test for exposure to/infection with Mtb. The tuberculin reaction can be boosted by repeated PPD tests as well as by BCG vaccination and by infection with environmental mycobacteria, all of which, depending on the epidemiologic circumstances, can complicate the interpretation of the TST. A recent meta-analysis indicated that in subjects without active tuberculosis, immunization with BCG significantly increases the likelihood of a positive tuberculin skin test and that the effect of BCG vaccination on PPD skin-test results was less after 15 years. Positive skin tests with indurations of >15 mm are more likely to be the result of tuberculous infection than of BCG vaccination (51). 5.4 Adverse events Although BCG vaccines are considered safe, i.e. to have a favourable benefit-torisk relationship, they are among the most reactogenic vaccines in common use. BCG induces a local ulcer which was more acceptable in the past when populations were accustomed to the local lesions produced by smallpox vaccine. The local lesion begins as a papule two or more weeks after vaccination. It generally proceeds to ulceration and heals after several months. A scar (typically round and slightly depressed) remains in most vaccinees and, is a useful albeit imperfect indicator of past vaccination. The probability that BCG vaccine leaves a lasting scar is lower after vaccination in early infancy than at older ages. BCG is not easy to administer as an intradermal injection in infants; the commonest mistake is to administer the injection too deep (subcutaneous), which may result in local injection-site abscesses. 11

22 Local reactogenicity differs between vaccines, varying with both sub-strains and dose of viable bacilli. Pasteur 1173P2, Danish 1331 and Sweden (Gothenburg) sub-strains have been found to be more reactogenic than the Tokyo 172, Glaxo 1077 or Brazilian (Moreau) strains (52). Several reports of outbreaks of BCG reactions, manifested as large ulcers and local lymphadenopa thy or suppurative lymphadenitis, followed changes, e.g. from less reactogenic Glaxo to more reactogenic Pasteur strain vaccines (53,54). Other complications of BCG vaccination include osteitis/ osteomyelitis and disseminated BCGosis, which is mainly seen in children with severe congenital or acquired immunodeficiencies, such as severe combined immunodeficiency (SCID), chronic granulomatous disease, Di George syndrome and homozygous complete or partial interferon gamma receptor deficiency, as well as HIV/AIDS. Osteitis/osteomyelitis has been reported in the past, particularly in Scandinavia and Eastern Europe/Russia, and has been associated with the Russian and Gothenburg strains of BCG (55 58) Management of reactions and complications Local injection-site lesions: It is generally considered that even large local lesions are best left untreated. Secondary infections at the injection site are unlikely. In extreme cases, systemic treatment with erythromycin (daily for up to one month) may be helpful. Keloids: Keloids are difficult to treat. Simple surgical removal is likely to make them worse. A combination of surgery, irradiation and drug treatment may be effective, but should only be undertaken by a specialized practitioner. Local lymph gland involvement: Axillary or cervical lymphadenitis will heal spontaneously and it is best not to treat the lesion if it remains unattached to the skin. An adherent or fistulated lymph gland may be drained and an anti-tuberculous drug may be instilled locally. Systemic treatment with antituberculosis drugs is ineffective. Rare severe complications: Rare complications, including lupus vulgaris, erythema nodosum, iritis, osteomyelitis and generalized BCGosis should be treated systemically with anti tuberculosis regimens, including isoniazid and rifampicin BCG and HIV/AIDS The relationship of HIV/AIDS to safety and efficacy of vaccination with BCG vaccines has been a concern for a number of years. WHO had previously recommended that, in countries with a high burden of tuberculosis, a single dose of BCG vaccine should be given to all healthy infants as soon as possible after birth unless the child presented with symptomatic HIV infection. However, recent reports showed that children who were HIV-infected when vaccinated with BCG at birth, and who later developed AIDS, were at increased risk of developing disseminated BCG disease (59,60,61). Among these children, the benefits of potentially preventing severe TB are outweighed by the risks associated with the use of BCG vaccine. Therefore, in November 2006, the WHO Global Advisory Committee on Vaccine Safety (GACVS) revised its previous recommendations concerning BCG vaccination of children infected with HIV (62), such that children who are known to be HIV-infected, even if asymptomatic, should no longer be immunized with BCG vaccine., WHO accordingly provided the guidance to facilitate national and local decisions on the use of BCG vaccine in infants at risk for HIV infection (63). 12 The Immunological Basis for Immunization Series - Module 5: Tuberculosis - Update 2011

