Historical Review of BCG Vaccine in Japan

Jpn. J. Infect. Dis., 60, 331-336, 2007

Invited Review Historical Review of BCG Vaccine in Japan Saburo Yamamoto1,2* and Toshiko Yamamoto1 1Department of Microbial and Molecular Pathogenesis, Texas A & M University Health Science Center, College of Medicine, Texas, USA, and 2Ex Laboratory of Tuberculosis Control, Department of Bacterial Pathogenesis and Infection Control, National Institute of Infectious Diseases, Tokyo 208-0011, Japan (Received June 29, 2007) CONTENTS: 1. Introduction 4. Quality control 2. BCG vaccine in Japan 5. Vaccine efficacy 2-1. Freeze-dried vaccine 6. Adverse reactions 2-2. Percutaneous administration 7. Genetic characterization of BCG strains 3. Vaccine production 8. Aspects of BCG vaccines

SUMMARY: Bacillus Calmette and Guérin (BCG) was introduced to Japan in 1924 by Kiyoshi Shiga and has been propagated for research purposes ever since propagation is accomplished using a glycerin-bile-potato mixture in the same manner used by Calmette and Guérin. To prepare a stable and safe freeze-dried BCG vaccine, several joint research projects were organized in 1949. At the National Institute of Infectious Diseases (formerly the National Institute of Health), the 172nd passage of BCG from the first culture was freeze-dried in 1961 and was used as the origin of the Japanese BCG strain, Tokyo-172. The Tokyo-172 was registered as an International Reference Strain in 1965 by the World Health Organization. In 1967, a multiple puncture method for BCG vaccination using a plastic cylinder implanted with nine fine needles at one end was introduced to Japan; thereafter, percutaneous administration replaced intradermal injection. The efficacy and adverse reactions of BCG vaccines as well as recent knowledge on the genetic characterization of BCG is also discussed.

lion children throughout the world have been vaccinated. 1. Introduction Tuberculosis (TB) is a pandemic disease that represents a 2. BCG vaccine in Japan major public health and economic problem throughout the world. Mycobacterium tuberculosis, the causative agent of 2-1. Freeze-dried vaccine TB, is estimated to have infected nearly one-third of the The BCG strain, which was donated by Calmette himself, world’s population, with an annual incidence of approximately was brought to Japan by Kiyoshi Shiga (1890-1957), one of eight million and two million tuberculosis from deaths each the discoverers of the dysentery bacillus, in November 1924. year. The strain was been maintained and utilized for research by Robert Koch (1843 -1910) first discovered M. tuberculosis Professor Imamura at Osaka University, while Professor Sato in 1882; his discovery prompted numerous lines of research was charged with the maintenance of the strain at the Insti- to create prophylactic and therapeutic vaccines against tuber- tute of Infectious Diseases at University of Tokyo. The strain culosis. In 1908, Albert Calmette (1863-1933) and Camille was removed from University of Tokyo to the Research In- Guérin (1872-1961) at the Pasteur Institute in Lille, France, stitute for Tuberculosis (RIT) of the Japan Anti-tuberculosis started working with a virulent strain of Mycobacterium bovis Association in 1940, where Ken Yanagisawa was charged that had been isolated by Edmond Nocard (1850-1903) from with the monthly propagation of the strain using a glycerin- a cow with tuberculous mastitis. Calmette and Guérin sub- bile-potato medium. In 1947, the strain was transported from cultured the organism every 3 weeks on potato slices cooked RIT to the National Institute of Infectious Diseases (NIID) in beef bile, supplemented with glycerol. In 1921, after 230 (formerly known as the National Institute of Health [NIH]) successive passages over the course of 13 years, they found and was maintained in the same manner as that used by that the resulting culture was stable to reversion to virulence Calmette and Guérin. The BCG strain has been utilized for but retained limited invasiveness in experimental animals. the production of vaccine and for research ever since. The first human vaccination using this attenuated strain, Tuberculosis became endemic in Japan after World War named the bacillus of Calmette and Guérin (BCG), was II as a result of social confusion