Bull. Org. mond. Sante' 1966, 34, 233-241Bull. Wld Hluh Org.

The Present Status of Trachoma Vaccination StudiesL. H. COLLIER, M.D.'

The control of trachoma is still an outstanding problem for many developing countries,despite the widespread use of antibiotics both for prevention and treatment. The isolationof the causal agent in 1957 gave hope of developing a prophylactic vaccine, and in recentyears much research has been done on this subject. This article summarizes the requirementsthat have to be met by a trachoma vaccine and problems arising in its manufacture andtesting. A review ofthe progress made by various workers in this field suggests that, althoughsome vaccines now being tested are partially effective, much remains to be learned aboutimmunity to trachoma and methods of inducing it artificially.

INTRODUCTION

Before considering the present position in regardto trachoma vaccine, it may be helpful to set outthe reasons for research on this subject, the require-ments that would have to be met by an " ideal "vaccine, and the more important basic investigationsthat are essential for placing these studies on asecure and rational foundation. The progress madeby various workers in this field will then be summa-rized; and finally, mention will be made of someof the main problems still to be solved.

NECESSITY FOR A TRACHOMA VACCINE

The case for a trachoma vaccine (and factors thatmust be considered in preparing it) has been statedpreviously (Collier, 1961b). In brief, trachoma is ingeneral susceptible to sulfonamides and antibiotics,and in some countries much has been done to controlit with tetracycline prophylaxis and therapy. Never-theless, to be fully effective, such measures must beapplied for long periods to large numbers of people,a requirement that may be prohibitively expensiveor indeed physically impossible in countries withscattered populations and poor communications.A prophylactic vaccine capable of inducing a solidand durable immunity would thus be preferableboth on logistic and on financial grounds.

1 Honorary Director, Trachoma Research Unit, MedicalResearch Council of Great Britain, London; and Head,Department of Virology, The Lister Institute, London,England.

In any event an effective vaccine would be auseful adjunct to chemoprophylaxis and chemo-therapy; it might even be used to treat establishedtrachoma by increasing antibody formation andthus accelerating the natural tendency to resolution.

BASIC REQUIREMENTS FOR VACCINES INTENDED FOR

LARGE-SCALE FIELD USE

Such preparations must not provoke generalreactions or severe local reactions, or cause sensitiza-tion to the antigen itself or to other constituents ofthe vaccine. A limited number of injections mustconfer reasonably long immunity against theantigenic varieties of trachoma-inclusion-con-junctivitis (TRIC) agent prevalent in the country ofuse. The vaccine must be easy to store and adminis-ter under field conditions in hot countries, and it isobviously desirable that production costs be lowenough to make mass distribution economicallypracticable.

THE VACCINE DEVELOPMENT PROGRAMME

As with any antigen intended for large-scale use,the development of a trachoma vaccine entails apreliminary series of interlinked epidemiologicaland laboratory studies, followed by pilot field trials.Should the latter prove successful, large-scale fieldtrials in various endemic areas would eventually befollowed by manufacture on a commercial basis.

1713 - 233

234 L. H. COLLIER

PRELIMINARY STUDIES

Epidemiological survey ofarea proposedforfield trialsSince most field work on trachoma is undertaken

in developing countries, the following factors mustbe assessed in choosing a suitable area:

Administrative aspects(a) Co-operation of central and local government and

of health authorities.(b) Stability and degree of co-operation of the popula-

tion under study.(c) Ease of access to study area; and communications

in general.(d) Availability and use of sulfonamides and anti-

biotics among the population under study.(e) Presence of other field projects that might assist or

interfere with the proposed investigation.(f) Possibility of establishing adequate laboratory

facilities.

Scientific aspects(a) Prevalence and age distribution of active trachoma,

with special attention to the usual age of onset.(b) Proof that the trachoma syndrome in the area

concerned is caused by TRIC agents."(c) If possible, serological classification of an adequate

sample of locally isolated TRIC agents, in terms of themouse toxin test (Bell & Theobald, 1962; Wang &Grayston, 1963) and/or the fluorescent antibody test(Hanna & Bemkopf, 1964; Nichols & McComb, 1964).

