Biologic&
(1990) 18, 135-141
REVIEW The Present and Future Role of BCG Vaccine in Tuberculosis Control BCG Laboratory
Marina Gheorghiu lnsfitut Pasteur, 25 rue du Dr Roux, 75724 Paris C6dex 15, France
Introduction This review is built around four commonly asked questions about BCG. The replies to these questions will define the role that the BCG has played in the control of tuberculosis to date and the possibilities for the development of improved vaccines. The (1) (2) (3) (4)
questions are: What do we mean by BCG? Are there immune responses to BCG? How effective is BCG in preventing tuberculosis? Can a better vaccine be developed?
What do we mean by BCG? The reply, although well known, is worth repeating; there is a bacterial strain called BCG; there is a BCG vaccine; there is BCG vaccination. The BCG strain The Calmette and Guerin bacillus (abbreviated to BCG) was derived from Mycobacterium bouis, a strain that causes bovine tuberculosis. Its virulence was attenuated and the strain stabilized in non-virulent form between 1908 and 1921 by 230 successive passages on media impregnated with beef bile. The resulting strain, the BCG variant, differed from the parent strain by losing its pathogenic potential. However, most importantly, the strain retained its immunogenicity since it could produce a delayed type hypersensitivity (DTH) to tuberculin and could protect against virulent strains of M. Louis.’ The growth, morphology and chemical composition of the daughter strain are similar to those of the virulent parent, but are sufficiently different to allow discrimination between them. BCG daughter strains The development of the BCG strain by Albert Calmette and Camille Guerin at the Pasteur Institute 1045-1056/90/020135+07
$03.00/O
resulted in the first vaccination in 1921. This was followed by the dissemination of the strain to many countries throughout the world. All the strains have been maintained for more than 30 years by passage on natural culture media every 3 weeks. The precise nature of these media varies from one country to another and may well be the cause of differences in the reactogenicity, first noticed in the 1950s. In the 19609, the WHO recommended the stabilization of the biological characteristics of these daughter strains by lyophilization and storage of the samples at low temperature. This was to avoid the production of less immunogenic mutants and their selection by successive passages on culture media. Inter-laboratory studies were undertaken under the aegis of the WHO and IABS (International Association of Biological Standardization) to delimit the differences between the BCG daughter strains better, which are generally known by the name of the country or laboratory in which they are kept. The results showed unequivocally; that nowadays the strains are not identical. The differences were in the morphology, biochemistry, growth and degree of immunogenicity, of which the cellular type immunity engendered by BCG is important.M There are four daughter strains from which large quantities of vaccine are produced worldwide and are supplied by UNICEF. The French strain 1173 P2 is used in France by ‘Pasteur Vaccins’ and in 14 other countries for their own production of vaccine. The upkeep and biological characteristics are known and have been described, since this strain has been used in many studies.3y6 The Glaxo strain 1077 was obtained from Copenhagen in the 1950s. It is, however, different from the Danish strain 1331 from which it is derived. Comparative studies have shown large differences between the two strains, resulting in the different names and the use of a larger bacterial mass per dose in the Glaxo vaccines.3*s*7*s These biological changes seem to be due to the mode of culture on media (Q1990 The International
Association ofBiological
Standardization
136
M. Ghemghiu
containing tensioactive agents. The strain is now used in England by ‘Evans’ and in France by ‘Merieux’. The Japanese strain 172 seems to have been selected for its high resistance to lyophilization. It is more resistant than other strains, and is more heat-stable. For this reason, 10 years ago, it became the reference strain for quality control of lyophilized vaccines. However, it contains so many viable units per unit weight that it is unlike any other strain. This led the WHO to renounce this strain in favour of ‘Working Reference No. 3’ based on the French strain 1173 P2.2,3xg Other daughter strains are in use worldwide. The best known include: Moreau (Brazil), Montreal (Canada, Institut Armand Frappier and Connaught), Russian (USSR) and Tice (USA). Laboratory studies and observations in man have shown that some strains are ‘strong’, like the French strain 1173 P2 and the Danish strain 1331 (Copenhagen), and some strains are ‘weak’ such as the Glaxo 1077 and Japanese 172 strains.2,3 It is very difficult to show that one strain is clearly superior to another in the protection of man against tuberculosis. No prospective comparative studies of the weak and strong strains have been done. However, animal studies of this type have been performed and show higher immunogenicity of the strong strains.