and the critical status of applied to a newborn whose grandmother had pulmonary national sanitation programs. Consequently, the mortality rate tuberculosis in Paris in 1921. The BCG vaccine was adopted of tuberculosis increased to more than 250 per 100,000 popula- by the Health Committee of the League of Nations in 1928, tion, peaking among individuals aged 25 to 29 years. Accord- and the mass vaccination of children was begun and was ingly, a BCG vaccination program was considered an urgent adopted by many countries, after newer and safer production priority, and a safe, stable and reliably potent vaccine--such processes were implemented. Since then, more than one bil- as a freeze-dried form--was needed. Research on a freeze- dried BCG vaccine had begun in Japan in 1943, and the mass *Corresponding author: Mailing address: 4-1-18 Nakacho, Koganei- production of dried BCG vaccine was initiated in 1947. BCG shi, Tokyo 184-0012, Japan. E-mail: [email protected] vaccination using the freeze-dried vaccine became compul-

331 sory under the Immunization Law in 1948 for all tuberculin- leaving tiny flat and sometimes depigmented slight scars. negative individuals aged 25 years or under. Subsequently, Lymph node involvement almost did not occur. A total of BCG vaccination emerged as a national policy for the control 114 newborn babies in a maternity hospital were vaccinated of tuberculosis; since 1949, the usage of all freeze-dried BCG using the multiple puncture method and were followed for vaccine has required official approval by the NIH in Tokyo. 1 year; an 89.5% tuberculin-positive rate and a reaction In 1961, the 172nd passage of BCG from the first culture diameter of 14.0 mm was obtained 1 year after vaccination was freeze-dried and selected as the origin of the Japanese in this cohort, with only one case of lymph node swelling. BCG strain, Tokyo-172. This lot has been kept at the NIID. The swollen lymph node was about the size of a soybean and Tokyo-172 was registered as an International Reference Strain was observed 3 months after vaccination; however, the swol- in 1965 by the World Health Organization (WHO). Thereafter, len lymph node was no longer palpable after 6 months. The commercial laboratories began producing and distributing the formation of keloid was not reported as a troublesome side BCG vaccine nationwide and also internationally, after in- reaction in this series, and the scars had disappeared in 40% house and national assessments. Today, only the Japan BCG of the vaccinated infants 1 year after vaccination. These Laboratory produces BCG vaccine in Japan. In 1951, BCG results suggested that newborn babies could be easily and vaccination was enforced by the Tuberculosis Prevention Law effectively vaccinated using a needle-implanted cylinder and so that all persons under the age of 30 years and employees percutaneous dried BCG vaccine made from the Japanese of all of ages were required to be vaccinated intradermally BCG strain (the multiple puncture method) without trouble- if they exhibited a negative to tuberculin reaction. In 1974, some side reactions. the Tuberculosis Prevention Law was revised so that all infants and children under the age of 4 years were primarily 3. Vaccine production vaccinated and all 1st grade students of elementary school or junior high school were revaccinated if they exhibited a nega- The tuberculosis mortality rate, which had been the leading tive tuberculin reaction. The Tuberculosis Prevention Law cause of death in Japan for several years, decreased steadily was further revised in 2003 so that only infants under the age after the war from over 200 per 100,000 population in 1951 of 6 months are primarily vaccinated, and no tuberculin tests to half that rate and falling as low as 1.8 per 100,000 popula- have been conducted since then. The Tuberculosis Preven- tion in 2004, to become the 25th leading cause of death in tion Law was integrated into Infectious Diseases Prevention Japan. The reduction in the mortality rate was most dramatic Law in 2007. among younger age groups. 