Laboratory studiesBefore subjecting a vaccine to an elaborate and

expensive field trial, it is obviously desirable to havesome laboratory evidence of potency. A potencytest measures some characteristic of the vaccineknown to be correlated with its power to protectagainst the naturally occurring infection, for exam-ple, capacity to protect a susceptible experimentalanimal against artificial challenge with the virulentorganism. In some instances, it is necessary onlyto do a simple quantitative test; thus titration ofsmallpox vaccine in rabbits or in chick embryosobviates the need for tedious protection experiments.As far as trachoma vaccine is concerned, we arestill at the stage at which none of the features thatcan be tested in the laboratory has been definitivelycorrelated with protective effect in the field; toremedy this position, it is therefore most important

1 The trachoma-inclusion-conjunctivitis subgroup of thepsittacosis-lymphogranuloma-trachoma (PLT) micro-organ-isms are referred to as TRIC agents. The strain designationsin this article are those used by the authors of the variouspapers reviewed; in most instances they follow the " Montrealsystem" proposed by Gear et al. (1963) and Collier (1963).

to record as much information as possible aboutevery batch of vaccine used for field trials.The following methods are available for testing

experimental trachoma vaccines.

Determination of ability to confer protectionagainst artificial challenge by the conjunctival route.Since only primates appear to be susceptible toinfection by this route, such tests must be done insimian species or in human volunteers. Althoughthese experiments are tedious and expensive, they are,short of an actual field trial, the most direct test ofpotency available.

Determination of antibody response to vaccinationof animals and man. Antibodies to TRIC agents canbe assayed by complement fixation (Collier et al.,1958; Grayston et al., 1960); neutralization ofinfectivity for cell cultures (Reeve & Graham, 1962)or mice (Bemkopf, 1959); agglutination (Bernkopfet al., 1960; Gancedo et al., 1961) and fluorescentmicroscopy (direct and indirect methods). Recentfindings by Parikh & Schechmeister (1964) suggestthat antibodies to PLT agents might also be studiedby gel-precipitin techniques. There is as yet no evi-dence about which, if any, of these methods is themost useful but, prima facie, antibodies that neu-tralize infectivity seem to be the most relevant to thepurpose under discussion.

Toxicity protection test in mice. By prior immuniza-tion with a suitable strain, mice can be protectedagainst the lethal toxic effect of TRIC agentsinjected intravenously; cross-protection tests of thiskind divide TRIC agents into at least two serologicalgroups which in general show some degree of anti-genic overlap. It is not known whether the abilityof a given vaccine to protect mice against intravenouschallenge with homologous antigen is related to itsperformance in protecting primates against con-junctival infection; there is, however, some evidencethat the results of cross-tests in monkeys challengedby the conjunctival route are related to the serolo-gical grouping indicated by the mouse test (Graystonet al., 1964).

Immunofluorescence testing. As well as measuringantibody, immunofluorescent techniques can dis-tinguish between TRIC antigens in terms of theirreactions with suitable conjugated antisera; thecomparatively small number of strains so far testedappear to fall into two main groups, roughlycorresponding to those indicated by the mouse toxintest. However, there is no information directly linkingthese findings with ability to protect against infection.

PRESENT STATUS OF TRACHOMA VACCINATION STUDIES

Determination of antigen content. Ultimately asimple assay of the amount of antigen in a givenbatch of vaccine would be the most satisfactoryindex to potency, provided, of course, that it can bedirectly related to protective effect. At present, theamount of TRIC agent in a given preparation isusually estimated by one or more of the followingmethods:

(a) Total particle count (Reeve & Taverne, 1962).(b) Determination of amount of complement-fixing

antigen.(c) Determination of amount of live agent, in terms of

egg lethal doses (ELD50), egg infective doses (EID50)and, for strains that grow in cell cultures, inclusion-forming units (Furness et al., 1960). In the case ofinactivated vaccines, the content of live agent should beestimated immediately before inactivation.

