INTERNATIONAL JOURNAL OF LEPROSY^ Volume 57, Number IPrinted in the U.S.A.

A Pilot Study of Three Potential Vaccinesfor Leprosy in Bombayl

R. Ganapati, Chandrakant R. Revankar, Diana N. J. Lockwood,Rowan C. Wilson, Janet E. Price, Penelope Ashton,

Leslie A. Ashton, Rowena M. Holmes, Chad Bennett,John L. Stanford, and Richard J. W. Rees2

Trials of the efficacy of BCG vaccinationagainst leprosy have produced variable re-sults, but the study carried out in Ugandashowed that under some conditions at leastit could be very effective (2). The develop-ment of soluble preparations of leprosy ba-eilli for skin testing (4 has meant thatmodified vaccincs could be assessed with-out the expense of direct protection trials,on the assumption that the induction of pos-itivity to these skin-test reagents indicatesan improved levei of immune protection.Thus, Convit and his colleagues have usedtheir soluble leprosy reagent (4) to assess theeffects of a combination of BCG plus killedMycobacterium leprae as a vaccine (3). Thisvaccine is currently under trial as to its di-rect protective effect in South America andMalâwi.

The soluble reagent leprosin A, preparedin London (14) from leprosy bacilli, has beenuscd to assess the effects of BCG in the In-dian towns of Agra ('°) and Ahmednagar(15), and to evaluate the combinations ofBCG with M. vaccae or M. leprae in TheLebanon (') and in Iran (5. 16). In the presentstudy, we have used this reagent together

Received for publication on 26 May 1988; acceptedfor publication in revised form on 3 November 1988.

= R. Ganapati, M.B.B.S, D.D.V.; C. R. Revankar,M.D., D.P.H., The Bombay Leprosy Project, Bombay,Maharashtra, India. D. N. J. Lockwood, M.B.Ch.B.,M.R.C.P.; R. C. Wilson, M.B.B.S., M.R.C.P.; J. E.Price, M.B.Ch.B.; P. Ashton, M.B.Ch.B.; L. A. Ashton,M.B.Ch.B.; R. M. Holmes, M.B.Ch.B.; C. Bennett,M.B.B.S.; J. L. Stanford, M.D., The School of Pathol-ogy, University College and Middlesex School of Med-icine, London, U.K. R. J. W. Rees, M.B.B.S., F.R.C.P.,F.R.C.Path., The Clinical Research Centre, WatfordRoad, Harrow, Middlesex, U.K.

Reprint requests to: Dr. J. L. Stanford, School ofPathology, University College and Middlesex Schoolof Medicine, Riding House Street, London W I P 7LD,U.K.

with three other new tuberculins (9) to de-termine the effects of these combinations inchildren attending schools in the slums ofBombay, India. Bombay is an enormous citywith 8-9 million inhabitants where bothleprosy and tuberculosis are endemie. In theslums where the children live environmen-tal mycobacteria abound, and even the pipeddrinking water may contam n up to 108 my-cobacteria per liter (6).

MATERIAI,S AND METHODSReagents used. The skin-test reagents used

wcre four of the series of new tuberculins(9)— tuberculin, leprosin A, scrofulin, andvaccin — prepared from AI. tuberculosis, AI.leprae, M. scroMlaceum, and AI. vaccae, re-spectively. They were given as 0.1 ml in-tradermal injections at least 10 cm aparttwo on each forearm. The doses injectedwere 0.2 pg of tuberculin and scrofulin, 1.0

of leprosin A, and 2.0 lig of vaccin, asused in previous studies ('' 5' '(). Diametersof induration were measured after 72 hr,and mcan reaction sizes of 2 mm or morewere considercd positive. Our experience inother studies (5. "1.14-16) led us to take thissmall size as positive. Proper use of the shortbevel, 26-gauge, intradermal needle, whichcauses minimal trauma (rarely more than1 x 1 mm) has not led to problems in theinterpretation of small-sized reactions. Thearbitrary decision that 4 x 5 mm is a neg-ative response and 5 x 5 mm a positiveresponse has never seemed sensible to us,especially when biopsy data show that thesame cens inffitrate very small reactions (anda proportion of zero reactions) as inffitratelarge reactions (17).