2-2. Percutaneous administration Freeze-dried BCG vaccine is one of the most beneficial In Japan, although intradermal BCG vaccination success- achievements of medical science in Japan. The purpose of fully achieved high tuberculin conversion rates, complaints BCG vaccination is to provide resistance to tuberculous of side reactions--mainly local lesions like ulcers, abscesses infection in the form of cellular immunity; this has been shown and ugly scars--mounted up to such an extent that an improved to be induced most effectively using living BCG, but not dead vaccination method was urgently requested. After more than bacilli, in humans. Therefore, each dose of BCG vaccine 10 years of cooperative studies examining percutaneous BCG should contain as many viable organisms and as few dead vaccination, a multiple puncture method using a plastic cyl- bacilli as possible to ensure a high potency. To perform the inder implanted with nine fine needles at one end was finally necessary basic and applied studies on freeze-dried BCG chosen in 1967. Thereafter, the multiple puncture vaccina- vaccine and its administration, several joint research projects tion method completely replaced intradermal injections in have been successively organized since 1949 to concentrate Japan. Local lesions noticeably decreased, both in intensity on the production, control and vaccination method for the and duration, compared with those produced by intradermal dried vaccine. vaccination. These research teams performed a joint study on the corre- To produce the vaccine, an ampoule containing 80 mg, 40 lation between viable numbers of dried BCG vaccine prepara- mg or 12 mg of dried BCG is reconstituted with 1.0 mL, 0.5 tions and the tuberculin conversion rate following intradermal mL or 0.15 mL of physiological saline, respectively. A drop vaccination after completing a pilot field study that had of suspended vaccine with a concentration of 80 mg BCG indicated a positive correlation. The researchers found that per mL is spread over an area about 15 × 30 mm in size on the tuberculin conversion rate at 1 and 3 months after the upper arm; then, two strong pushes are separately made inoculation was well correlated with the number of injected using the needle-implanted cylinder, producing a total of 18 viable units. On the basis of these results, they concluded punctures. The intensity of the skin punctures was found to that one dose of BCG vaccine should contain as high a num- be correlated with the tuberculin-positive rate and the appear- ber of viable bacilli and as few dead organisms as possible to ance of local lesions following vaccination. The viability of increase the vaccine’s immunizing ability. With this in mind, the vaccine was also correlated with the tuberculin conver- the researchers concentrated their efforts toward the mass sion rate and the mean size of the tuberculin reaction 1 production of dried BCG vaccine so as to achieve the highest month after vaccination: an 80% positive rate and a tuberculin proportion of viable bacilli in 1 dose of BCG vaccine as reaction with a mean size of 15 mm would be expected when possible and to maintain this level of viability until inocula- a vaccine with 15 million culturable particles in 1 mg of BCG tion. The joint research projects exploring these production was used, and a 60% positive rate and a mean size of 12 mm objectives concentrated on the following points. would be expected for a vaccine with a 10 million viable Resistance of young BCG cultures to freeze-drying: To count. Although small pustules and crusts were induced by improve the viability of the freeze-dried BCG vaccine, the the multiple puncture method at 1 month after vaccination, sensitivity of growing BCG to the freeze-drying process was hypertrophic scars--particularly ugly keloids--were relatively examined. As a result, younger cultures in the logarithmic infrequent (less than 1%), even after revaccinations using the growth phase were found to be highly resistant to the freeze- new puncture method because most of the pustules healed drying process, compared with cultures in the stationary