Preparation of vaccineThe following factors have to be considered.Choice of host cell. The trachoma vaccines so far

used in man have been prepared from chick-embryoyolk sacs, in which all known TRIC agents can begrown, and which have the merit of yieldinglarge quantities of antigen. The disadvantages areprobable contamination with avian leucosis virus,unless RIF-tested eggs are used; relative difficulty inpurification; and the possibility that vaccine willinduce sensitization to egg protein.Although antigens grown in cell cultures have as

yet been used only in animal experiments, they arepotentially useful for vaccine production. A con-siderable number of cultured cells will be needed toyield the same amount of antigen as a chick embryo,but such preparations are more readily purified andare free from avian leucosis virus. On the otherhand, primary explants are liable to carry othercontaminants, such as vacuolating agents; and theuse of diploid cell lines has not so far received officialapproval because of doubts about the significanceof chromosomal anomalies in serial subcultures.

Choice of vaccine strains. The TRIC agent(s) tobe incorporated in a vaccine must produce a high

yield in the culture system employed and mustpossess satisfactory immunogenicity in relation tothe strains prevalent in the country of use.

Choice of live or inactivated antigen. Most investi-gators have used inactivated antigens both forlaboratory studies and for field trials. There is,however, some evidence from animal experimentsthat live antigens are more effective, and suchpreparations have been used in man without un-toward effects.

Use of adjuvants. Trachoma vaccines containingadjuvants-usually aluminium hydroxide or mineraloil-have been tested both in man and in simianspecies. Although some workers claim good results,there is as yet no general agreement as to whetheradjuvants assist the induction of immunity; and thereis evidence that in certain circumstances their usemay actually enhance the response to subsequentchallenge by the conjunctival route.

Tests for innocuity and purity, and freedom fromcontaminants. There are as yet no national or inter-national regulations specifically dealing with tra-choma vaccine; unless or until such regulationscome into force, manufacture and testing of anyantigens destined for use in man should conformwith the relevant official requirements relating toviral vaccines in the countries in which they are tobe manufactured and/or used, and with the recom-mendations of the WHO Expert Committee onTrachoma in its third report (1962).

CONDUCT OF FIELD TRIALS

The principles to be observed in the conduct oftrachoma vaccine field trials were set out in the thirdreport of the WHO Expert Committee on Trachoma(1962) and need not be repeated here, except tore-emphasize the absolute necessity for valid ran-domization and " double-blind" procedures, forpreliminary definition of diagnostic criteria, and,should more than one ophthalmologist be involved,for mutual agreement on methods of examinationand recording of physical signs.

REVIEW OF PROGRESS IN TRACHOMA VACCINATION STUDIES

It will be appreciated from what has been said is so voluminous that it cannot be reviewed in thethat a vaccine development programme entails space of this article. I shall, therefore, deal onlymuch fundamental research; indeed, the literature with key papers directly concerned with preliminarydealing with trachoma vaccine and cognate subjects tests of efficacy, and with actual field trials. For

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L. H. COLLIER

convenience, the investigations of the various groupsknown to be working on this subject are consideredseparately.

HARVARD SCHOOL OF PUBLIC HEALTH,BOSTON, MASS., USA

All the antigens prepared by these workers foruse in man consisted of formol-inactivated elemen-tary body suspensions extracted from yolk sacs byether treatment and centrifugation (Bell & Theobald,1962).An early study by Snyder et al. (1962) suggested

that the clinical course of experimental trachoma ina single vaccinated volunteer was somewhat lesssevere than that in a control subject receiving adummy vaccine; the antigen used was SA-2, laterdescribed as TRIC/2/SAU/HAR-2/OT. Nevertheless,there was no detectable response to vaccination interms of complement-fixing antibody, and TRICagent was demonstrated throughout the experimentalinfection in both vaccinated and control subjects;this " double-blind " study is noteworthy for its useof a scoring system to assess the response tochallenge.The method was later improved by using challenge

inocula of known titre (Bell, Murray et al., 1963);for strain SAU/HAR-2 the infectivity end-pointfor the human conjunctiva proved similar to thatfor the chick embryo. Three more volunteers receivedfour injections of aqueous SAU/HAR-2 vaccine over aperiod of 10 months, and 26 months later a dose ofthis strain combined with TRIC/l/ET/HAR/2/OT in amineral oil adjuvant. The results of subsequentchallenge with small doses of SAU/HAR-2 were againinconclusive. Increases in the serum titre of antibodyfixing complement with group antigen were demon-strated following vaccination, and in the controlsubjects after challenge; in no instance did the titreexceed 1/160.

