HEPATITIS B VACCINE IN THE EXPANDED PROGRAMME OF IMMUNISATION: THE GAMBIAN EXPERIENCE

1057 TABLE I-THE IMMUNISATION SCHEDULE

Preventive Medicine HEPATITIS B VACCINE IN THE EXPANDED PROGRAMME OF IMMUNISATION: THE GAMBIAN EXPERIENCE

THE GAMBIA HEPATITIS STUDY GROUP*

As part of the Gambia Hepatitis Intervention Study, hepatitis B vaccine has been integrated into the national Expanded Programme of Immunisation (EPI) without major changes to the schedule of immunisation or to the mechanism of delivery. Serological results on a sample of vaccinated children at one year of age show that the strategy has been effective in reducing the prevalence of persistently infected children. The number of non-responders to the vaccine is low ( < 2%) and the antibody concentrations attained in responding children should give adequate protection when they are at high risk of persistent hepatitis B infection. Integration of hepatitis B vaccine into the EPI is feasible and effective in Africa.

BCG = bacille Calinette-Guerin. HB = hepatitis B. DPT - diphtheria-pertussis-tetanus.

Summary

INTRODUCTION

THE Gambia Hepatitis Intervention Study (GHIS) is a collaborative project of the Gambia Government, the International Agency for Research on Cancer (WHO), and the Medical Research Council of the UK. GHIS aims to evaluate whether the prevention of persistent infection with hepatitis B virus by immunisation will prevent chronic liver disease and, especially hepatocellular carcinoma. The study, which has been described in detail elsewhere,! has three phases: first, integration of hepatitis B immunisation into the Expanded Programme of Immunisation (EPI); second, evaluation of the immunogenicity of the vaccine and the duration of protection which requires the long-term followup of a cohort of 1000 immunised children; and third, determination of the incidence of chronic liver disease in the vaccinated and unvaccinated. One reason for choosing The Gambia as the site for this study was that the EPI in The Gambia has been one of the most successful in Africa. The small size of the country, a high population density by African standards, and a reasonably good road network have contributed to this success. The high coverage is achieved by delivery of immunisation at both fixed and mobile maternal child health clinics. Another reason for choosing The Gambia was the high prevalence of endemic hepatitis B infection in the country: childhood infection in the Gambia2 and in neighbouring Senegal3 is very common-more than 90% of the population have been infected with hepatitis B virus by the age of 15 years. Although the force of infection seems to vary from one village to Anothermother-to-child transmission is uncommon.4 Child-to-child transmission by an unidentified route seems to be the predominant means of

spread. *A. J. Hall, H. M.

Inskip, F. Loik, J. Chotard, M. Jawara, M. Vail Mayans (International Agency for Research on Cancer [WHO], Fa)ara, The Gambia); B. M. Greenwood, H. Whittle (MRC Laboratories, Fajara, The Gambia); A. B. H. Njie, K. Cham (Medical and Health Department, Government of The Gambia, Banjul, The Gambia); and F. X. Bosch, C. S. Muir (International Agency for Research on Cancer [WHO], Lyon, France).

This paper describes how the study has achieved into the EPI, and the short-term effects of the vaccination programme on the hepatitis B status of children.

integration

MATERIALS AND METHODS

Vaccination Schedule

Twenty-five teams were delivering vaccine at the beginning of the programme, but with the acceleration of the EPI the number has increased to forty. The schedule of immunisation at the beginning of integration of hepatitis B vaccine included BCG (bacille Calmette-Guerin), three doses of triple antigen (diphtheria, pertussis, tetanus) and poliomyelitis (oral), measles, and yellow fever vaccines. However, a severe epidemic of poliomyelitis in 1986 led to a revision of this schedule (table i). Standard WHO cluster surveys, which have been done annually since the inception of the EPI, show that 95% of children receive the initial BCG and that coverage decreases to 80% with measles immunisation at nine months old. Nevertheless, the proportion of children who are fully immunised has consistently been 50% or more. A coverage survey for hepatitis B vaccinations of children aged 12 to 18 months was done in 1988. Enough plasma-derived vaccine was donated to immunise the 60 000 children required by the sample size determination.1 Serological data from the population surveysZ.3 indicated that neither vaccination at birth nor the use of hepatitis B immunoglobulin were necessary: perinatal transmission contributed little to overall infection. Thus, prevention of persistent infection may be possible without a radical change in the delivery of EPI vaccines. Because the schedule had to match existing ages of immunisation in the current schedule, a series of pilot trials was undertaken. The initial trials evaluated various doses given intradermally both with and without BCG.5 The results of these trials were regarded as unacceptable for a public health programme. Hepatitis B vaccine was given in 10 ug doses by the intramuscular deltoid route: seroconversion is poor when the vaccine is administered in the gluteal muscle.6 Clearly, immunisation as soon after birth as possible was important, and so the first dose was given at the same time as BCG-ie, during the first month of life. This was the earliest feasible time because 90% of births in The Gambia are home deliveries and the children are only brought to the clinics after being named at 7 days old. The second dose of hepatitis B vaccine was scheduled to be given with the first dose of triple antigen at two months of age or later (see table I), and the third dose of vaccine with the third dose of triple antigen at four months of age or later. The minimum interval between doses was fixed at four weeks. In a pilot study, a four-dose regimen was more immunogenic than a three-dose regimen (Whittle HC, Eccles MJ, unpublished). Thus, a fourth dose of hepatitis B vaccine was given with measles vaccine. If we could improve coverage with hepatitis B vaccine at nine months, we could likewise improve measles coverage, which is perhaps the most important component of the EPI in West Africa. The addition of hepatitis B vaccine to the existing vaccines does not reduce their efficacy in terms of antibody response.10,11