“*‘l It has been shown that does Ptimes higher than the strong strains must be used with weak strains to obtain the same degree of delayed type hypersensitivity (DTH) and protection. Other immunity indicators such as local granuloma and residual virulence are also more apparent after vaccination with the strong strains314 The greater degree ofprotection afforded by the strong strains can be seen in the guinea-pig model proposed by D. Smith. This model is the closest to the physiopathology of infection in man although the results of the two systems may not entirely correlate.10-12 Table
1. Protection
Country Brazil (Sao Paulo) Thailand (Bangkok) Togo (Lome) Burma (Rangoon) Argentina (Buenos-Aires) Israel
against
GIOXO
Severe and extropulmonaryforms of TB
50% 60 70 80 90 ~~~~~-~~‘~~~~~~~~~‘~~~~~~~~~t~~~~~~~~~~~~~-~~~~~
TTokyo
TGICXOT
TTokyo
All forms of TB
Severe forms of TB
Case-control Contact Contact Case-control
53% 50% 39%
87-90% 60% 80% 80%
Case-control TBcohort
74% 24%
Retrospective Contact
72%
Tokyo
The experimental results which indicate that strong strains are more immunogenic in animal experiments are now supported by findings in followup case-control studies in children, and/or casecontacts of smear-positive cases. Indeed, the protective role of BCG against serious forms of tuberculosis in young children (95%) and globally against all forms of tuberculosis (74%) is confirmed by the data published in the WHO Weekly Epidemic Record13 and illustrated in Fig. 1 and Table 1. There is, however, evident variation between the strains used. The overall protection for less severe forms of tuberculosis conferred by strain Tokyo 172 is 39% to 53%, for strain Glaxo 1077 24% to 50% and for Pasteur 1173 P2 72% to 74%. Other studies, such as the one performed in Hong Kong, showed higher protection with the Pasteur than with the Glaxo strain,37 and the Seoul (South Korea) study has again shown good protection (72%) with the Pasteur strain.14 As concluded in the WHO Weekly Epidemic Record I3 the results obtained in children, even if less amb&uous than those of control trials, still may have been biased towards milder forms of tuberculosis which are more difficult to diagnose. However, animal experiments generally show higher protection with strong strains, and research into cell-mediated
tuberculosis Studies
TMo4
Do %
Figure 1. BCG strains ranking by percentage protection in man.
(pulmonary) Japan Korea (Seoul)
30% 40 50 60 70 80% 1~~~~~~~~~1~~~~~~~~~‘~~~~~~~~~1~~~~~~~~~~~~~~~~~~~ Tokyo T GlW.0 TGlaro lP;s,eu, ’ 11
AllformsofT8
64%
(extrapulmonary) 95% -
BCG strain used Moreau Tokyo Glaxo Tokyo Pasteur Glaxo
Tokyo Pasteur
(?I
137
BCG vaccine in TB control
immunity also often uses strong BCG strains. These facts, together with the findings in child case-control studies, have to be taken into account when choosing a given strain for vaccine production, even if it is not clearly proved that strong strains are superior to weak ones. For the latter, there is even less favourable data except that they produce a reduced percentage of lymphadenitis after vaccination. The side effect of the strong strains are the higher incidence of lymphadenitis, especially in neonates, as happened in Germany with the Danish strain,15 and more recently in Mozambique and Zimbabwe with the Pasteur strain.‘6,17 Studies performed with reduced doses of vaccine solved this problem in Germany.15 Other studies recently performed with the Pasteur strain showed that, when vaccinating neonates with a reduced dose of O-025 mg, the percentage of this lymphadenitis falls to O-7%, levels considered tolerable. The DTH was maintained over 80%, the amount needed to assure good immunization.18 The use of a weaker BCG strain is tempting for both vaccine producers and users, since immediate results without side effects assure tranquility for both. Some countries, such as the Ivory Coast, Algeria, etc. used such strain vaccines for decades, but with practically no results, so have switched to the Pasteur strain. After a short period with improved information and accuracy of vaccination, the side effect percentage was tolerable. The major aim of vaccination programmes is protection against tuberculosis, a long term goal. Vaccines prepared with strong strains therefore are not to be eliminated, and vaccination accuracy has to be very carefully thought out. The BCG vaccine