In a small-scale collaborative trial in Portugal,Sampaio and co-workers (1963) showed that tra-choma attack rates in subjects receiving eithersAU/HAR-I or SAU/HAR-2 antigens were less thanthat in a control group receiving a dummy vaccine,but the differences were not statistically significant.There was no correlation between the serologicaltitres and resistance to trachoma.

Full-scale field trials in Saudi Arabia were startedin 1962, preceded by studies of the epidemiology andmicrobiology of trachoma in the area concerned(Bell, Nichols & Haddad, 1963; Snyder et al., 1964).The bivalent vaccine used was prepared from

strains sAU/HAR-2 and ET/HAR-2, representing the twoserological groups defined by the mouse toxin test;it was given either as an aqueous suspension atzero time, two weeks and six months, or emulsifiedwith Drakeol-6/Arlacel-A at zero time and at sixmonths. The dummy antigen consisted of normalyolk-sac extract containing monovalent typhoidvaccine. Vaccines were tested on a strict " double-blind" basis both in children who had not yetacquired trachoma and in those with established dis-ease. The latest report by Snyder and his colleagues(1964) shows that six months after the last doseof vaccine, 52% of 82 originally non-trachomatouschildren receiving adjuvant vaccine had acquiredthe disease, compared with 55% of 97 in the controlgroup. At the most recent follow-up examination40% of 98 children receiving aqueous vaccine hadacquired trachoma compared with 61 % of 99 con-trols. This difference is statistically significant(R.L. Nichols, personal communication).Of the vaccinated children who subsequently

acquired trachoma slightly fewer were inclusion-positive as compared with the control subjects inthis category; there was a significant reduction inthe average number of inclusions per child receivingvaccine proper.

Neither aqueous nor adjuvant vaccine influencedthe course of the disease in children suffering fromtrachoma at the time of vaccination.The dummy adjuvant material containing typhoid

vaccine provoked local reactions in about 5% ofsubjects and its use had to be discontinued.

US NAVAL MEDICAL RESEARCH UNIT NO. 2, TAIWANWITH THE

UNIVERSITY OF WASHINGTON, SEA1TLE, WASH., USA

A preliminary study (Grayston et al., 1960)showed that complement-fixing antibodies wereelicited in man and monkeys by injections of purifiedtrachoma agent grown in the yolk sac, given either(a) as formolized elementary bodies, alone orattached to aluminium hydroxide particles, or(b) as live antigen. There was some evidence fromsmall numbers of subjects that vaccine modified theresponse of monkeys to conjunctival challenge, andthe course of experimental trachoma in previouslyinfected volunteers. Later, it was found that thealuminium hydroxide vaccine induced a higher andmore prolonged antibody response in man thaneither plain formolized or live antigens (Grayston,Woolridge & Wang, 1962). However, large singledoses of aluminium hydroxide vaccine resulted in

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PRESENT STATUS OF TRACHOMA VACCINATION STUDIES

worsening of experimentally induced infection,whereas repeated small doses of vaccine without thisadjuvant gave some protection. The deleteriouseffect was ascribed to hypersensitivity to the micro-organism itself rather than to egg material. Bycontrast, no such reaction was seen in vaccinatedchildren who subsequently became naturally in-fected. This paper drew attention to the difficulty ofdiagnosing early trachoma in children included in thefield trial, and to variations in the diagnosis of a givenchild seen at different times by the same observer.

Grayston, Wang and co-workers (1962) alsostudied the influence on experimental trachoma of analuminium hydroxide vaccine prepared from strainsTw-l and Tw-3 (formerly Tw-29) inactivated byformol. Three volunteers infected in one eye twomonths previously with strain Tw-I each receivedsix injections of vaccine during the next four months;three similarly infected controls received dummyvaccine. In all three control subjects there was cross-infection of the uninoculated eye, and relapsefollowing subsequent treatment with oxytetracyclineand sulfonamide; neither occurred in the vaccinatedsubjects.