Logistics Hepatitis B vaccine is delivered to The Gambia by air from the USA in insulated containers which have temperature-sensitive

1058 immunised with hepatitis B vaccine at each of these then recruited for serological follow-up. Finger-prick blood samples were taken from all children and their mothers before the first dose of vaccine. A further blood sample was taken from the children at four months of age at two centres, and at one year of age at all centres. Blood samples will be taken every year until the children are nine years old. Serum samples were stored at - 70’C within 24 h of collection. They were then tested for hepatitis B surface antigen (HBsAg) by reverse passive haemagglutination (’Hepatest’, Burroughs Wellcome), and for hepatitis B surface antibody (anti-HBs), core antibody (anti-HBc), and hepatitis B e that

were

centres were

antigen (HBeAg) by radioimmunoassay (’Sorin’, Biomedica). Anti-HBs was quantitated in mIU/ml with the WHO reference preparation as a standard. This study has been approved by the joint Gambia Government/ MRC and the International Agency for Research on Cancer ethical committees. RESULTS

Percentage of children receiving dose of hepatitis B vaccine by age.

HB,, HB2, HB3, HB4 = first, second, third, fourth doses, respectively. Numbers in

parentheses

=

no

of children

receiving each dose.

indicators. Because potency is destroyed by freezing, the vaccine must be handled in the same way as the triple antigen; thus, we expanded refrigeration capacity in the 4-8°C range at the regional vaccine stores. Otherwise the vaccine was handled in the same way as in the normal cold chain. Workshops were held for staff involved in immunisation: they included an introduction to the epidemiology and clinical aspects of hepatitis B infection in The Gambia and world wide, and discussion about the handling of the vaccine and about the importance of the deltoid muscle as a site of injection. These meetings also provided a forum to update knowledge of other aspects of the EPI. The vaccine was originally provided in 20 flg vials (ie, two doses). However, difficulties with refrigerator storage space outweighed savings on wastage of vaccine: it has since been ordered in 60 flg (six doses) vials. The vaccine is administered with re-sterilisable syringes and needles following the principle of one needle, one syringe, one child. The use of re-sterilisable syringes and needles is routine practice in the developing world and is not associated with hepatitis B transmission. Further storage difficulties have arisen because the vaccine must be packed in individual boxes to comply with Food and Drug Administration regulations: therefore, every vial on receipt in The Gambia has to be repacked to fit the vials into the available fridges.

Documentation The GHIS requires the precise identification of each child entering the study: each immunisation team had a clerk to assist in the keeping of registers. The infant welfare card given to each mother at the child’s first attendance at the clinic has been adapted: a tear-off sheet with a carbon paper allows a copy of all identification details--ie, child’s name, date of birth, mother’s name, father’s name, village of birth, clinic attended, date of attendance, and registration number-that can be obtained easily for the project. The registration numbers have been prefixed with a two letter code, unique to each immunisation point, which gives the child a unique identity number. This information is returned to a central office where it is computerised. The clerk also records all subsequent vaccinations given to that child; the child’s name and registration number are used to link these vaccinations to the original registration in the computer. Thus, a complete national immunisation register has been created.