The vaccine is prepared from a given BCG strain, and composed of live attenuated bacilli capable of inducing protection against tuberculosis and delayed type hypersensitivity in the intradermal tuberculin test. The quality of the vaccine depends on several factors: the immunogenicity of the strain, and the ratio of live to dead bacilli in the preparation. If the ratio is high, the local reactions are lower and the immunogenicity higher, allowing use of lower doses to give satisfactory immunization with minimal side effects. The ratio of live to killed bacteria varies enormously (three logarithms) depending on the manufacture and culture methods used. Dispersed grown BCG avoids clumping of the bacilli and the destruction of the cells by the grinding of the bacillary mass common to the classic method.lg Similarly, the
method of lyophilization and preparation also influence the ratio of live cells. A percentage higher than 10% of that of a fresh suspension is required, with a stability and heat-stability of at least 20% after exposure to 37°C for one month. The BCG vaccination
Having chosen the most effective vaccine, the method of vaccination must be selected. The protection conferred depends not only on the vaccine, but also on: the route of vaccination; the dose; the time between vaccination and exposure to infection. Although it is generally accepted that the best method of vaccination is intradermal injection, this is difflcult in new-born infants who require: precision in the injection technique; precision in the dose; background data on the age, vaccine and doses to give DTH within the accepted norms (>50 to 100%) with a tolerable minimum of suppurative adenitis (
(1990) 18, 135-141
REVIEW The Present and Future Role of BCG Vaccine in Tuberculosis Control BCG Laboratory
Marina Gheorghiu lnsfitut Pasteur, 25 rue du Dr Roux, 75724 Paris C6dex 15, France
Introduction This review is built around four commonly asked questions about BCG. The replies to these questions will define the role that the BCG has played in the control of tuberculosis to date and the possibilities for the development of improved vaccines. The (1) (2) (3) (4)
questions are: What do we mean by BCG? Are there immune responses to BCG? How effective is BCG in preventing tuberculosis? Can a better vaccine be developed?
What do we mean by BCG? The reply, although well known, is worth repeating; there is a bacterial strain called BCG; there is a BCG vaccine; there is BCG vaccination. The BCG strain The Calmette and Guerin bacillus (abbreviated to BCG) was derived from Mycobacterium bouis, a strain that causes bovine tuberculosis. Its virulence was attenuated and the strain stabilized in non-virulent form between 1908 and 1921 by 230 successive passages on media impregnated with beef bile. The resulting strain, the BCG variant, differed from the parent strain by losing its pathogenic potential. However, most importantly, the strain retained its immunogenicity since it could produce a delayed type hypersensitivity (DTH) to tuberculin and could protect against virulent strains of M. Louis.’ The growth, morphology and chemical composition of the daughter strain are similar to those of the virulent parent, but are sufficiently different to allow discrimination between them. BCG daughter strains The development of the BCG strain by Albert Calmette and Camille Guerin at the Pasteur Institute 1045-1056/90/020135+07
$03.00/O
resulted in the first vaccination in 1921. This was followed by the dissemination of the strain to many countries throughout the world. All the strains have been maintained for more than 30 years by passage on natural culture media every 3 weeks. The precise nature of these media varies from one country to another and may well be the cause of differences in the reactogenicity, first noticed in the 1950s. In the 19609, the WHO recommended the stabilization of the biological characteristics of these daughter strains by lyophilization and storage of the samples at low temperature. This was to avoid the production of less immunogenic mutants and their selection by successive passages on culture media. Inter-laboratory studies were undertaken under the aegis of the WHO and IABS (International Association of Biological Standardization) to delimit the differences between the BCG daughter strains better, which are generally known by the name of the country or laboratory in which they are kept. The results showed unequivocally; that nowadays the strains are not identical. The differences were in the