Purified formol-inactivated TRIC agent emulsi-fied in mineral oil adjuvant (Bayol F with Arlacel)induced a prolonged rise in the titre of complement-fixing antibody in volunteers (Khaw et al., 1963).However, it failed to immunize against conjunctivalchallenge, although the incubation period of theinfection was longer and the symptoms were milderthan in control subjects. Unlike the aluminiumhydroxide vaccine, it did not exacerbate the ophthal-mological response to challenge. As in previousstudies, the titre of complement-fixing antibodyinduced by vaccination was unrelated to immunity.

Vaccines of the aluminium hydroxide type madefrom strains TRIc-Taiwan-l (formerly Tw-10) andTRIc-Taiwan-3 (formerly Trw-29) were tested inchildren of preschool age in Taiwan (Grayston etal., 1963); these strains are prototypes of the twoserological types present in Taiwan (Wang & Gray-ston, 1963). Both monovalent (Tw-3) and bivalentvaccines were tested, but in different areas. Vaccinesand dummy preparations were given at random, andthe trials were made on a " double-blind " basis.Over a 2 l2-year observation period, both mono-valent and bivalent vaccines significantly reducedthe attack rate, and delayed the average time ofonset in those children who acquired trachoma.It is interesting that the monovalent antigen per-formed better than the bivalent vaccine. With the

former, 4.5% of 67 vaccinated children eventuallybecame trachomatous, compared with 21 % of 62in the control group; the respective figures for biva-lent vaccine were 9.9% of 121 and 16.8% of 155.This paper also mentions, without giving details ofpreparation, use of an ultraviolet-irradiated vaccine.In 36 children receiving it, the attack rate and averageduration of disease were significantly less than in53 control subjects.

Later evidence suggested that monovalent vaccinesprepared from the prototype strains used (Tw-1and Tw-3) failed to protect against each other inmonkeys challenged by the conjunctival route(Grayston et al., 1964). This paper also reports theoccurrence of pannus, Herbert's pits and cicatriza-tion in a proportion of monkeys infected with theBour strain of trachoma and the Ic-Cal-3 strainof inclusion conjunctivitis. Pannus was seen onlyin previously immunized or infected animals, andwas ascribed to hypersensitivity.

In these monkey experiments, vaccine purifiedby the method used hitherto (trypsinization andpolymyxin precipitation) proved less effective than(a) crude antigen given with mineral oil adjuvant,(b) adjuvant vaccines containing virus purified byGenetron extraction and sucrose density gradientcentrifugation, and (c) adjuvant vaccines containingvirus purified by ultrasonic treatment and sucrosedensity centrifugation. The authors comment parti-cularly that Genetron treatment-which kills thevirus and extracts soluble complement-fixing anti-gen-does not remove the protective antigen.

FRANCIS I. PROCTOR FOUNDATION FOR RESEARCHON OPHTHALMOLOGY,

AND THE

DEPARTMENTS OFOPHTHALMOLOGY AND MICROBIOLOGY,UNIVERSITY OF CALIFORNIA MEDICAL CENTER,

SAN FRANCISCO, CALIF., USA

In a quantitative study of the response of monkeysto infection with several TRIC agents, strain Bourinduced the most severe disease (Dawson et al., 1961).The conjunctival response to infection with Bourwas modified, but not completely suppressed, byprior immunization with crude live yolk-sac sus-pensions of the homologous strain, or of strainsAsghar, Apache-l and Ic-Cal-3.

E. Jawetz (personal communication) found thatconjunctival inoculation of human volunteers withthe inclusion conjunctivitis strains Ic-Cal-3 oric-Cal-8 conferred a moderate degree of resistance

6

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L. H. COLLIER

to a subsequent inoculation with the homologousstrain. However, there is evidence that such inocula-tions do not confer protection against challenge by aheterologous strain.

