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Preventive Medicine

HEPATITIS B VACCINE IN THE EXPANDEDPROGRAMME OF IMMUNISATION:THE GAMBIAN EXPERIENCE

THE GAMBIA HEPATITIS STUDY GROUP*

Summary As part of the Gambia HepatitisIntervention Study, hepatitis B vaccine has

been integrated into the national Expanded Programme ofImmunisation (EPI) without major changes to the scheduleof immunisation or to the mechanism of delivery.Serological results on a sample of vaccinated children at oneyear 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 respondingchildren should give adequate protection when they are athigh risk of persistent hepatitis B infection. Integration ofhepatitis B vaccine into the EPI is feasible and effective inAfrica.

INTRODUCTION

THE Gambia Hepatitis Intervention Study (GHIS) is acollaborative project of the Gambia Government, theInternational Agency for Research on Cancer (WHO), andthe Medical Research Council of the UK. GHIS aims toevaluate whether the prevention of persistent infection withhepatitis B virus by immunisation will prevent chronic liverdisease and, especially hepatocellular carcinoma. The study,which has been described in detail elsewhere,! has threephases: first, integration of hepatitis B immunisation into theExpanded Programme of Immunisation (EPI); second,evaluation of the immunogenicity of the vaccine and theduration of protection which requires the long-term follow-up of a cohort of 1000 immunised children; and third,determination of the incidence of chronic liver disease in thevaccinated 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 themost 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 thissuccess. The high coverage is achieved by delivery ofimmunisation at both fixed and mobile maternal childhealth clinics. Another reason for choosing The Gambia wasthe high prevalence of endemic hepatitis B infection in thecountry: childhood infection in the Gambia2 and in

neighbouring Senegal3 is very common-more than 90% ofthe population have been infected with hepatitis B virus bythe age of 15 years. Although the force of infection seems tovary from one village to Another mother-to-childtransmission is uncommon.4 Child-to-child transmission byan unidentified route seems to be the predominant means ofspread.

*A. J. Hall, H. M. Inskip, F. Loik, J. Chotard, M. Jawara, M. Vail Mayans(International Agency for Research on Cancer [WHO], Fa)ara, TheGambia); B. M. Greenwood, H. Whittle (MRC Laboratories, Fajara, TheGambia); 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).

TABLE I-THE IMMUNISATION SCHEDULE

BCG = bacille Calinette-Guerin.

HB = hepatitis B.DPT - diphtheria-pertussis-tetanus.

This paper describes how the study has achieved

integration into the EPI, and the short-term effects of thevaccination programme on the hepatitis B status of children.

MATERIALS AND METHODS

Vaccination Schedule

Twenty-five teams were delivering vaccine at the beginning ofthe programme, but with the acceleration of the EPI the number hasincreased to forty. The schedule of immunisation at the beginningof integration of hepatitis B vaccine included BCG (bacilleCalmette-Guerin), three doses of triple antigen (diphtheria,pertussis, tetanus) and poliomyelitis (oral), measles, and yellowfever vaccines. However, a severe epidemic of poliomyelitis in 1986led to a revision of this schedule (table i). Standard WHO clustersurveys, which have been done annually since the inception of theEPI, show that 95% of children receive the initial BCG and thatcoverage decreases to 80% with measles immunisation at ninemonths old. Nevertheless, the proportion of children who are fullyimmunised has consistently been 50% or more. A coverage surveyfor hepatitis B vaccinations of children aged 12 to 18 months wasdone in 1988.

Enough plasma-derived vaccine was donated to immunise the60 000 children required by the sample size determination. 1

Serological data from the population surveysZ.3 indicated thatneither vaccination at birth nor the use of hepatitis B immuno-globulin were necessary: perinatal transmission contributed little tooverall infection. Thus, prevention of persistent infection may bepossible without a radical change in the delivery of EPI vaccines.Because the schedule had to match existing ages of immunisation inthe current schedule, a series of pilot trials was undertaken. Theinitial trials evaluated various doses given intradermally both withand without BCG.5 The results of these trials were regarded asunacceptable for a public health programme.