332 phase--during which large quantities of bacterial mass were dried BCG vaccine, the vaccine container must be sealed being harvested to prepare the liquid BCG vaccine. When under a high vacuum for maximum viability. The replace- younger Sauton cultures of 8 or 9 days were used for the ment of air with dried nitrogen has been useful for maintain- mass production of vaccine, the potency of the Japanese ing viability. The viability of freeze-dried BCG decreases very freeze-dried BCG vaccine improved. The dried vaccine pro- rapidly under normal air pressures, even when stored at 4°C. duced from the younger BCG cultures was found to have Consequently, sealing freeze-dried BCG vaccine ampoules a superior immunizing potency in humans. In addition, the in vacuo is indispensable. Japanese strain of BCG (Tokyo 172) was shown to be con- Procedure for freeze-dried BCG vaccine production: siderably more resistant to freeze-drying, compared with other At the BCG production laboratories, the Tokyo 172 strain is BCG strains used in various countries. used in a seed lot system to transfer the BCG culture to a Addition of preservatives to the dried BCG vaccine to Sauton-potato medium, from which it is further transferred protect viable bacilli from freeze-drying: The discovery that to a Sauton medium for vaccine culturing. The second Sauton sodium glutamate could be used to produce heat-stable, culture is usually harvested after 7 to 10 days. After washing freeze-dried BCG vaccine in place of sucrose or lactose was with the suspension fluid, the dehydrated bacterial mass a remarkable advance in stabilizing the potency of freeze- (approximately 70% moisture content) is homogenized in a dried BCG vaccine. BCG vaccine can remain sufficiently round flask containing crystal or stainless-steel balls rotating viable when stored at 5°C for 2 years or even at 37°C for 4 at a high speed. The moist bacteria is triturated homo- weeks, with typical survival rates of over 50%. Thus, dried geneously, and suspended in a solution of sodium glutamate glutamate BCG vaccine can be transported and used even at not higher than 15 w/v% to make the moist bacterial con- without refrigeration for short periods of time, even in tropical centration to 80 mg/mL to serve as the final bulk. The final environments. The effect on the allergenic potency of storing suspension is dispensed into brown-glass ampoules in aliquots dried sucrose or glutamate BCG vaccines at 5°C or 37°C of 0.15 mL, 0.5 mL or 1.0 mL, depending on the desired doses, was compared. When both vaccines were stored at 5°C, the prior to pre-freezing. The freeze-drying is carried out in a resulting tuberculin allergy was similar. However, when the chamber-type freeze-dryer at freeze-dried and hermetically two vaccines were stored at 37°C for the same period of time, at a pressure not higher than 13.3 Pa. After being taken out of the allergy induced by vaccination with the glutamate vaccine the freeze-dryer, the dried ampoules are handled under pre- was much stronger. cautions against moisture absorption and contamination and Moisture content of dried BCG vaccine: The storage of are then sealed hermetically. Suitable numbers of ampoules dried BCG vaccine for long periods of time, even at low extracted at random from the final products are submitted by temperatures, requires a low moisture content in the freeze- lot to in-house and national assessments before use. dried product. The optimal moisture content for living BCG Characteristics of the Japanese freeze-dried BCG vac- organisms to survive in mass-produced vaccines in Japan was cine: The freeze-dried BCG vaccine produced from the less than 3%, as measured using the Abderhalden method. At Tokyo 172 strain has a high potency in humans, as demon- higher moisture contents, the dried vaccines lost their viabil- strated by the size of the tuberculin reaction and the local ity more rapidly, especially at higher temperatures. Dried lesion. However, the Japanese strain has been reported to have glutamate vaccine with a moisture content of less than 3.0% a lower residual virulence and a weaker immunogenicity. could be maintained at a satisfactory level of viability at 5°C Since the Japanese strain characteristically has a much higher for 2 years and at 37°C for 1 month. During the freeze- viability before and after freeze-drying, compared with other drying process, the BCG vaccine can be heated up to around strains, the weaker antigenicity of the living vaccine might 37°C in vacuo to reduce the residual moisture to less than have been supplemented quantitatively by the relatively high 3.0%. For freeze-dried