ISTITUTO SUPERIORE DI SANITA, ROME, ITALY

AND THE

CLINICA OCULISTICA DELL'UNIVERSITA DI ROMA, ITALYWITH THE

CENTRO OFTALMICO HAILE SELASSIE I, ASMARA,ETHIOPIA

Findings in Ethiopia by Felici & Vozza (1960)suggested that formolized yolk-sac vaccines madefrom strain Asmara 3/1960 (Bietti et al., 1962),alone or combined with mineral oil or aluminiumhydroxide adjuvants, protected volunteers againstconjunctival challenge with the homologous strain;aqueous vaccine appeared the most effective. Theresults of similar experiments in monkeys aredifficult to interpret since only a proportion of thecontrols were infected by the challenge dose used.It is stated, without giving clinical details, that bothtypes of adjuvant vaccine favourably modified thecourse of established trachoma in children. Sub-sequent use of less concentrated challenge inocula involunteers gave more clear-cut evidence of pro-tection by both oil and aluminium hydroxidevaccines. Like others, these investigators noteddiscrepancies between the titre of circulating anti-body (measured by complement-fixation or micro-agglutination) and susceptibility to experimentalinfection.A total of 4100 subjects were included in field

trials started in Ethiopia between 1960 and 1962(Bietti, 1964), but since many could not be tracedover a Three-year follow-up period, there wasdifficulty in interpreting some of the results. Thetrials were made on a " double-blind " basis, usingmonovalent vaccine (strain Asmara 3/1960) usuallycombined with mineral oil adjuvant. Evaluations ofresults were based only on changes in degree offollicular hyperplasia. The Italian workers are of theopinion that a single dose of oil adjuvant vaccinehad no effect, but that use of a second dose ofaqueous vaccine 45 days later diminished the attackrate in healthy subjects and favourably affectedthe course of established trachoma. In babies, oiladjuvant vaccine sometimes gave rise to nodulesthat remained palpable for long periods.

Preliminary evidence suggested that the beneficialeffect of vaccination on established trachoma wasenhanced by subsequent parenteral treatment with

a long-acting sulfonamide (3-sulfanilamidomethoxy-pyrazine).

DR GRAHAM SCOTT, JOHANNESBURG, SOUTH AFRICA

(IN COLLABORATION WITH SOUTH AFRICAN INSTITUTE

FOR MEDICAL RESEARCH)

Scott (1964) described experiments with livevaccine made from yolk sacs infected with strainTRIC/ /ZA/SAIMR 1 '59/oT. Few details of pre-paration are given, and the content of live TRICagent is not stated. One volunteer was immunizedand later repeatedly challenged with dilutions ofhomologous strain varying from 1/300 to 1/30(infectivity titres not stated). The course of infectionwas compared with that in a control subject chal-lenged with dilutions of 1/300 and 1/200. It is dif-ficult to be sure from the published details whether,as claimed, significant protection was achieved.Another volunteer infected with a second strain ofTRIC agent was vaccinated during the course of theexperimental disease; it is stated, without givingclinical details, that marked improvement wasobserved after each of three spaced injections,although TRIC agent could be cultivated from theconjunctiva throughout the observation period.A trial was undertaken in two nursery schools in

an African township; each contained 100 childrenaged 2-5 years. In both schools, approximately 20%of children suffered from mild trachoma and TRICagent was isolated from about 30% of clinical cases.All the children in one school were vaccinated sub-cutaneously with two or three spaced doses atmonthly intervals; the other school served as control.Over a 20-month observation period the attack ratein the control children was considerably greaterthan that in the vaccinated group; it is suggestedthat vaccination also had a limited therapeuticeffect. Unfortunately, this trial is open to theobjection that the vaccines were not given at random.

MEDICAL RESEARCH COUNCIL TRACHOMA RESEARCH

UNIT, LONDON, ENGLAND, AND FAJARA, GAMBIA

Live vaccines made from yolk sacs infected withinclusion blennorrhoea strain TRIC/ /GB/MRC-4/ON(formerly LB4) protected baboons against con-junctival challenge with the homologous strain(Collier, 1961a). Two subcutaneous doses and oneintravenous dose at weekly intervals gave almostcomplete immunity; three subcutaneous dosesconferred partial protection. The technique waslater refined by assigning numerical scores to the

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PRESENT STATUS OF TRACHOMA VACCINATION STUDIES

physical signs of infection and presence of inclusions,and by submitting the scores to analysis of varianceto determine the significance of differences withinand between groups in response to challenge. Incollaboration with workers from Pfizer Ltd, a seriesof such tests were made, mostly with MRC-4 asboth vaccine and challenge strain (Collier et al.,1964). Animals immunized by the subcutaneous/intravenous method still showed evidence of pro-tection up to 15 months later, although immunitywas obviously waning. Live antigens emulsified withDrakeol-6/Arlacel-A gave variable results; withinthe same group of animals some were partiallyprotected whereas the response of others to challengewas similar to or significantly more severe than thatof controls.