The vaccines used were the same as thosepreviously employed in studies in The Leb-anon ('), and in Iran (5. 16). BCG Glaxo (freeze

33

34^ International Journal of Leprosy^ 1989

TABLE 1 Residis ofskin tests Inumber positive/total (9á positive), mean ± S. D. indur-ation] inunediately before and 1-3 years after vaccination with BCG alone (vaccine A),BCG plus 10' killed M. vaccae (vaccine 13), or BCG idas 10' M. leprae (vaccine C)."

Vaccine A Vaccine 13 Vaccine C

Tubercul inTime 0 17/64 (27%)

3.24 ± 1.00 mm18/61 (30%)2.90 ± 0.71 mm

17/61 (28%)2.94 ± 0.93 mm

p < 0.00001h p < 0.00001 p < 0.000011-3 years 49/64(77%) 44/61 (72%) 51/61 (84%)

12.74 ± 5.90 mm 12.49 ± 7.51 mm 12.04 ± 6.92 mm

Leprosin ATime O 19/64 (30%) 10/61 (16%) 19/61 (31%)

4.19 ± 3.49 mm 4.00 ± 2.60 mm 4.53 ± 3.76 mmNS p < 0.002 NS

1-3 years 22/64 (34%) 26/61 (43%) 24/61(39%)6.38 ± 5.03 mm 4.92 ± 3.27 mm 5.62 ± 3.76 mm

Scroful inTime O 36/64 (56%) 27/61(44%) 29/61(48%)

5.35 ± 3.93 mm 4.52 ± 2.30 mm 5.62 ± 3.90 mmNS NS NS

1-3 ycars 35/64 (55%) 30/61 (49%) 35/61 (57%)4.92 ± 3.86 mm 5.77 ± 3.85 mm 6.40 ± 3.80 mm

VaccineTime O 29/64 (45%) 26/61 (43%) 25/61 (41%)

4.39 ± 2.33 mm 4.74 ± 2.46 mm 4.89 ± 2.84 mmNS NS NS

1-3 years 22/64 (34%) 22/61 (36%) 30/61 (49%)5.30 ± 4.33 mm 4.74 ± 2.63 mm 5.64 ± 3.33 mm

" Mean age of the children was 7.2 years prior to vaccination.Prohabilities calculated hetween O time and 1-3 years by Fisher's exact test.NS = difference hetween O time and 1-3 years not statistically significant.

dried) was made up with the water providedby the manufacturer (vaccine A), or the sameBCG was made up with a suspension of 108/ml of í. vaccae in saline (vaccine• B), orwith a suspension of 108/m1 of ,1/. leprae insaline (vaccine C). The organisms in thesesuspensions had been killed by exposure to2.5 megarads from a 60cobalt source. Vac-eines were administered by an intradermalinjection of 0.1 ml high over the left deltoidmuscle.

Study plan. The children studied wereaged 3-17 years and attended a number ofschools in several different slum districts ofthe city. They were arbitrarily selected forvaccination on the basis of the lack of a scarfrom earlier BCG vaccination and a skin-test reaction to tuberculin ofless than 5-mmmean diameter of induration. Out of 1595children tested, 395 were suitable for vac-cination, and 292 were vaccinated-96 weregiven vaccine A, 99 were given vaccine B.and 97 were given vaccine C. Among the1200 children who were not suitable for

vaccination, 775 had BCG scars (523 or 67%of them were tuberculin positive) (H), and425 had responses to tuberculin of 5 mmor more despite their lack of a BCG scar.