23 5.5 Protective efficacy and duration of immunity The protective efficacy of a vaccine is measured in terms of the percentage reduction in disease among vaccinated individuals that is attributable to vaccination. The best method for determining the protective efficacy of a vaccine is a prospective, randomized, double-blind, placebo-controlled trial in previously unvaccinated individuals. These studies are difficult and expensive; the last such trial was the Chingleput trial in the 1960s (64). Many recent studies have used case-control and contact follow-up methods (65,66,67). Both randomized controlled trials and case-control studies have been evaluated on several occasions by meta-analyses and/or systematic reviews (68 72). Although the WHO currently emphasizes BCG s utility in prevention of severe childhood TB disease, e.g. miliary tuberculosis and tuberculous meningitis, the main public-health burden and source of spread of tuberculosis is adult pulmonary disease. It is therefore reasonable to consider BCG vaccine efficacy against severe childhood tuberculosis separately from that against adult tuberculosis, leprosy and other mycobacterial diseases. BCG and TB infection. There is some evidence to suggest that BCG does prevent TB infection. In a recent outbreak of TB in a junior school in the United Kingdom, the rate of TB infection, as detected by a reactive gamma interferon test, enzyme-linked immunospot assay (ELISPOT), was 47% in unvaccinated children compared to 29% in vaccinated children (73). Another study, in Istanbul, investigated risk factors for tuberculosis infection in 979 child household contacts of 414 adult index patients with sputum smear-positive pulmonary tuberculosis, and reported that BCG-vaccinated children had an odds ratio of 0.60 (95% CI , p=0.003) for tuberculosis infection (74). Childhood tuberculosis, tuberculosis meningitis and miliary TB: There is evidence that BCG provides consistent and appreciable protection against tuberculous meningitis and miliary disease. A meta-analysis of five randomized controlled trials and eight case-control studies indicated no significant heterogeneity and an average protection of approximately 80% (86% with 95% CI: 65% to 95% for controlled trials and 75% with 95% CI: 61% to 84% for case-control studies) (75). This was confirmed by a meta-analysis of protection associated with vaccination in infancy as currently recommended (76). Due to the rarity of these severe childhood TB events, most of the data are from observational studies. For example, no cases of TB meningitis or miliary TB were seen in the South India Chingleput trial (77,78), and only five cases of miliary disease (all in the placebo group) were identified in the British Medical Research Council (MRC) trial (78). Evidence of protection against pulmonary disease in children, which is rarely smear-positive and hence difficult to diagnose is less consistent, and appears to suggest lower protection in tropical than in temperate regions. Adult pulmonary tuberculosis: This form of TB is responsible for the major public-health burden of tuberculosis and is also associated with the greatest controversy related to efficacy of BCG. A wide range of efficacy estimates, from 0 to approximately 80%, have come from prospective trials and observational case-control and contact studies. This variability is highly significant (p<0.0001), indicating that the variation reflects true biological differences and not just sampling errors. The reason, or reasons, for the great differences remain unclear despite several hypothetical explanations (see below). However, in a cohort of individuals vaccinated at ages 1 20 in the 1930s, long-term follow-up revealed approximately 52% efficacy of a single BCG vacccination 13