Hepatitis B vaccine was given in 10 ug doses by the intramusculardeltoid route: seroconversion is poor when the vaccine is

administered in the gluteal muscle.6 Clearly, immunisation as soonafter birth as possible was important, and so the first dose was givenat the same time as BCG-ie, during the first month of life. Thiswas the earliest feasible time because 90% of births in The Gambiaare home deliveries and the children are only brought to the clinicsafter being named at 7 days old. The second dose of hepatitis Bvaccine was scheduled to be given with the first dose of tripleantigen at two months of age or later (see table I), and the third doseof vaccine with the third dose of triple antigen at four months of ageor later. The minimum interval between doses was fixed at fourweeks. In a pilot study, a four-dose regimen was more immunogenicthan a three-dose regimen (Whittle HC, Eccles MJ, unpublished).Thus, a fourth dose of hepatitis B vaccine was given with measlesvaccine. If we could improve coverage with hepatitis B vaccine atnine months, we could likewise improve measles coverage, which isperhaps the most important component of the EPI in West Africa.The addition of hepatitis B vaccine to the existing vaccines does notreduce their efficacy in terms of antibody response.10,11

Logistics

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

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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 vaccinemust be handled in the same way as the triple antigen; thus, weexpanded refrigeration capacity in the 4-8&deg;C range at the regionalvaccine stores. Otherwise the vaccine was handled in the same wayas in the normal cold chain.

Workshops were held for staff involved in immunisation: theyincluded an introduction to the epidemiology and clinical aspects ofhepatitis B infection in The Gambia and world wide, and discussionabout the handling of the vaccine and about the importance of thedeltoid muscle as a site of injection. These meetings also provided aforum 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 outweighedsavings on wastage of vaccine: it has since been ordered in 60 flg (sixdoses) vials. The vaccine is administered with re-sterilisable

syringes and needles following the principle of one needle, onesyringe, one child. The use of re-sterilisable syringes and needles isroutine practice in the developing world and is not associated withhepatitis B transmission. Further storage difficulties have arisenbecause the vaccine must be packed in individual boxes to complywith Food and Drug Administration regulations: therefore, everyvial on receipt in The Gambia has to be repacked to fit the vials intothe available fridges.

Documentation

The GHIS requires the precise identification of each childentering the study: each immunisation team had a clerk to assist inthe keeping of registers. The infant welfare card given to eachmother at the child’s first attendance at the clinic has been adapted: atear-off sheet with a carbon paper allows a copy of all identification

details--ie, child’s name, date of birth, mother’s name, father’sname, village of birth, clinic attended, date of attendance, andregistration 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 uniqueidentity number. This information is returned to a central officewhere it is computerised. The clerk also records all subsequentvaccinations given to that child; the child’s name and registrationnumber are used to link these vaccinations to the originalregistration in the computer. Thus, a complete nationalimmunisation register has been created.

The Cohort

The 1000 children that will be followed up long term wererecruited from routine immunisation clinics. The country wasdivided into four geographic zones and in each zone an

immunisation team was chosen at random. The first 250 children

that were immunised with hepatitis B vaccine at each of thesecentres were then recruited for serological follow-up. Finger-prickblood samples were taken from all children and their mothers beforethe first dose of vaccine. A further blood sample was taken from thechildren at four months of age at two centres, and at one year of ageat all centres. Blood samples will be taken every year until thechildren are nine years old. Serum samples were stored at - 70’Cwithin 24 h of collection. They were then tested for hepatitis Bsurface antigen (HBsAg) by reverse passive haemagglutination(’Hepatest’, Burroughs Wellcome), and for hepatitis B surfaceantibody (anti-HBs), core antibody (anti-HBc), and hepatitis B eantigen (HBeAg) by radioimmunoassay (’Sorin’, Biomedica).Anti-HBs was quantitated in mIU/ml with the WHO referencepreparation as a standard.

This study has been approved by the joint Gambia Government/MRC and the International Agency for Research on Cancer ethicalcommittees.

