immunity in syphilis studies in active immunity · immunityin syphilis studies in active immunity...

Brit. J. vener. Dis. (1976) 52, 300

Immunity in syphilisStudies in active immunity

H. T. AL-SAMARRAI* AND W. G. HENDERSONtFrom the Department of Pathology and the Department of Microbiology, The Royal Free Hospital, London

SummarySupport for the concept of the development ofimmunity during the course of syphilis is availablein the literature. In experimental syphilis in rabbits,some immunity is present approximately 3 weeksafter infection with Treponema pallidum. Resistanceto re-infection increases to a maximum at approxi-mately 3 months after infection. Termination of thisstate by penicillin treatment within this 3-monthperiod may enable re-infection to be accomplished.

Attempts to reproduce this state of immunityexperimentally by injection of T. pallidum itself,or protein derivatives, or ultrasonic disintegratesobtained from T. pallidum or non-pathogenictreponemes, have been unsuccessful. However,promising results in rabbits have resulted frominjecting T. pallidum suspensions attenuated bystorage at 40C, penicillin, or gamma irradiation,and also by suspensions preserved by glutaral-dehyde. In the present study, partial resistance tointratesticular challenge in rabbits with T. pallidumhas been obtained by immunization with a varietyofnon-pathogenic treponemes, as exemplified by thestrains Nichols, Kazan 2, 4, 5, and 8, Treponemaminutum, Treponema ambigua, Treponema refringensand Treponema microdentium. Success is attributedto the processing of immunizing antigens at 40C andstorage until use at -20°C. Attempts to attenuateT. pallidum by immunological means, namely,passage through a limited number of immunizedrabbits, were unsuccessful.

Received for publication April 4, 1976*Present address: Department of Bacteriology, The Medical School,University of Baghdad, Baghdad, IraqtPresent address and address for reprints: Department of Bacteriology,Royal Postgraduate Medical School, Hammersmith Hospital, DuCane Road, London W12 OHS

IntroductionIt is well known that immunity to Treponema

pallidum develops in the course of syphilis in rabbitsand human beings. In experimental syphilis in rabbitssome immunity is present approximately 3 weeksafter infection with T. pallidum. Resistance tore-infection increases to a maximum approximately3 months after infection. Termination of this stateby penicillin treatment within this 3-month periodmay enable re-infection to be accomplished(Magnuson and Rosenau, 1948; Magnuson, Rosenau,and Clark, 1949; Turner and Nelson, 1950).

Attempts to produce immunity in rabbits againstexperimental infection with T. pallidum using T.pallidum vaccine have been made by a number ofinvestigators. Some have reported failure, includingMagnuson, Halbert, and Rosenau (1947), Eagle andFleischman (1948), Waring and Fleming (1951),McLeod and Magnuson (1953), McLeod (1962),and Miller, Fazzan, and Whang (1963). Others havereported success including Miller (1967, 1971),Metzger, Michalska, Podwin'ska, and Smog6r (1969),and Jones, Zeigler, and Jones (1976).

Similarly, attempts at inducing immunity inrabbits by 'avirulent' cultivable strains of T. pallidum,or non-pathogenic treponemes, have given variableresults. Thus, Knox, Dacres, Short, and Glicksman(1967) reported success with an avirulent cultivabletreponemes, whereas Gelperin (1951), with theReiter treponemes, and more recently Izzat, Dacres,Knox and Wende (1970), and Izzat, Smith, Jacksonand Knox (1971), with the Nichols non-pathogenictreponemes, reported failure.The present study records attempts to produce

immunity in rabbits against T. pallidum usingcultivable non-pathogenic treponemes. In experi-ment 1, intratesticular challenge was applied atintervals of 3 to 9 weeks after immunization, and inexperiment 2, at 7 months. Experiment 3 wasdesigned to test simultaneously immunity to challenge,the virulence of the challenging T. pallidum, andpossible attenuation through passage.

copyright. on F

ebruary 23, 2020 by guest. Protected by

http://sti.bmj.com

/B

r J Vener D

is: first published as 10.1136/sti.52.5.300 on 1 October 1976. D

ownloaded from

http://sti.bmj.com/

Immunity in syphilis 301

TABLE I Human treponemes

Treponemal strains

T. pallidum(Nichols pathogenic)Nichols(non-pathogenic)Kazan 2

Kazan 4Kazan 5

Kazan 8

T refringens

T. minutum

T. ambigua

T. microdentium

Biological origins

C.S.F. of patient with neurosyphilis (Nichols and HSough, 1913)