24 against adult TB lasting for 50 to 60 years (79), and a report from Brazil described substantial protection (39% efficacy, CI: 9% 58%) lasting years following BCG given to newborns against all forms of TB (80). A major current TB control concern is increasing multi drug-resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB). Interestingly, an observational study in Brazil found a decreased risk of TB disease in contacts of MDR-TB cases was associated with previous BCG vaccination, including in persons with prolonged exposure to MDR-TB cases (81). More information is needed regarding BCG protection against drug-resistant strains of TB. Tuberculosis and HIV/AIDS: Protection against clinical TB appears to be diminished. Infection with HIV severely impairs the BCG-specific T-cell response during the first year of life. Thus BCG provides little, if any, vaccine-induced benefit in HIV-infected infants (82). Leprosy and other mycobacterioses: Numerous controlled trials, cohort and case-control studies and observational studies have shown some protection against leprosy associated with BCG. A recent meta-analysis of six controlled trials, two cohort studies and 14 case-control studies has reported summary protective effects of 43 (27% 55%), 62 (53% 69%) and 58 (47% 67%), respectively (83). The highest efficacy estimates have been reported from Kenya, Malawi and Uganda) (84,85,86). Three different studies have evaluated protection by the same BCG vaccines against tuberculosis and leprosy in the same population, and in each case the protection was appreciably greater against leprosy (87,88,89). Importantly, there is evidence that BCG vaccines impart as much protection against lepromatous as they do against the tuberculoid forms of the disease, although this was not observed in the Chingleput trial population (89). This has important public-health implications, given that lepromatous disease is the most severe and is thought to be responsible for most transmission of M. leprae in the community. Protection against leprosy provided by BCG given to infants, may last for 30 years or longer (90). There is also evidence that BCG provides some protection against Buruli ulcer (M. ulcerans infection) (91,92), and against glandular disease attributable to various other environmental mycobacteria, in particular M. avium-intracellulare. This evidence is based upon observations in Sweden (93) and the Czech Republic (94), where increases in childhood glandular mycobacterioses were identified in cohorts born after infant BCG was discontinued. In Sweden among children under five years of age the protection appeared to be in the order of 85%. Non-specific immune stimulation: Mycobacteria and their cell-wall constituents have long been known to exert non-specific immune stimulation. Complete Freund s adjuvant, a potent, albeit highly reactogenic, immune stimulator used in experimental animal systems, contains killed mycobacteria (95,96). Live BCG is being used to successfully treat non muscle-invasive bladder carcinoma, with an efficacy superior to chemotherapy in terms of completeness of response and disease-free survival period (97). Moreover, there are indirect indications that BCG provides a general survival benefit to individuals who respond to vaccination with the development of a positive tuberculin skin test (TST) or a BCG scar (98). 14 The Immunological Basis for Immunization Series - Module 5: Tuberculosis - Update 2011

25 Booster doses: Despite the widespread use of boosters in many countries, there has been very little formal evaluation of their utility. Analyses of data from Hungary (99) and Poland (100,101) were consistent with revaccination providing some protection, but were based on small numbers and inappropriate controls and were therefore not convincing. A case-control study in Chile failed to find evidence of increased protection associated with an increased number of BCG scars (102). In Finland, no increase in tuberculosis was observed since that country discontinued revaccination of schoolchildren in 1990, although overall case numbers are too small for convincing analysis (103). A controlled trial to evaluate the efficacy of a BCG booster in protection against tuberculosis was carried out in Malawi, and found no evidence for protection (104). On the basis of such data, WHO did not encourage revaccination in 1995 (105). A more recent controlled trial in Brazil (106) failed to show evidence of increased protection by revaccination in schoolchildren aged 7 to 14 years despite evidence of an increase in immune response following revaccination (107). However, it has been shown that oral administration of BCG, Moreau sub-strain, does boost the cell-mediated immune response measured in PBMCs by ELISPOT in adults who received intradermal BCG in childhood or adolescence (108). Although there is no convincing evidence that BCG boosters are effective in preventing tuberculosis, several studies have supported their utility against leprosy. A randomized trial in Malawi and case-control studies in Venezuela (109) and Myanmar (110) have shown an increase in protection associated with increasing numbers of BCG doses (or BCG vaccine scars). The Malawi study is of interest because it was a formal trial and because no protection against tuberculosis was observed by either an initial