RESULTS

The pilot trials that had been done before the main studyinvolved administration of vaccine precisely at the agesintended. However, this does not happen in an EPI. Thefigure shows the actual ages at which children received eachdose of vaccine: some children receive the full course at amuch older age than intended. Nevertheless, the coveragesurvey showed that 98% of children aged 12-18 months hadreceived the first dose of hepatitis B vaccine, 94% the seconddose, 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 thechildren who were rebled had had at least three doses ofvaccine compared with only 58% of those who could not betraced. This is inevitable because the major reason for failureto trace was migration. Some of these untraced children hadprobably died because the observed mortality rate in thiscohort is considerably less than that for The Gambia as awhole.The hepatitis B marker status of the children has been

related to the mother’s hepatitis B status at the time of birthof the child (table ll): for 6 children this was unknown-allwere anti-HBs-positive and anti-HBc-negative. Only 2 ofthe 710 children had a potential persistent hepatitis Binfection and only 15 (2%) had no surface antibody (ie, < 10

mIU/ml). 37 children had positive core antibody, whichmight point to natural infection or to long persistence ofmaternal antibody. The highest proportion with core

antibody was in children whose mothers were HBsAg-positive 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

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

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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 themother’s status is unknown plus 2 children in whom there was insufficientblood to measure core antibody status.

mothers were HBsAg-positive but HBeAg-negative, andlowest in children of non-carrier mothers. 4 children were

HBsAg-positive: 2 were the possible carriers describedabove and the other 2 had surface antibody but no coreantibody.Table III shows the anti-HBs concentrations of the cohort

children according to the number of doses of vaccinereceived. The concentration of antibody increased with anincrease in the number of doses of vaccine in terms of the

proportion in each stratum. There were no differences bysex. All children had received the first dose of vaccinebecause this was a condition of entry to the cohort.

DISCUSSION

The integration of hepatitis B vaccination into the EPI hasbeen highly successful. There were no special logisticdifficulties and the health staff were enthusiastic about thenew topic and vaccine. This reflected beneficially on moraleand on the general interest and enthusiasm for the EPI.Some operational decisions were necessary: the firstimmunisation was limited to children presenting forvaccination for the first time under the age of one year. Thiswas determined partly by the limited supply of vaccine andpartly by the rapid decline in risk of persistent infection afterthe age of one year.12 There was no contraindication tovaccination.

In 94% of children, immunisation has produced what isbelieved to be a protective level of antibody at one year ofage. An additional 4% of children have core antibody whichmay represent natural infection without persistent carriage.We would expect that 8-10% of the cohort children wouldbe HBsAg-positive at one year of age,&mdash;ie, 60-70children-but only 2 children were positive. If the

protection is long-lasting then the adult prevalence ofhepatitis B carriage of 20% will be substantially reduced.The high concentrations of antibody in children who havereceived four doses points to long-lasting protection.However, there may be a risk of a rapid decline of antibodyand a return to susceptibility in the children who were nottraced and who did not have all the doses of vaccine. This

emphasises the need for high coverage with every dose ofthis vaccine. Remarkably, 4 of the 12 children born toHBsAg-positive, HBeAg-positive mothers have protectiveamounts of antibody, 6 have core and surface antibody, andonly 2 are potential carriers, despite the fact that they did notreceive the first dose of vaccine until they were 3 weeks old.According to Asian data, there is a 90% probability thatsuch children born to HBeAg-positive mothers will becomecarriers if they are not vaccinated. The 2 children who hadHBsAg with anti-HBs but without anti-HBc may have been

infected with the hepatitis B variant described in Senegal.13These children will be followed up. However, a definitiveanswer will require a long-term marker of this infectionbecause, if they subsequently become HBsAg-negative withno core antibody as predicted by the Senegalese data, we willnot know whether this is a true fmding or a laboratory error.The amount of blood taken from these children does notallow 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 betweendoses. Whether this is an important determinant of hepatitisB antibody concentration in addition to the number of dosesin this population is unknown. A more importantimplication of this age variability is the scope for preventionof persistent infection. The probability of persistence is veryhigh if infection occurs in the first year of life. If a highproportion of children are still infected at this age, because oflate immunisation, the present schedule will fail. Thenumber of persistent infections will not be known until theresults of the serological survey at 2 years of age but thecurrent serological results are encouraging. Nevertheless,variable age at immunisation is a factor which othercountries considering hepatitis B immunisation should takeinto account in their baseline serological surveys. Will abooster dose be necessary later in life? Immunised childrenshould have passed the ages at which risk of persistence ishigh by the time that the antibody decays. If immunisationalters the age dependency of risk, the children would then besusceptible and still at risk of persistence. This point canonly be answered by following up children who werevaccinated early in life for many years. The efficacy of thefourth dose to attain high titre antibody will also have to waitfor longitudinal data.The evaluation of the success of integrating hepatitis B