Alleged cultural adaptation of the above strain (Nichols and Hough, 1913)

Alleged cultural adaptation of pathogenic T. pallidum strain isolated fromsyphilitic papule (Aristofski and Gelzer, 1926: see Christiansen, 1963)Alleged cultural adaptation of pathogenic T. pallidum (see Christiansen, 1963)Alleged cultural adaptation of pathogenic T. pallidum isolated from blood ofsecondary syphilitic (Karimova and Kondratjev, 1940: see Christiansen, 1963)Alleged cultural adaptation of pathogenic T. pallidum (see Christiansen, 1963)

Genital saprophyte (Schaudinn and Hoffmann, 1905)

Genital saprophyte (Dobell, 1912)

Mouth saprophyte (Seguin and Vinzent, 1936)

Mouth saprophyte (Noguchi, 1912)

Present source

VDRL, London Hospital

VDRL, Atlanta, Georgia, USA


VDRL, Atlanta, Georgia, USAVDRL, Atlanta, Georgia, USA


VDRL, London Hospital

Pasteur Institute, Paris



Materials and methodsTreponemes examined in this study The treponemalstrains of this study are listed in Table I, together withtheir origins and their present source. T. pallidum itselfis represented by the non-cultivable Nichols pathogenicstrain which is maintained in the rabbit testicle. Cultivablenon-pathogenic treponemes are represented by five strainsof alleged cultivable strains of T. pallidum, and by fouroral or ano-genital strains saprophytic to man.

Non-pathogenic treponemes, cultivation and harvestCulture medium All strains of non-pathogenic treponemeswere cultivated in a modified Brewer (1940) thioglycollatemedium using a modified culture system described byGelperin (1949). The medium was prepared from com-

mercially available dehydrated products-Spirolate Broth(BBL) and Brain Heart Infusion (BBL) (Hanson andCannefax 1964). The complete reconstituted mediumconsisted of 14-5 g. Spirolate Broth and 18-5 g. BrainHeart Infusion to each 100 ml. of distilled water. Trypton,asparagine, and sodium thioglycollate were added to themixture as suggested by Hanson and Cannefax (1964) inquantities of 25 mg. of each. The mixture was autoclavedat 121°C for 15 min. Sterile calf serum inactivated at 56°Cfor 30 min. was added to the autoclaved and cooled mediumto a final concentration of 10 per cent. Semi-liquid mediumwas made by adding 0-1 g. ion agar No. 2 (Oxoid) to thebasal medium. Solid medium was made by adding 0-75 g.ion agar No. 2 (Oxoid) to the basal medium. Serum was

usually added while the medium was still very warm afterautoclaving.

Semi-liquid and solid media were used only to restorethe activity of inactive or sluggish stock cultures. Bulkcultures in liquid medium were inoculated from theseprimary cultures. Media were prepared fresh and usedwithin 48 hrs. Serum was added immediately beforeinoculation.

Maintenance and seed cultures Treponemes were receivedfrom the reference laboratories either lyophilized or as

cultures in solid medium, or in gelatinized serum con-

taining a portion of meat at the bottom of the tube.Lyophilized organisms were rehydrated with distilledwater. Primary cultures were made in 20 ml. semi-solid

or solid medium in screw-capped bottles of 30 ml. capacity,by deep inoculation with a Pasteur pipette of approxi-mately 0-2 ml. of culture. Incubation of semi-solid mediumcultures was allowed to proceed at 35°C in 5 per cent.carbon dioxide and 95 per cent. nitrogen mixture. Growthusually became apparent in 3 to 10 days as a slight haze inthe depths of the medium. Maintenance cultures, whichwere passaged every 2 weeks, were not incubated in thecarbon dioxide and nitrogen mixture, but were incubated inscrew-capped bottles of30 ml. capacity filled with mediumso as to exclude as much air as possible. The amountpassaged each time was approximately 1 ml. Under theseconditions the treponemes do not multiply at theirmaximum rates. For the preparation of seed culturesmultiplying at maximum rates, larger inocula of 3-5 ml.were passaged at 2 to 3 day intervals for 6 to 8 passages,as described above. For mass cultivation the entire contentsof the 30 ml. bottles were used as inocula, as describedbelow.

Mass cultivation 5 to 10 litres of liquid medium wereprepared for each strain, and distributed in aliquots of450 ml. in screw-capped bottles of 500 ml. capacity.Each large bottle of medium was inoculated with approxi-mately 30 ml. of the seed culture. Bottles were then filledto the top with fresh medium so as to exclude as much airas possible, tightly closed, and incubated at 35°C. Cultureswere gently agitated once or twice a day.