or a repeated dose of BCG, despite the dose-dependent protection against leprosy. However, in a more recent Brazilian study, no increase in protection against leprosy was observed associated with a booster BCG vaccination (111). Taken together the evidence is not entirely consistent but suggests that BCG boosters may give increased protection sometimes, where an initial dose is effective, but not otherwise. The question of utility of boosters is thus referred to the more basic issue of the inconsistent behaviour of an initial BCG vaccination. Duration of immunity The duration of protection after neonatal BCG vaccination is not well known but it is commonly believed to decline gradually to non-significant levels over 10 to 20 years (71). BCG vaccine is not thought to prevent reactivation of latent TB, a main source of adult pulmonary disease and Mtb dissemination in the community. Hence, neonatal BCG vaccination is not considered to be effective in preventing acquisition or transmission of adult pulmonary TB. However, a long-term follow-up of a randomized, prospective, controlled BCG trial in the 1930s revealed approximately 52% efficacy of a single BCG vaccination against adult TB lasting for 50 to 60 years in a cohort of individuals vaccinated at ages 1 20 (79), and Barreto et al (2005) described substantial protection (39% efficacy, CI: 9% 58%) lasting years following BCG given to newborns against all forms of TB (79). Studies of the immune response produced by MVA85A, a new TB vaccine candidate for boosting neonatal BCG later in life, have also produced evidence of very durable immunogical memory following childhood or adolescent BCG vaccination (112). 15

26 Delaying BCG vaccination for a few weeks appears to be associated with a better immune response. A study from South Africa showed that infants who received delayed BCG vaccination demonstrated higher frequencies of BCG-specific CD4 T-cells, particularly polyfunctional T-cells co-expressing IFN-gamma, TNF-alpha and IL-2, and most strikingly at one year of age (113). However, the immune benefit of delaying BCG vaccination must be weighed against the interim risk of an infection with Mtb, in particular in TB high endemicity areas in general, or in children born to households with active TB cases. Reasons for variable BCG efficacy The variation in protection by BCG in clinical trials against pulmonary tuberculosis and against leprosy has attracted much attention, but few clear answers (114,115). Several of the suggested hypotheses are below. Differences between BCG vaccines, both in genetics of multiple sub-strains and physical properties of vaccine preparations, including effects of manufacturing method, multiple passages in production, freeze-drying and ratio of killed to live mycobacteria. Although there is clear evidence of BCG strain variation association with reactogenicity and adverse effects, there is no firm evidence based on limited data that strain differences are a significant factor contributing to variable BCG vaccine efficacy against tuberculosis (29). Environmental mycobacteria are ubiquitous. There is evidence (a) from human and animal studies, that exposure to environmental mycobacteria can provide some degree of protection against subsequent challenge with tubercle bacilli (116), i.e. masking of BCG-mediated protection; (b) from animal studies, that protection by BCG is reduced in animals that have already received some protection by prior exposure to environmental mycobacteria (117), and (c) that superinfection with environmental mycobacteria can impair established BCG immunity (118), i.e. blocking of BCG-mediated protection. Furthermore, BCG efficacy tends to be lower in populations living in warmer and wetter regions (closer to the Equator), in particular in rural areas where exposure to environmental mycobacteria is greater. This circumstantial evidence suggests that exposure to environmental mycobacteria may be responsible for some of the variation in BCG efficacy. Human genetics. There is evidence that several genes that control cellular immune mechanisms, including human leukocyte antigen DR (HLA-DR) and HLA-DQ, vitamin D receptor and IFN-γ receptor polymorphisms, and natural resistance associated monocyte protein (NRAMP) influence susceptibility to tuberculosis and other mycobacterial infections ( ). Hence it has been conjectured that population genetic differences might explain some of the variable efficacy behaviour of BCG. However, meta-analysis has shown that many of these studies are methodologically flawed (123). Moreover, systematic review has demonstrated that these genetic associations may be limited to certain populations (124)., Appropriate studies are therefore needed, for example, comparing the frequency of genetic determinants between protected and unprotected vaccinees. 16 The Immunological Basis for Immunization Series - Module 5: Tuberculosis - Update 2011