The pilot trials that had been done before the main study involved administration of vaccine precisely at the ages intended. However, this does not happen in an EPI. The figure shows the actual ages at which children received each dose of vaccine: some children receivethe full course at a much older age than intended. Nevertheless, the coverage survey showed that 98% of children aged 12-18 months had received the first dose of hepatitis B vaccine, 94% the second dose, 92% the third dose, and 74% the fourth dose. At one year of age, 764 of the 1041 children recruited into the cohort were traced and blood was taken. 87% of the children who were rebled had had at least three doses of vaccine compared with only 58% of those who could not be traced. This is inevitable because the major reason for failure to trace was migration. Some of these untraced children had probably died because the observed mortality rate in this cohort is considerably less than that for The Gambia as a whole. The hepatitis B marker status of the children has been related to the mother’s hepatitis B status at the time of birth of the child (table ll): for 6 children this was unknown-all were anti-HBs-positive and anti-HBc-negative. Only 2 of the 710 children had a potential persistent hepatitis B infection and only 15 (2%) had no surface antibody (ie, < 10 mIU/ml). 37 children had positive core antibody, which might point to natural infection or to long persistence of maternal antibody. The highest proportion with core antibody was in children whose mothers were HBsAgpositive and HBeAg-positive; it was lower in children if the TABLE II-HEPATITIS B MARKERS IN VACCINATED CHILDREN AT ONE YEAR OF AGE ACCORDING TO MOTHER’S HEPATITIS B STATUS

The Cohort The 1000 children that will be followed up

long

term were

recruited from routine immunisation clinics. The country divided into four

immunisation

geographic

team was

chosen

at

was

and in each zone an random. The first 250 children

zones

*2 children HBsAg +. t2 children HBsAg + . hepatitis B status of 6 children NK not known. =

not

known.

1059 TABLE III-ANTI-HBS CONCENTRATIONS IN IMMUNISED CHILDREN AT ONE YEAR OF AGE BY DOSE

*This represents all of the children in table 11 plus the 6 children for whom the mother’s status is unknown plus 2 children in whom there was insufficient blood to measure core antibody status.

mothers were HBsAg-positive but HBeAg-negative, and lowest in children of non-carrier mothers. 4 children were HBsAg-positive: 2 were the possible carriers described above and the other 2 had surface antibody but no core

antibody. Table III shows the anti-HBs concentrations of the cohort children according to the number of doses of vaccine received. The concentration of antibody increased with an increase in the number of doses of vaccine in terms of the proportion in each stratum. There were no differences by sex. All children had received the first dose of vaccine because this was a condition of entry to the cohort. DISCUSSION

The integration of hepatitis B vaccination into the EPI has been highly successful. There were no special logistic difficulties and the health staff were enthusiastic about the new topic and vaccine. This reflected beneficially on morale and on the general interest and enthusiasm for the EPI. Some operational decisions were necessary: the first immunisation was limited to children presenting for vaccination for the first time under the age of one year. This was determined partly by the limited supply of vaccine and partly by the rapid decline in risk of persistent infection after the age of one year.12 There was no contraindication to vaccination. In 94% of children, immunisation has produced what is believed to be a protective level of antibody at one year of age. An additional 4% of children have core antibody which may represent natural infection without persistent carriage. We would expect that 8-10% of the cohort children would be HBsAg-positive at one year of age,—ie, 60-70 children-but only 2 children were positive. If the protection is long-lasting then the adult prevalence of hepatitis B carriage of 20% will be substantially reduced. The high concentrations of antibody in children who have received four doses points to long-lasting protection. However, there may be a risk of a rapid decline of antibody and a return to susceptibility in the children who were not traced and who did not have all the doses of vaccine. This emphasises the need for high coverage with every dose of this vaccine. Remarkably, 4 of the 12 children born to HBsAg-positive, HBeAg-positive mothers have protective amounts of antibody, 6 have core and surface antibody, and only 2 are potential carriers, despite the fact that they did not receive the first dose of vaccine until they were 3 weeks old. According to Asian data, there is a 90% probability that such children born to HBeAg-positive mothers will become carriers if they are not vaccinated. The 2 children who had HBsAg with anti-HBs but without anti-HBc may have been