Experiments with inactivated vaccines weredisappointing. The performance of formolizedantigens was variable, and enhancement of responseto challenge was occasionally encountered. Vaccinesthat were highly immunogenic when given as livesuspensions failed to protect after inactivation byheating at 37°C, or by exposure to twice the dose ofultraviolet light needed to abolish infectivity.Good immunity was conferred by a " fast-

killing" variant of MRc-4 (Reeve & Taverne, 1963)grown in HeLa cells, against challenge with theoriginal " slow-killing " strain grown in the yolk sac.A recent unpublished experiment suggests that

MRc-4 and a Gambian strain TRTC/ /WAG/MRC-187/OT) failed to cross-protect against conjunctivalinfection.

In parallel with these animal studies, the TrachomaResearch Unit made an intensive survey of theepidemiology, clinical features and microbiologyof trachoma in two neighbouring Gambian villages(Sowa et al., 1965). The prevalence and attack ratesso determined were later used to compute the num-bers of children to be used in vaccine field trials.The first field investigation was a study of the

influence on established trachoma of a live antigenmade from a locally isolated strain (TRIC/ /WAG/MRc-221/oT) (Collier et al., 1963). Vaccine properand dummy vaccine were assigned at random to

38 children with virologically proved, active tra-choma. Statistical analysis of changes in score forphysical signs and presence of TRIC agent in theconjunctiva showed that about one-third of thosevaccinated were benefited, but this effect was ofshort duration. Small increases in circulating com-plement-fixing antibody to the group antigen wereobserved in some subjects, but bore no relation tothe clinical or virological response to vaccination.

In a subsequent trial (to be published) a liveyolk-sac vaccine was prepared at the Lister Institutefrom strain TRIC/ /WAG/MRC-187/oT. It was heldat -600C until just before use and then emul-sified with Drakeol-6/Arlacel-A. Altogether 278 non-trachomatous children in two Gambian villageswere selected by clinical examination, followed inevery doubtful case by egg inoculation of con-junctival scrapings and examination for inclusions.Vaccine proper and dummy vaccine were assignedat random to equal numbers of children and injectedintramuscularly in 0.25-ml doses; a few transientminor local reactions were observed. Seven monthslater, all children were re-examined and given asecond similar dose; those with physical signssuggestive of trachoma were tested for TRIC agentas before. At this follow-up 8.2% of the 122 vaccin-ated subjects examined had acquired trachoma,compared with 13% of 123 controls. Six monthsafter the booster dose, the respective figures were37.3% and 29.8%. These differences were not sta-tistically significant.

In these field experiments all clinical examinationswere made by the same ophthalmologist, who hadno knowledge of the identity of the vaccines orcontrol preparations, and, at each follow-up, noaccess to the results of previous examinations.

In collaboration with Pfizer Ltd, Evans MedicalLtd and the Lister Institute, a vaccine prepared fromstrains Asghar and SAU/HAR-2 is being tested for pro-phylactic efficacy in the Gambia; and in collabora-tion with these organizations and with the Instituteof Ophthalmology, London, and Teheran University,a large trial using both live and formol-inactivatedantigens has been started in Iran.

CONCLUSIONS

This paper does not pretend to give a critical viewof trachoma vaccine research, but rather to sum-marize the more important problems, and theresults of recent investigations. The latter was not

an easy task, and the drawing of more than the mostgeneral conclusions is made even more difficult bythe virtual impossibility of comparing the findingsof different workers. There are variations in the

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L. H. COLLIER

clinical features of trachoma and in the methodsused for recording them and for assessing changes;in epidemiology and presence of intercurrent infec-tions; in methods of testing for TRIC agent in theconjunctiva; in types of vaccine, vaccine strains,purification methods and immunization schedules;and in the methods used to test vaccines in thelaboratory. The main conclusion that emerges isthat TRIC agents are comparatively poor antigens,and that a fully effective vaccine has not yet beenreported. Although a measure of protection can beobtained against both experimental and naturallyoccurring trachoma, and there is some evidence oftherapeutic effect, there is no assurance of solidimmunity; and several reports suggest that anybeneficial effect is of comparatively short du-ration.