vaccination into the EPI is more complex than it is for otherdiseases. Because children do not have any recognisabledisease following infection, serological surveys, rather thandisease surveillance, will be needed to monitor the success ofthe immunisation programme; the appropriate field

methodology for these surveys must be developed andstandardised. The true benefit of this programme also

requires measurement of the reduction in long-termsequelae of infection. We hope that the GHIS will providethis. As the cost of hepatitis B vaccine decreases, othercountries are starting programmes of integrated hepatitis Bimmunisation. Therefore, multiple antigen vaccines areneeded because mothers and children are not fond of threeneedles at a clinic visit. We hope that manufacturers willpursue 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 theEPI.

This study is generously funded by a grant from the Department forCooperation and Development of the Ministry of Foreign Affairs of Italy.The vaccine for the study was donated by Merck, Sharp and Dohme. Wethank Fiona Shenton, Fatou Joof, and Maimuna Mendy, MRCLaboratories, Fajara, The Gambia, for technical assistance. The success ofthe programme is entirely dependent on the health workers of The GambianDepartment of Medicine and Health and on the parents of the Gambianchildren. We would especially like to thank all of the children and motherswho make up the cohort.

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

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1. Hall AJ, Inskip HM, Loik F, et al. The Gambia Hepatitis Intervention Study. CancerRes 1987; 47: 5782-87.

2. Whittle HC, Bradley AK, McLoughlan K, et al. Hepatitis B virus infection in twoGambian villages. Lancet 1983, ii: 1203-06.

3. Barin F, Perrin J, Chotard J, et al. Cross sectional and longitudinal epidemiology ofhepatitis 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 Bvirus infection in Senegal, West Africa. Trop Paediatr 1985; 106: 843-48.

5. Whittle HC, Lamb WH, Ryder RW. Tnals of intradermal hepatitis B vaccines inGambian chidren. Ann Trop Paediatr 1987; 7: 6-9.

6 CDC Suboptimal response to hepatitis B vaccine given into the buttock MMWR1985; 34: 105-08

7. Jilg W, Schmidt M, Deinhardt F, Zachoval R. Hepatitis B vaccination: how long doesprotection last? Lancet 1984; ii: 458

8. Coursaget P, Yvonnet B, Chotard J, et al. Seven year study of hepatitis B vaccineefficacy in infancy from an endemic area (Senegal). Lancet 1986; ii: 1143-44

9. Hadler SC, Francis DP, Maynard JE, et al. Long-term immunogenicity and efficacyof 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 JPSimultaneous administration of diphtheria-tetanus-pertussis-polio and hepatitis Bvaccines m a simplified immunisation programme: immune responses to diphtheriatoxoid, tetanus toxoid, pertussis, and hepatitis B surface antigen Infect Immun1986, 51: 784-87.

11. Yvonnet B, Coursaget P, Deubel V, Diop-Mar I, Digoutte JP, Chiron JPSimultaneous administration of hepatitis B and yellow fever vaccines Dev BiolStand 1986; 65: 205-07.

12 Coursaget P, Yvonnet B, Chotard J, et al. Age- and sex-related study of hepatitis Bvirus chronic carrier state in infants from an endemic area (Senegal) J Med Virol1987, 22: 1-5

13 Coursaget P, Yvonnet B, Bourdil C, et al HBsAg positive reactivity in man not due tohepatitis B virus. Lancet 1987; ii: 1354-58.