morphology, biochemistry, growth and degree of immunogenicity, of which the cellular type immunity engendered by BCG is important.M There are four daughter strains from which large quantities of vaccine are produced worldwide and are supplied by UNICEF. The French strain 1173 P2 is used in France by ‘Pasteur Vaccins’ and in 14 other countries for their own production of vaccine. The upkeep and biological characteristics are known and have been described, since this strain has been used in many studies.3y6 The Glaxo strain 1077 was obtained from Copenhagen in the 1950s. It is, however, different from the Danish strain 1331 from which it is derived. Comparative studies have shown large differences between the two strains, resulting in the different names and the use of a larger bacterial mass per dose in the Glaxo vaccines.3*s*7*s These biological changes seem to be due to the mode of culture on media (Q1990 The International
Association ofBiological
Standardization
136
M. Ghemghiu
containing tensioactive agents. The strain is now used in England by ‘Evans’ and in France by ‘Merieux’. The Japanese strain 172 seems to have been selected for its high resistance to lyophilization. It is more resistant than other strains, and is more heat-stable. For this reason, 10 years ago, it became the reference strain for quality control of lyophilized vaccines. However, it contains so many viable units per unit weight that it is unlike any other strain. This led the WHO to renounce this strain in favour of ‘Working Reference No. 3’ based on the French strain 1173 P2.2,3xg Other daughter strains are in use worldwide. The best known include: Moreau (Brazil), Montreal (Canada, Institut Armand Frappier and Connaught), Russian (USSR) and Tice (USA). Laboratory studies and observations in man have shown that some strains are ‘strong’, like the French strain 1173 P2 and the Danish strain 1331 (Copenhagen), and some strains are ‘weak’ such as the Glaxo 1077 and Japanese 172 strains.2,3 It is very difficult to show that one strain is clearly superior to another in the protection of man against tuberculosis. No prospective comparative studies of the weak and strong strains have been done. However, animal studies of this type have been performed and show higher immunogenicity of the strong strains.“*‘l It has been shown that does Ptimes higher than the strong strains must be used with weak strains to obtain the same degree of delayed type hypersensitivity (DTH) and protection. Other immunity indicators such as local granuloma and residual virulence are also more apparent after vaccination with the strong strains314 The greater degree ofprotection afforded by the strong strains can be seen in the guinea-pig model proposed by D. Smith. This model is the closest to the physiopathology of infection in man although the results of the two systems may not entirely correlate.10-12 Table
1. Protection
Country Brazil (Sao Paulo) Thailand (Bangkok) Togo (Lome) Burma (Rangoon) Argentina (Buenos-Aires) Israel
against
GIOXO
Severe and extropulmonaryforms of TB
50% 60 70 80 90 ~~~~~-~~‘~~~~~~~~~‘~~~~~~~~~t~~~~~~~~~~~~~-~~~~~
TTokyo
TGICXOT
TTokyo
All forms of TB
Severe forms of TB
Case-control Contact Contact Case-control
53% 50% 39%
87-90% 60% 80% 80%
Case-control TBcohort
74% 24%
Retrospective Contact
72%
Tokyo
The experimental results which indicate that strong strains are more immunogenic in animal experiments are now supported by findings in followup case-control studies in children, and/or casecontacts of smear-positive cases. Indeed, the protective role of BCG against serious forms of tuberculosis in young children (95%) and globally against all forms of tuberculosis (74%) is confirmed by the data published in the WHO Weekly Epidemic Record13 and illustrated in Fig. 1 and Table 1. There is, however, evident variation between the strains used. The overall protection for less severe forms of tuberculosis conferred by strain Tokyo 172 is 39% to 53%, for strain Glaxo 1077 24% to 50% and for Pasteur 1173 P2 72% to 74%. Other studies, such as the one performed in Hong Kong, showed higher protection with the Pasteur than with the Glaxo strain,37 and the Seoul (South Korea) study has again shown good protection (72%) with the Pasteur strain.14 As concluded in the WHO Weekly Epidemic Record I3 the results obtained in children, even if less amb&uous than those of control trials, still may have been biased towards milder forms of tuberculosis which are more difficult to diagnose. However, animal experiments generally show higher protection with strong strains, and research into cell-mediated
tuberculosis Studies
TMo4
Do %
Figure 1. BCG strains ranking by percentage protection in man.