Thus, although the considerable progress nowbeing made in knowledge of the PLT group giveshope of improved trachoma vaccines, many impor-tant problems call for rapid solution. More researchis needed on methods of potency testing; on thenature of the immunizing antigen of TRIC agents;

on methods for enhancing and prolonging artificialimmunity and on the use of adjuvants for thispurpose; on the significance of the antibodiesdetected by various methods; on the extent of cross-protection against conjunctival infection by differentserological types of TRIC agents; and on the relativemerits of live and inactivated antigens. In additionto these fundamental studies, we have still to deter-mine the minimum immunizing dose, the hosttissue most suitable for production purposes, theoptimum conditions for storing vaccines, and thepossibility of inducing sensitization to the antigenitself or to other constituents of the vaccine.Because there are so many problems still to be

solved, it is of the first importance that every effortbe made to avoid premature and overoptimisticreports appearing in public communication media.Since trachoma is still a major world health problem,its conquest is a matter of great general interest;but there is a distinct possibility that such reportsmay influence public health authorities to relaxprematurely their efforts to control it by meansother than vaccination.

ACKNOWLEDGEMENTS

I am grateful to Dr S. Bell, Professor G. B. Bietti, Professor T. Grayston, Professor E. Jawetz and ProfessorP. ;Thygeson for providing reprints of published work and additional information, which were most helpful in thepreparation of this paper.

RtSUMIt

L'isolement, en 1957, de l'agent du trachome (virusTr-IC, sous-groupe des virus PLT: psittacose-lympho-granulomatose-trachome) avait fait esperer la realisationd'un vaccin prophylactique. La lutte contre le trachomereste un probleme primordial dans beaucoup de pays envoie de developpement, malgre la prevention et le trai-t&hent au moyen des antibiotiques et des sulfamides.' L'auteur resume les conditions auxquelles doit satis-faire un vaccin antitrachomateux et les problemes queposent sa fabrication et son contr6le. Le programmed'etude de tels vaccins comporte des enquetes pr6limi-naires, epidemiologiques et de laboratoire, suivies d'unessai pilote sur l'homme. D'apres ces resultats, onentreprendra des essais A grande echelle dans diverseszones end6miques avant- d'aborder la fabrication com-mrciale. Les enquetes doivent pr6ciser la prevalence et ladistdibution selon l'age dq, trachome evolutif et l'agehabituel des sujets nouvellement atteints; prouver que le

syndrome est bien du a un virus Tr-IC; si possible classerserologiquement un exemplaire du virus trachomateuxisole localement. L'efficacit6 du vaccin est recherchee aulaboratoire puis par experimentation humaine. Enl'absence de normes nationales ou internationales, lesvaccins contre le trachome doivent etre conformes auxnormes sur les vaccins viraux du pays et aux recommanda-tions 6nonc6es dans le troisieme rapport du Comit6OMS d'experts du Trachome (1962). Les essais humainsdoivent etre menes suivant la methode du double ano-nymat.

I1 existe une abondante litterature sur les essais devaccins antitrachomateux. Plusieurs de ces vaccins sontpartiellement efficaces et il reste beaucoup a apprendresur l'immunologie du trachome et les methodes propresA conferer une immunite. Les virus Tr-IC sont de mauvaisantigenes qui n'ont pas encore permis d'obtenir unvaccin pleinement efficace. Des recherches sont necessaires

240

PRESENT STATUS OF TRACHOMA VACCINATION STUDIES 241

pour mesurer I'activit6 des vaccins, pr6ciser l'importancedes anticorps mis en evidence par des methodes diff&rentes, juger le degr6 d'immunite crois6e obtenu pardiff6rents types s6rologiques du virus, et comparer lesantigenes vivants et inactives. D'autres problemes sont

encore a r6soudre: determiner la dose immunisanteminimale, le tissu le plus favorable a la production devaccin, les conditions optimales de conservation et lesrisques de sensibilisation i l'antigEne ou a d'autrescomposants du vaccin.

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