27 Differences in Mtb were first suggested as an explanation for the failure of two different BCGs in the Chingleput trial (125), but this hypothesis could not be supported by animal challenge studies. Interest in the hypothesis has been sustained by increased evidence of genetic heterogeneity of Mtb strains (126,127), but data from appropriate studies are not available to address this possible factor in variable BCG efficacy. Natural history of tuberculosis. It has also been suggested that protection by BCG might reflect the local natural history of tuberculosis and, as such, be greater against primary infection or (endogenous) reactivation disease than against disease attributable to exogenous reinfection. The relevance of this hypothesis involves not only immune mechanisms but also the level and temporal trend infection risk; a declining risk of infection, as has occurred in many communities, will be associated with an increase in the average age at infection and a decrease in the probability of reinfection (127). Each of these trends may have important implications for protection by BCG. Despite the existence of a large body of literature discussing these various hypotheses, there is still no consensus regarding the variable efficacy of BCG in different studies and populations. 17

28 6. Future prospects In recent years there has been a dramatic increase in the number of candidate TB vaccines evaluated in research laboratories. Better understanding of the immunological deficits of BCG and impressive progress in knowledge of mycobacterial genomics have paved the way for promising new products. What is needed in terms of new TB vaccines is probably not one, but more likely two, or even three products with different profiles: a priming vaccine to replace BCG to be given early in life and before exposure to MTB; another one to boost antimycobacterial immune responses either early or later in life when latent TB is potentially installed, and possibly a therapeutic vaccine against active TB. It is unlikely that a vaccine can be identified which covers several or all of these functional profiles. Priming vaccines are intended for use in newborns or young infants, i.e. at a time-point when the individual s immune system has not yet been exposed to natural infection with Mtb or other mycobacteria. Booster TB vaccines are vaccines that ideally can be given together with other childhood vaccines during the first year of life, but also at almost any time-point, to schoolchildren, adolescents or adults. TB vaccines to be used against firmly established latent TB may require a different set of antigens than the ones that are expected to be active against primary infection, and the type of immune response induced may differ. Also, the fact that later in life TB can arise both from endogenous reactivation and from exogenous reinfection may need to be reflected in the antigenic composition of a potent booster TB vaccine. At least one of the new booster TB vaccine candidates has been shown to boost pre-existing anti-mycobacterial T-cell immune responses, but it is currently unknown if these booster responses will actually translate into improved protection against TB-induced pathologies. Therapeutic vaccines, i.e. those that are to be given to individuals with active TB disease, are usually intended as adjunct to antibiotic treatment, with the aim of shortening the duration of anti-tb chemotherapy (128). An argument can be made that since BCG is widely used, has a good safety record and provides some protection against non-pulmonary forms of TB in infants, we should develop a better BCG. Several approaches are currently underway to genetically improve BCG (129,130,131). Currently however, it is unknown which antigenic shortcomings render conventional BCG suboptimal as a vaccine. The fact that BCG s parent organism, M. bovis, has primarily evolved in an adaptation to bovine rather than human hosts has sparked numerous efforts to attenuate the actual human pathogen Mtb. Examples include vaccine candidates carrying mutation in genomic loci responsible for metabolic, regulatory or virulence pathways (132,133). Non-living TB vaccine candidates include protein subunit and virus-vectored vaccines. Protein subunit vaccines have been shown to be powerful vaccines against other diseases, e.g. hepatitis B or human papillomavirus and, due to their stability, ease of standardization and safety in the immunocompromized host, are certainly the first choice of the vaccine industry. In this context, proteins secreted by Mtb have received special attention as subunit vaccines because such antigens are among the first molecules 18 The Immunological Basis for Immunization Series - Module 5: Tuberculosis - Update 2011