infected with the hepatitis B variant described in Senegal.13 These children will be followed up. However, a definitive answer will require a long-term marker of this infection because, if they subsequently become HBsAg-negative with no core antibody as predicted by the Senegalese data, we will not know whether this is a true fmding or a laboratory error. The amount of blood taken from these children does not allow for extensive retesting of samples. One specific difficulty encountered with the integration of hepatitis B vaccine into EPI is the variable age at immunisation which leads to variable intervals between doses. Whether this is an important determinant of hepatitis B antibody concentration in addition to the number of doses in this population is unknown. A more important implication of this age variability is the scope for prevention of persistent infection. The probability of persistence is very high if infection occurs in the first year of life. If a high proportion of children are still infected at this age, because of late immunisation, the present schedule will fail. The number of persistent infections will not be known until the results of the serological survey at 2 years of age but the current serological results are encouraging. Nevertheless, variable age at immunisation is a factor which other countries considering hepatitis B immunisation should take into account in their baseline serological surveys. Will a booster dose be necessary later in life? Immunised children should have passed the ages at which risk of persistence is high by the time that the antibody decays. If immunisation alters the age dependency of risk, the children would then be susceptible and still at risk of persistence. This point can only be answered by following up children who were vaccinated early in life for many years. The efficacy of the fourth dose to attain high titre antibody will also have to wait for longitudinal data. The evaluation of the success of integrating hepatitis B vaccination into the EPI is more complex than it is for other diseases. Because children do not have any recognisable disease following infection, serological surveys, rather than disease surveillance, will be needed to monitor the success of the immunisation programme; the appropriate field methodology for these surveys must be developed and standardised. The true benefit of this programme also requires measurement of the reduction in long-term sequelae of infection. We hope that the GHIS will provide this. As the cost of hepatitis B vaccine decreases, other countries are starting programmes of integrated hepatitis B immunisation. Therefore, multiple antigen vaccines are needed because mothers and children are not fond of three needles at a clinic visit. We hope that manufacturers will pursue this and also reduce the cost of vaccine since this programme in The Gambia has clearly shown that persistent infection with hepatitis B in Africa can be prevented by integration of the hepatitis B vaccine into the EPI. This study is generously funded by a grant from the Department for Cooperation and Development of the Ministry of Foreign Affairs of Italy. The vaccine for the study was donated by Merck, Sharp and Dohme. We thank Fiona Shenton, Fatou Joof, and Maimuna Mendy, MRC Laboratories, Fajara, The Gambia, for technical assistance. The success of the programme is entirely dependent on the health workers of The Gambian Department of Medicine and Health and on the parents of the Gambian children. We would especially like to thank all of the children and mothers who make up the cohort.

Correspondence should be addressed to A. J. H., International Agency for on Cancer, PO Box 273, Banjul, The Gambia.

Research

1060

predominant subtype in a mixtures; natural and recombinant IFN-(3; and recombinant IFN-y. The transient production of IFNs in the body is usually under strict inductional control Common inducing stimuli for IFN-a and IFN-[3 include viruses, bacteria, and double-stranded RNA. IFN-as are most efficiently induced in peripheral blood mononuclear cells, and IFN-P in fibroblasts and epithelial cells. IFN-y, in keeping with its more extensive role in the control of immune responses, is induced by mitogens and antigens. In addition, IFNs can be induced by other protein regulators such as the cytokines tumour necrosis factor, IL-1, IL-2, and colony stimulating factors.3 IFNs may also be produced constitutively in some sites of the body, particularly the peritoneum, and render cells involved in front line defence against viral infection permanently resistant to such infection most

Peptide Regulatory Factors INTERFERONS FRANCES R. BALKWILL

Biological Therapy Laboratory, Imperial Cancer Research Fund, PO Box 123, Lincoln’s Inn Fields, London WC2A 3PX THE original concept of interferons at the time of their discovery in 1957 was simple but unique. The term "interferon" identified a factor produced by cells in to viral infection that could protect other cells of the same species from attack by a wide range of viruses.The first twenty years of research on interferons (IFNs) were hampered by difficulties with production and purity, which were solved in the late 1970s by use of recombinant DNA technology and monoclonal antibodies. Meanwhile, sufficient data had been generated to suggest that the original concept of one interferon with one biological function was too simple and that IFNs are members of a large family of regulatory proteins.2 In the past 10 years, laboratory observations have been accompanied by clinical trials of IFNs in infectious and malignant diseases. Such trials have identified some disorders in which IFNs have a therapeutic role, have given us insights into their mechanisms of action, and have provided the foundation for trials of other cytokines and peptide regulatory factors in human disease. I shall now review the molecular and cellular biology of human IFNs and discuss their clinical potential.