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 theirdiscovery in 1957 was simple but unique. The term"interferon" identified a factor produced by cells in

response to viral infection that could protect other cells ofthe same species from attack by a wide range of viruses. Thefirst twenty years of research on interferons (IFNs) werehampered by difficulties with production and purity, whichwere solved in the late 1970s by use of recombinant DNAtechnology and monoclonal antibodies. Meanwhile,sufficient data had been generated to suggest that theoriginal concept of one interferon with one biologicalfunction was too simple and that IFNs are members of alarge family of regulatory proteins.2 In the past 10 years,laboratory observations have been accompanied by clinicaltrials of IFNs in infectious and malignant diseases. Suchtrials have identified some disorders in which IFNs have a

therapeutic role, have given us insights into theirmechanisms of action, and have provided the foundation fortrials of other cytokines and peptide regulatory factors inhuman disease. I shall now review the molecular and cellular

biology of human IFNs and discuss their clinical potential.

FAMILY OF IFN MOLECULES

In man, as in other species, IFNs are a multigene familywhose protein products can be divided into three maintypes-IFN-a, IFN-0, and IFN-y.2 Whilst there is onlyone gene for IFN-P, or IFN-y, there are at least twenty-three different IFN-a genes that code for fifteen functional

proteins. The IFN-a genes are closely related and are allclustered on chromosome 9, close to the IFN-(3 gene; theIFN-y gene is on chromosome 12. The IFN proteins consistof 165-187 aminoacids, with molecular weights of17-25 kD. It is unclear why there are so many IFN-as; thereare few functional differences between the a subtypes, andthe clinical activity of single a subtypes and naturallyproduced mixtures is identical. Several IFN are nowavailable for clinical use, including a mixture of IFN-asubtypes made by stimulating lymphoblastoid cells withinactivated Sendai virus; recombinant IFN-o, which is the

most predominant subtype in a mixtures; natural andrecombinant IFN-(3; and recombinant IFN-y.The transient production of IFNs in the body is usually

under strict inductional control Common inducing stimulifor IFN-a and IFN-[3 include viruses, bacteria, anddouble-stranded RNA. IFN-as are most efficiently inducedin peripheral blood mononuclear cells, and IFN-P infibroblasts and epithelial cells. IFN-y, in keeping with itsmore extensive role in the control of immune responses, isinduced by mitogens and antigens. In addition, IFNs can beinduced by other protein regulators such as the cytokinestumour necrosis factor, IL-1, IL-2, and colony stimulatingfactors.3 IFNs may also be produced constitutively in somesites of the body, particularly the peritoneum, and rendercells involved in front line defence against viral infectionpermanently resistant to such infection

IFN RECEPTORS

To exert their regulatory role on cells, IFNs, in commonwith other cytokines and growth factors, must first interactwith specific cell membrane receptors. Two distinct IFNcell-surface receptors are widely distributed in the body.4IFN-a and IFN-P share a receptor which is thought to beabout 110-130 kD. The receptor for IFN-y has now beencloned.s It shows no similarities with known proteins, ismost likely the product of a single mRNA, and a molecularweight of approximately 54 kD would be predicted from thededuced aminoacid sequence. This estimate is considerablylower than the apparent molecular weight of the purifiednatural receptor protein, a difference that may be accountedfor by glycosylation. The receptor genes for the a/ (3 and yreceptors are located on chromosomes 21 and 6,respectively.

MOLECULAR EVENTS IN IFN-TREATED CELLS

After binding to cell surface receptors, IFNs act byrapidly and transiently inducing or up-regulating somecellular genes and down-regulating others.Z,6 This alterationin 50-100 cellular proteins is responsible for IFN-inducedchanges in cell behaviour. Little is known about the signalsthat are transmitted from the IFN receptor complex to thenucleus, but there is some information about control of theIFN-induced genes that have common IFN responsesequences upstream from their coding regions. MostIFN-induced genes are activated by all three types, althougha few are specific for either IFN-a and IFN-P or IFN-y.2,6Although the functions of very few IFN-induced genes areknown, this incomplete knowledge gives some insight intomechanisms responsible for the various effects of IFNs.