BCG vaccine intended for vaccina- number of living bacilli present in the vaccine at the time tions using the multiple puncture method, which requires a of vaccination. In addition, the Japanese freeze-dried BCG high bacterial content of 12, 40 or 80 mg per ampoule, a higher vaccine generally produces less lymph node involvement, moisture content may be recommended for the preparation probably because of the reduced residual virulence. As vac- of vaccines with a high viability and sufficient stability dur- cines for human use should be evaluated with regard to both ing storage. efficacy and side reactions, the Japanese freeze-dried BCG Protection of dried BCG vaccine from sunlight: BCG is vaccine is considered to exhibit favorable characteristics. especially sensitive to both direct and indirect sunlight. Viable BCG in both liquid and freeze-dried vaccines is killed 4. Quality control by sunlight within a short period of time: the viability decreases to 1/15 in 60 min in reconstituted BCG vaccine and to 1/10 Extracted samples of bulk BCG suspension prior to freeze- in 60 min in dried BCG vaccine. Therefore, the interior of drying and ampoules of freeze-dried BCG vaccine repre- the vaccine production facility should not be exposed to any senting each lot are submitted to the National Institute of sunlight. To prevent the exposure of the dried vaccine to sun- Infectious Diseases for national quality-control checks, in light during transport or at the vaccination clinic, the vaccine addition to in-house assays by the manufacturer in according should be either covered with black paper or sealed in brown- to the Minimum Requirements for Biological Products (Min- colored ampoules. These countermeasures can successfully istry of Health, Labour and Welfare, Japanese Government). prevent the harmful effects of indoor daylight on reconstituted The crude bulk BCG suspension is subjected to a guinea pig BCG vaccine. Although the effect of sunlight on freeze-dried test to ensure the safety of the vaccine and the absence of BCG vaccine seems to be comparatively mild, its bacteri- virulent mycobacteria. Control tests on the freeze-dried BCG cidal effect was much more obvious in the reconstituted BCG vaccines, including the above-mentioned safety test, were first suspension if it was not protected. established in 1949 in Japan and include the following tests. Necessity of vacuum-sealing of vaccine ampoules: Since Freeze-dried BCG vaccines have been examined using this normal air pressure has been found to harm the viability of national assay for almost 60 years.

333 Color and physical characteristics of BCG vaccine: coagulated egg slant medium. The contents of 10 ampoules Dried BCG vaccine is a white powder-like substance that can are reconstituted with physiological saline to the vaccine con- be easily moved by shaking; it should be easily dissolved centration, as indicated on the label, and further diluted in when diluted with physiological saline to produce a homoge- sterile water to a concentration of 0.5 × 10–5 mg of moist neous suspension. The pH of the dissolved vaccine should be bacteria per mL. within the range of 5.5 to 7.0. Each dilution is inoculated in a medium tube at a dose of Staining test: Smears are made directly from undiluted 0.1 mL; the cultures are incubated at 37.5 ± 0.5°C for 4 weeks. suspensions or from diluted specimen in physiological saline. At the end of the culture period, the number of colonies Smears are stained by Gram’s procedure and the acid-fast appearing in one medium tube is counted. The sum and un- procedure, and examined microscopically to verify the mor- biased variance of the square root of the value are calculated. phological appearance of the bacteria. The Ziehl-Neelsen The number of culturable particles contained in the test staining test is applied to identify and determine the homoge- sample should be within a range specified upon statistical neity of the bacteria in the vaccine under a microscope. The comparison of the results using the sum and the unbiased Japanese BCG vaccine usually shows many aggregates of variance. acid-fast bacilli in various sizes in addition to single bacilli Total bacterial content test: The test sample is diluted to after reconstitution. a concentration of 1 mg/mL