(pulmonary) Japan Korea (Seoul)
30% 40 50 60 70 80% 1~~~~~~~~~1~~~~~~~~~‘~~~~~~~~~1~~~~~~~~~~~~~~~~~~~ Tokyo T GlW.0 TGlaro lP;s,eu, ’ 11
AllformsofT8
64%
(extrapulmonary) 95% -
BCG strain used Moreau Tokyo Glaxo Tokyo Pasteur Glaxo
Tokyo Pasteur
(?I
137
BCG vaccine in TB control
immunity also often uses strong BCG strains. These facts, together with the findings in child case-control studies, have to be taken into account when choosing a given strain for vaccine production, even if it is not clearly proved that strong strains are superior to weak ones. For the latter, there is even less favourable data except that they produce a reduced percentage of lymphadenitis after vaccination. The side effect of the strong strains are the higher incidence of lymphadenitis, especially in neonates, as happened in Germany with the Danish strain,15 and more recently in Mozambique and Zimbabwe with the Pasteur strain.‘6,17 Studies performed with reduced doses of vaccine solved this problem in Germany.15 Other studies recently performed with the Pasteur strain showed that, when vaccinating neonates with a reduced dose of O-025 mg, the percentage of this lymphadenitis falls to O-7%, levels considered tolerable. The DTH was maintained over 80%, the amount needed to assure good immunization.18 The use of a weaker BCG strain is tempting for both vaccine producers and users, since immediate results without side effects assure tranquility for both. Some countries, such as the Ivory Coast, Algeria, etc. used such strain vaccines for decades, but with practically no results, so have switched to the Pasteur strain. After a short period with improved information and accuracy of vaccination, the side effect percentage was tolerable. The major aim of vaccination programmes is protection against tuberculosis, a long term goal. Vaccines prepared with strong strains therefore are not to be eliminated, and vaccination accuracy has to be very carefully thought out. The BCG vaccine
The vaccine is prepared from a given BCG strain, and composed of live attenuated bacilli capable of inducing protection against tuberculosis and delayed type hypersensitivity in the intradermal tuberculin test. The quality of the vaccine depends on several factors: the immunogenicity of the strain, and the ratio of live to dead bacilli in the preparation. If the ratio is high, the local reactions are lower and the immunogenicity higher, allowing use of lower doses to give satisfactory immunization with minimal side effects. The ratio of live to killed bacteria varies enormously (three logarithms) depending on the manufacture and culture methods used. Dispersed grown BCG avoids clumping of the bacilli and the destruction of the cells by the grinding of the bacillary mass common to the classic method.lg Similarly, the
method of lyophilization and preparation also influence the ratio of live cells. A percentage higher than 10% of that of a fresh suspension is required, with a stability and heat-stability of at least 20% after exposure to 37°C for one month. The BCG vaccination
Having chosen the most effective vaccine, the method of vaccination must be selected. The protection conferred depends not only on the vaccine, but also on: the route of vaccination; the dose; the time between vaccination and exposure to infection. Although it is generally accepted that the best method of vaccination is intradermal injection, this is difflcult in new-born infants who require: precision in the injection technique; precision in the dose; background data on the age, vaccine and doses to give DTH within the accepted norms (>50 to 100%) with a tolerable minimum of suppurative adenitis (