Follow-up skin testing with the four re-agents was carried out 1 to 3 years aftervaccination in as many children as possible,without the person doing the tests knowingwhich child had received which vaccine.Thus, the results were recorded "blind." Thesizes of the vaccine scars were also mea-sured at these times. Most of the skin testingand vaccinating for the study was carriedout by a series of students in the course oftheir elective studies.

RESULTSEffects of the three vaccines. The mean

age at the time of vaccination of the chil-dren who were followed up was 7.2 ± 1.7(mean ± S.D.) years (range 3-15 years), andthe mean age of those among them who hadresponses to leprosin A at the time of vac-

57, 1^Ganapati, et al.: Pilot Study of Three Potential Vaccines^35

TABLE 2.^Percentages of persons, mainly chddren, converting to leprosin A positivityafiei. BCG or BCG pias M. vaccac, and the differences between them.

BCG Difference BCG +^vaccae

Leprosy-endemic situations

Iran

Children of patients (16) 63% (31%) 94% (8 years)"Village children (5)

lndia

33% (16%) 49% (8 years)

Bombay children (this study)" 27% (14%) 41%Contacts of patients in South India' 47% (12%) 59% (1 year)

Situations non-endemic for leprosy

Irai]

Village children (s) 53% (3%) 56% (8 years)

Lebanon

Village children (') 12% (7%) 19% (3 years)

"The time in years is the period between vaccination and follow-up skin tests." To make them comparable with data from other studies, percentages shown from the present study are those

for children who had zero responses to leprosin A prior to vaccination.Preliminary results from an ongoing study.

cination was 7.0 ± 1.6 years (range 5-13years). The skin-test results for these chil-dren before and from 1-3 years after vac-cination with each of the three vaccines areshown in Table 1. It can be seen that alithree vaccines significantly increased tu-berculin positivity (p < 0.00001), and thatonly vaccine B increased leprosin A posi-tivity (p <0.002). There were no significanteffects on responsivcness to scrofulin orvaccin.

The postvaccination scar sues were thesame for ali three vaccines, and the meanscar size for ali childrcn was 6.1 ± 2.5 mm;two children had no visible scar, and thelargest scar measured 17 mm. There wereno significant differcnces between the dif-ferent schools in the effects of the vaccinesor bctween the scar sizes (data not shown).

DISCUSSIONAli three vaccines very significantly in-

creased tubcrculin positivity (p < 0.00001)to a similar degrce, showing that the addi-tions did not adversely affcct tubcrculinconvcrsion. The most important observa-tion of the study is that the incorporationofM. vaccae with BCG (vaccine B) produceda significant enhancement of postvaccina-tion leprosin-A positivity, which was notseen after BCG alone or after the combi-nation of BCG with M. leprae. However, ithas to be noted that there was no significant

difference in the final percentage positivityto leprosin A after each of the three vac-eines. Inclusion of an unvaccinated groupin our study might have been desirable, butwas not considered ethical.

Leprosy is highly endcmic for the slumarcas where the schools were situated, andthe source of sensitization to leprosin A islikely to be from casual contact with leprosypatients, rather than a direct effect of thevaccine. Thus, the data obtained in urbanBombay support the observations made inrural Iranian Averbaijan (5- '() that the com-bination of BCG Glaxo with M. vaccae maybc a bctter vaccine for leprosy than is BCGalone (Table 2). Although the numbcrs vac-cinated wcre small, it is interesting to notethat the addition of M. leprae to BCG didnot significantly improve recognition oflep-rosin A over the period of the study.

The mcchanism of action of vaccinescontaining BCG is interesting in that post-vaccination positivity dircctly caused by thevaccinc wanes very considerably within 3years unless boosted from the environment.This is clearly seen in the comparison be-tween results obtaincd in The Lebanon ('),where contact with environmental myco-bacteria appears to be minimal, and thoseobtained in Agra (1") and Ahmednagar (''),where environmental mycobacteria abound.BCG acts by priming the individual to rec-ognize small cnvironmental challenges andto develop delayed-type hyperscnsitivity,

36^ International Journal of Leprosy^ 1989

which may be directed to antigens of groupi (common mycobacterial), group iv (7. '2' 13)

(species specific), or even ofgroup ii (8)(slow-grower associated antigens).