in saline, and the absorbance of Safety test for free from virulent mycobacteria: A test the dilution is measured using a spectrophotometer at 470 sample of 2.5 mg/mL is injected into six normal guinea nm and a light path of 10 mm. The absorbance should be no pigs weighing between 300 and 400 g; the injections are per- more than the determined value for each spectrophotometer. formed intramuscularly into the thighs of three animals and Moisture content test: The moisture content is determined subcutaneously into the lateral abdomens of the other three by measuring the sample’s weight loss as a result of water animals. The sample is injected at a dose of 1.0 mL (2.5 mg evaporated during heating and reduced pressure, according of moist bacteria), and all the inoculated animals are observed to the Abderhalden method. A portion of the dried vaccine for at least 6 weeks before autopsy. None of the animals should powder collected from several final products is further dried show any abnormal signs, other than lesions at the site of over phosphorus pentoxide (P2O5) or silica gel at a pressure injection and regional lymph nodes with an apparent tendency of not more than 0.6 kPa at 58-62°C for 3 h to measure the toward recovery at the time of autopsy. No animal should residual water content of the vaccine. The moisture content show progressive tuberculous lesions or other abnormal should be no more than 3.0%. changes, except mild lesions with a tendency toward recovery at autopsy performed at the end of the observation period. At 5. Vaccine efficacy least two-third of the animals must survive the observation period. All of the lots examined during the past nearly 60 BCG is the only vaccine against tuberculosis being used years have been proven to be safe and free from virulent throughout the world. BCG induces specific immune responses mycobacteria based on the above criteria. to mycobacterial antigens and appears to be able to elicit pro- Sterility test: The sterility test confirms the absence of tective immunity against tuberculosis. In general, the protec- microorganisms that can be detected using a sensitive me- tive effect of a vaccine is determined by an epidemiological dium. The fluid thioglycolate and the soybean casein digest study to compare the incidence of the disease with and with- medium have been used as test media for many years. The out vaccination. A total of 17 controlled trials demonstrating ampoules of dried BCG vaccine are sampled from each filling the efficacy of BCG vaccine in humans have been reported lot and are inoculated into the fluid thioglycolate medium since 1935 (1). Table 1 summarizes the results of the major and the soybean casein digest medium, the sensitivity of which trials. The first large-scale field trial of BCG efficacy was has been confirmed. Each inoculated medium is incubated at evaluated in tuberculin-negative children and adolescents both 30-35°C or 20-25°C, respectively, for 7 days, then between the ages of 0-20 years and belonging to eight North checked at intervals by visual examinations. American Indian tribes. Pulmonary tuberculosis occurred in Potency test: The potency test is performed by determin- 238 of the 1,457 unvaccinated persons and in 64 of the 1,551 ing the number of culturable particles in Ogawa’s solid persons given BCG vaccination, with a protective efficacy

Table 1. Summary of major randermized trials Area Strain Efficacy (%) NTM exposure* Reference Haiti Montreal 80 Vandiviere, H.M. et al. (5) Canada Montreal 80 low Ferguson and Simes (32) British Danish 77 low Hart and Sutherland (4) North American Indians Phipps 75 low Aronson, J.D. et al. (2) Chicago Tice 75 low Rosenthal, S.R. et al. (3) South Africa Glaxo 37 Coetzee and Berjak (33) Puerto Rico Birghaug 29 high Comstock, G.W. et al. (9) India Madras 14 high Frimodt-Moller, J. et al. (6) Georgia Tice 0 high Comstock and Webster (7) Georgia, Alabama Tice 14 high Comstock, G.W. et al. (8) South India French, Danish 0 high Tripathy, S.P. (10) *NTM, nontuberculosis mycobacteria. Revised from Bloom, B.R. and Fine, P.E.M. (1994): The BCG experience: Implications for future vaccines against tuberculosis. p. 531-557. In Bloom, B.R. (ed), Tuberculosis - Pathogenesis, Protection and Control. ASM Press, Washington D.C.