29 of the pathogen to be encountered by the human immune system after infection. Several adjuvanted protein subunit vaccines were in clinical trials during 2010 (134,135). Virus-vectored approaches are currently receiving attention employing vectors that have already been used for development of HIV/AIDS and malaria vaccines. One of these, a vaccine based on a genetically modified version of the vaccinia virus, is the most advanced of all the new TB vaccine candidates, with a test-of concept (ToC) clinical trial ongoing in 2010 to establish preliminary efficacy (136). Another vaccine candidate, based on an adenovirus platform (137), had also entered a Phase IIb ToC trial in 2010 and, if proven efficacious, was expected to be ready for licensure sometime between 2018 and The current plans for use of virus-vectored vaccines or protein subunit vaccines imply their clinical evaluation and eventual use as either early boosters of a neonatal vaccination with live mycobacteria at one to six months of age, or as late boosters in a post-exposure situation in schoolchildren, adolescents or adults. However, there are also proponents for an inversed sequence, i.e. a non-living vaccine at birth followed by a booster vaccination with BCG or a new live vaccine at 3 4 months of age (138). Such an approach aims to avoid the severe adverse events observed in some HIV-infected infants who had been vaccinated with BCG at birth a time-point at which HIV infection cannot be diagnosed. 19

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40 The World Health Organization has provided technical support to its Member States in the field of vaccine-preventable diseases since The office carrying out this function at WHO headquarters is the Department of Immunization, Vaccines and Biologicals (IVB). IVB s mission is the achievement of a world in which all people at risk are protected against vaccine-preventable diseases. The Department covers a range of activities including research and development, standard-setting, vaccine regulation and quality, vaccine supply and immunization financing, and immunization system strengthening. These activities are carried out by three technical units: the Initiative for Vaccine Research; the Quality, Safety and Standards team; and the Expanded Programme on Immunization. The Initiative for Vaccine Research guides, facilitates and provides a vision for worldwide vaccine and immunization technology research and development efforts. It focuses on current and emerging diseases of global public health importance, including pandemic influenza. Its main activities cover: i ) research and development of key candidate vaccines; ii ) implementation research to promote evidence-based decision-making on the early introduction of new vaccines; and iii ) promotion of the development, evaluation and future availability of HIV, tuberculosis and malaria vaccines. The Quality, Safety and Standards team focuses on supporting the use of vaccines, other biological products and immunizationrelated equipment that meet current international norms and standards of quality and safety. Activities cover: i ) setting norms and standards and establishing reference preparation materials; ii ) ensuring the use of quality vaccines and immunization equipment through prequalification activities and strengthening national regulatory authorities; and iii ) monitoring, assessing and responding to immunization safety issues of global concern. The Expanded Programme on Immunization focuses on maximizing access to high quality immunization services, accelerating disease control and linking to other health interventions that can be delivered during immunization contacts. Activities cover: i ) immunization systems strengthening, including expansion of immunization services beyond the infant age group; ii ) accelerated control of measles and maternal and neonatal tetanus; iii ) introduction of new and underutilized vaccines; iv ) vaccine supply and immunization financing; and v ) disease surveillance and immunization coverage monitoring for tracking global progress. The Director s Office directs the work of these units through oversight of immunization programme policy, planning, coordination and management. It also mobilizes resources and carries out communication, advocacy and media-related work. Department of Immunization, Vaccines and Biologicals Family and Community Health World Health Organization 20, Avenue Appia CH-1211 Geneva 27 Switzerland vaccines@who.int Web site: ISBN