AI. vaccae was specifically addcd to BCGto improve recognition of casually encoun-tered organisms, not to induce positivity toleprosin A by some crossreactivity inspecies-specific (group iv) antigens betweenit and the leprosy bacillus. The prescnt studyis one of a series in which the effects of theaddition of AI. vaccae are being assessed insituations of differing leprosy endemicity.The results of studies completed or still inprogress are shown in Table 2. In each ofthe studies in leprosy-endemic regions, skin-test rccognition of the leprosy bacillus liasbeen successfully enhanced.

SUMMARYThree vaccines, BCG Glaxo alone (vac-

cine A), BCG Glaxo plus 10 killed Myco-bacterium vaccae (vaccine B), and BCGGlaxo plus 107 killed M. leprae (vaccine C),were given to groups of selectcd children.The effects of thesc vaccincs on subsequentquadruple skin testing 1-3 ycars after vac-cination were comparcd. Ali three vaccinesequally and significantly (p < 0.00001) in-creased positivity to tuberculin, but onlyvaccine B was found to significantly en-hance development ofskin-test positivity toleprosin A (p < 0.002). The data supportthe evidence previously obtained in ruralIran that the combination of BCG with killedM. vaccae is likely to be a better vaccine forleprosy than is BCG alone.

RESUMENSe seleccionaron 3 grupos de Mitos a los cuales se

les administró la vacuna BCG Glaxo sola (vacuna A),BCG Glaxo más 107 Mycobacterium vaccae muertos(vacuna 13), o BCG Glaxo más 10' M. leprae muertos

(vacuna C). Se compararon los efectos de estas tresvacunas sobre las respuestas intradérmicas de los nifios

a 4 antígenos de prueba, 1-3 afios después de la va-

cunación. Las tres vacunas incrementaron significati-vamente (p < 0.00001) la reactividad ala tuberculina,pero sOlo la vacuna B incremente' significativamentela reactividad a la leprosina A (p < 0.002). Los datos

apoyan la evidencia obtenida previamente en Irán deque la combinación de BCG con ,If. vaccae muerto esprobablemente una mejor vacuna contra Izt lepra quecl BCG sólo.

RÉSUMÉOn a administre à des groupes sélectionnés d'enfants

trois vaccins, à savoir le IICG Glaxo seul (vaccin A),

le IICG Glaxo accompagné de 107 Mycobtreterium vae-cae tués (vaccin 13) et le BCG Glaxo plus 10' .1/. lepra('tués (vaccin C). On a compare les resultais d'une épreuve

cutanée quadruple, pratiquée I à 3 ans après la vac-cination, dans les trois groupes. Les trois vaccins ont,

tous, eu comine ellet d'aceroitre Izt réaction positive à

la tuberculine, de maniere statistiquement significative(p < 0.00001). Nézminois, seul le vaccin 13 renforcait

significziti ventem le développement d'une positivitécutanée à Izt léprosine A (p < 0.002). Ces données

confirmem les résultats obtenus précédemment dans

les régions rurales de l'Iran, qui montraient qu'unecombinaison de IICG et de M. vaccue tué parait consti-tuer un meilleur vaccin pour la lepre que le BCG seul.

Acknom ledgments. We should like to thank the chil-

dren, their parents, and the staff of the schools for theirparticipation in this study. Without the assistance of

members of the staff of the Bombay Leprosy Projectwe could not have carried out the work, and we arevery grateful for their assistance.

We also wish to thank LEPRA for financial support

for preparation of the reagents, and Glaxo Ltd. for the

donation of IICG. Money was kindly provided by LEP-RA which, together with bursarics from the Universityof Leicester and from the Medical Research Council,

U.K., allowed the students to carry out their electivcstudics. We thank our supporters most sincerely.

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