334 against active tuberculosis of 75% during the 9 -11 year lymphadenitis is greater among newborns than among older follow-up period (2). Another trial was performed in high-risk infants and children, especially when a full dose of vaccine is infants in Chicago by Rosenthal and colleagues. They found given; therefore, the WHO recommends using a reduced dose that tuberculosis developed in 59 of the 1,665 unvaccinated in children younger than 30 days. persons, while active disease occurred in 16 of the 1,716 vac- The mean risk of osteitis after BCG vaccination has varied cinated individuals (efficacy = 75%) after a follow-up period from 0.01 per million in Japan to 300 per million in Finland of 12-23 years (3). The Medical Research Council of Great (15,18-21). Fatal disseminated BCG infection has been Britain (MRC) conducted a large controlled trial of the Dan- reported to occur at a rate of 0.19 to 1.56 cases per million ish strain of BCG vaccine in 27,400 school children between vaccines, occurring mostly in patients with severe defects in the ages of 14-16 years. After a 15-year follow-up period, cell-mediated immunity, such as severe combined immuno- 243 cases of tuberculosis had developed among 13,300 per- deficiency and human immunodeficiency virus (HIV) infec- sons in the unvaccinated group, compared with 56 cases tion (22). The safety of the BCG vaccine in children and adults among the 14,100 persons in the vaccinated group (4). who are infected within HIV is unknown. Currently, WHO Vandiviere and colleagues also found that BCG conferred a recommends giving the BCG vaccine to asymptomatic HIV- high degree of protection. A total of 3,174 villagers in Haiti infected infants who live in high-risk areas for tuberculosis. were randomly assigned to either receive the BCG vaccine BCG is not recommended for symptomatic HIV-infected or to remain unvaccinated. The length of the follow-up period infants or for persons known to be or suspected of being was 3 years. The efficacy of BCG vaccination was 80% (5). HIV-infected if they are at minimal risk for infection with M. On the other hand, several trials have reported that the efficacy tuberculosis (23). of BCG vaccination was low or none (6-10). One of those, co-sponsored by the Indian Council of Medical Research, 7. Genetic characterization of BCG strains WHO and the United States Public Health Service, was carried out in Chingleput, India, between 1968 and 1971. More BCG vaccines are divided into the early strains, including than 300,000 villagers older than 1 month of age were ran- the Japan, Birkhaug, Russia and Brazil BCG strains, that were domly divided into either a BCG-vaccinated or a saline- obtained between 1924 and 1926, and the late strains, such inoculated group. During the 15-year follow-up period, vacci- as the Pasteur, Danish, Glaxo and Connaught BCG strains nation provided some protection against tuberculosis in that were obtained after 1931 (24). Recently, the genomic children; among adults, however, the vaccinated group had a compositions of the various BCG daughter strains have been higher prevalence of tuberculosis than the unvaccinated group. studied by performing comparative hybridization experiments Thus, it is difficult to draw a clear conclusion regarding the using a DNA microarray (25-28). Regions deleted from the efficacy of the BCG vaccine based on the results of random- BCG vaccines, relative to the virulent M. tuberculosis H37Rv ized trials alone because the results of these trials have reference strain, were found by sequencing across the miss- varied from 0 to 80%. Several case-controlled studies of BCG ing segment of the H37Rv genome. Eleven regions of H37Rv vaccination have also been performed to assess the efficacy were found that were absent from one or more virulent strains of the BCG vaccine for tuberculosis control. The results of of M. bovis; five additional regions were present in M. bovis these studies also varied widely, with the effectiveness of but absent from some or all BCG strains, and the presence of vaccination ranging between 2-84% (11). 