response

FAMILY OF IFN MOLECULES

In man, as in other species, IFNs are a multigene family whose protein products can be divided into three main types-IFN-a, IFN-0, and IFN-y.2 Whilst there is only one gene for IFN-P, or IFN-y, there are at least twentythree different IFN-a genes that code for fifteen functional proteins. The IFN-a genes are closely related and are all clustered on chromosome 9, close to the IFN-(3 gene; the IFN-y gene is on chromosome 12. The IFN proteins consist of 165-187 aminoacids, with molecular weights of 17-25 kD. It is unclear why there are so many IFN-as; there are few functional differences between the a subtypes, and the clinical activity of single a subtypes and naturally produced mixtures is identical. Several IFN are now available for clinical use, including a mixture of IFN-a subtypes made by stimulating lymphoblastoid cells with inactivated Sendai virus; recombinant IFN-o, which is the

1. Hall AJ, Inskip HM, Loik F, et al. The Gambia Hepatitis Intervention Study. Cancer Res 1987; 47: 5782-87. 2. Whittle HC, Bradley AK, McLoughlan K, et al. Hepatitis B virus infection in two Gambian villages. Lancet 1983, ii: 1203-06. 3. Barin F, Perrin J, Chotard J, et al. Cross sectional and longitudinal epidemiology of hepatitis B m Senegal. Proc Med Virol 1981; 27: 148-67. 4. Marinier E, Barrois V, Larouze B, et al. Lack of perinatal transmission of hepatitis B virus infection in Senegal, West Africa. Trop Paediatr 1985; 106: 843-48. 5. Whittle HC, Lamb WH, Ryder RW. Tnals of intradermal hepatitis B vaccines in Gambian chidren. Ann Trop Paediatr 1987; 7: 6-9. 6 CDC Suboptimal response to hepatitis B vaccine given into the buttock MMWR 1985; 34: 105-08 7. Jilg W, Schmidt M, Deinhardt F, Zachoval R. Hepatitis B vaccination: how long does protection last? Lancet 1984; ii: 458 8. Coursaget P, Yvonnet B, Chotard J, et al. Seven year study of hepatitis B vaccine efficacy in infancy from an endemic area (Senegal). Lancet 1986; ii: 1143-44

IFN RECEPTORS

To exert their regulatory role on cells, IFNs, in common with other cytokines and growth factors, must first interact with specific cell membrane receptors. Two distinct IFN cell-surface receptors are widely distributed in the body.4 IFN-a and IFN-P share a receptor which is thought to be about 110-130 kD. The receptor for IFN-y has now been cloned.s It shows no similarities with known proteins, is most likely the product of a single mRNA, and a molecular weight of approximately 54 kD would be predicted from the deduced aminoacid sequence. This estimate is considerably lower than the apparent molecular weight of the purified natural receptor protein, a difference that may be accounted for by glycosylation. The receptor genes for the a/ (3 and y receptors are located on chromosomes 21 and 6,

respectively. MOLECULAR EVENTS IN IFN-TREATED CELLS

After

binding to cell surface receptors, IFNs act by transiently inducing or up-regulating some rapidly cellular genes and down-regulating others.Z,6 This alteration in 50-100 cellular proteins is responsible for IFN-induced changes in cell behaviour. Little is known about the signals that are transmitted from the IFN receptor complex to the nucleus, but there is some information about control of the and

IFN-induced genes that have common IFN response sequences upstream from their coding regions. Most IFN-induced genes are activated by all three types, although

few are specific for either IFN-a and IFN-P or IFN-y.2,6 Although the functions of very few IFN-induced genes are known, this incomplete knowledge gives some insight into mechanisms responsible for the various effects of IFNs. a

9. Hadler

SC, Francis DP, Maynard JE, et al. Long-term immunogenicity and efficacy of hepatitis B vaccine m homosexual men. N Engl J Med 1986; 315: 209-14 10. Coursaget P, Yvonnet B, Relyveld EH, Barres JL, Diop-Mar I, Chiron JP Simultaneous administration of diphtheria-tetanus-pertussis-polio and hepatitis B

a simplified immunisation programme: immune responses to diphtheria toxoid, tetanus toxoid, pertussis, and hepatitis B surface antigen Infect Immun 1986, 51: 784-87. 11. Yvonnet B, Coursaget P, Deubel V, Diop-Mar I, Digoutte JP, Chiron JP Simultaneous administration of hepatitis B and yellow fever vaccines Dev Biol Stand 1986; 65: 205-07. 12 Coursaget P, Yvonnet B, Chotard J, et al. Age- and sex-related study of hepatitis B virus chronic carrier state in infants from an endemic area (Senegal) J Med Virol 1987, 22: 1-5 13 Coursaget P, Yvonnet B, Bourdil C, et al HBsAg positive reactivity in man not due to hepatitis B virus. Lancet 1987; ii: 1354-58. vaccines m