16 deleted regions (RD1 - RD16) were confirmed (24). RD1 Several explanations have been offered for these conflict- is missing from all BCG strains and is speculated to coincide ing results, including differences in trial design, different with the attenuated virulence of M. bovis. RD2 is present in immunogenicity of the vaccine strains, or genetic inconsist- the early BCG vaccines but is missing from the late BCG encies between individual strains. However, the most plausible vaccines. The loss of RD2 has been confirmed to coincide explanation seems to be differences in exposure to non- with the deletion of the mpt64 gene, which encodes the anti- tuberculous mycobacteria as environmental mycobacteria be- genic protein MPT64. Some of these regions are missing from tween individuals living in hot climates versus those living only one strain; RD8 is missing from Canadian BCG strains in cold climates (12). A meta-analysis of BCG vaccination like the Frappier and Connaught strains, while RD14 is miss- for the prevention of tuberculosis has been performed using ing from only the Pasteur strain, and RD16 is missing from prospective randomized trials and case control studies (13,14). only the Moreau strain. A small deletion of 22 base pairs in They concluded that on average, BCG vaccination was about RD16 in the Japanese strain was uncovered by chance in a 50% protective in preventing tuberculosis. multiplex PCR-based analysis of BCG strains and was confirmed to coincide with a morphological difference (29, 30). Recently, BCG Pasteur 1173P2 has been subjected to a 6. Adverse reactions comparative genome and transcriptome analysis, and the For more than 80 years, BCG vaccines have been admin- 4,374,522-bp genome has been concluded to contain 3,954 istered to billions of individuals throughout the world, and protein-coding genes, 58 of which are present in two copies today BCG is considered one of the safer vaccines available. as a result of two independent tandem duplications, DU1 and Regional lymphadenitis most commonly occurs within the DU2 (31). first 5 months after vaccination. Several factors contribute to its occurrence, including the vaccine strain, the total number 8. Aspects of BCG vaccines of viable and nonviable bacilli in the vaccine preparation, and the dose of BCG given, in addition to the age and immune Although the current vaccine strains are all descendants of status of the vaccinee. Certain BCG strains, such as the Japa- the original M. bovis isolate, subsequent passages under nese strain and the Moreau strain, are rarely associated with different laboratory conditions have resulted in a variety of lymphadenitis, whereas the French (Pasteur) strain gives rise BCG strains including the French strain Pasteur 1173 P2, the to a much higher incidence (15-17). The risk of suppurative Danish strain 1331, the Glaxo strain 1077 and the Japanese

335 strain Tokyo 172; these various strains exhibit phenotypic as 14. Colditz, G., Brewer, T., Berkey, C., et al. (1994): Efficacy of BCG well as genotypic differences, as mentioned above. In terms vaccine in the prevention of tuberculosis. JAMA, 271, 698-702. 15. Lotte, A., Wasz-Hockert, O., Poisson, N., et al. (1984): BCG complica- of efficacy, no consensus exists as to which strain of BCG is tions: estimates of the risks among vaccinated subjects and statistical appropriate for general use. Reconstituted vaccines contain- analysis of their main characteristics. Adv. Tuberc. Res., 21, 107-193. ing both living and dead bacilli, and the number of culturable 16. Gheorghiu, M. (1978): Potency and suppurative adenitis in BCG vacci- particles and the biochemical composition of the vaccines, nation. Dev. Biol. Stand., 41, 79-84. vary considerably depending on the strain and production 17. Muzy, S.G.R., Sant’Anna, C.C., Lapane, S.P.R., et al. (1983): Intradermal BCG vaccination complications -- analysis of 51 cases. Tubercle, 64, method of the vaccines. A clinical trial in South Africa was 23-27. recently performed to examine the immune responses of vac- 18. Kroger, L., Korppi, M., Brander, E., et al. (1995): Osteitis caused by cinated infants after percutaneous or intradermal administra- bacillus Calmette-Guerin vaccination: A retrospective analysis of 222 tion of the Japanese BCG or intradermal administration of cases. J. Infect. Dis., 172, 574-576. the Danish BCG. The results suggested that the BCG strain 19. Bergdahl, S., Fellander, M. and Robertson, B. (1976): BCG osteo- myelitis: experience in the Stockholm region over the years 1961-1974. and route of delivery may determine the clinical outcome of J. Bone Joint Surg., 58B, 212-216. vaccination by influencing the immune response that leads 20. Vanicek, H. (1988): Complications after initial BCG vaccination in a 5- to protection against tuberculosis (34,35). 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