Conditions for incubation and growth Experience showedthat treponemal growth was better when air was excludedfrom cultures and they were incubated tightly closed at35°C. For maximum growth the duration of incubationvaried with different strains. The longer the period forwhich cultures were incubated the greater the tendency forsome organisms to produce cyst-like structures. For thisreason, organisms were harvested before the end of theexponential phase. Optimum times for harvesting differenttreponemes are shown in Table II.

Harvesting and washing of treponemes Treponemes wereharvested by centrifuging cultures at 8000 x G for10 min. at 5°C in an MSE centrifuge. The deposit, whichresembled a thick paste, was washed by re-suspending inapproximately 10 to 20 ml. of buffered saline depending

copyright. on F


http://sti.bmj.com

/B

r J Vener D


ownloaded from

http://sti.bmj.com/

302 British 7ournal of Venereal Diseases

on the bulk of the deposit, using repeated aspiration andexpulsion with a syringe and 20-gauge needle to homo-genize the suspension. The suspension was then madeup to 250 ml. with buffered saline and recentrifuged at8000 x G for 30 min. at 5°C. Washing and centrifugationwere repeated four times in all, using 250 ml. each time,except in the final instance in which a pre-weighed 100 ml.polythene tube, which was re-weighed after deposition ofthe cells and discarding of the supernatant fluid, was used.The weights of organisms per litre of culture medium arepresented in Table II. After the final centrifugationtreponemal deposits were re-suspended in buffered salineand the volume made up to contain 10 g. net weight per100 ml.

Preparation and storage of preparations for immunizationFor disruption, 30 ml. of the 10 per cent. suspension weretransferred to a wide-mouthed glass tube, and subjectedto ultrasonic vibration with a Bronwill Biosonil IIIsonifer (10 Kc 300 W), at approximately 100 Watts persq. cm. using a probe of 3/4 in. (1 -90 cm.) diameter,giving a total energy of 284 W. The tube was cooled witha jacket of crushed ice. The temperature was not allowedto exceed 8°C.

Antigen preparations in Freund adjuvant, and wholetreponemal suspensions for injection, were stored at-20°C before injection.

Immunization procedure for non-pathogenic treponemesAntigens for injection were composed of treponemeswhich had been washed four times in saline (as previouslydescribed) before being re-suspended in saline to forma 10 per cent. wet W/V suspension. Suspensions oftreponemes were exposed to ultrasonic disintegration for60 sec. at 4°C, and then emulsified with an equal volumeof complete Freund adjuvant. Animals were injected with1-5 ml. of this emulsion of which 0 5 ml. was given intra-muscularly into each gluteal region and 0.5 ml. sub-cutaneously in the back of the neck. This procedure wascarried out weekly for 3 wks. After 2 wks respite, injectionswere recommenced using treponemal suspensions withoutadjuvant. Injections were made intradermally 0 5 ml. in

each gluteal region and in the back of the neck, weekly for4 wks. 10 days after the 4th injection rabbits were bled2 ml. from the ear, and the antibody content estimated byimmunodiffusion. At the same time three intradermalinjections were given as before and repeated at interals of10 to 14 days with bleeding until titres of 1 in 32 or morewere obtained with homologous antigens at a concentra-tion of 1 g./100 ml. For such titres the latter schedulerequired to be repeated 1 to 5 times.

Propagation, maintenance, and harvesting of T. pallidumThe particular strain used in these studies was the Nicholsstrain of virulent T. pallidum (Nichols and Hough, 1913).This organism is routinely used in the Treponema pallidumImmobilization Test (TPI) and was obtained from theVenereal Diseases Reference Laboratory, The LondonHospital, Whitechapel, London, El, by the courtesy ofDr. A. E. Wilkinson.

Animals Adult male New Zealand white rabbits weighingapproximately 3 to 4 kg. were obtained from commercialsuppliers. Before inoculation animals were allowed toacclimatize to the animal house for 3 weeks or more. Theywere kept in individual cages with food and water alwaysavailable. They were fed on pellet diet (Oxoid laboratoryanimal diets). The temperature of the room was usuallybelow 22°C.

Before inoculation approximately 5 ml. of blood wasdrawn from the middle artery of the ear of each rabbit.The serum was separated, inactivated at 56°C for 30 min.,and tested for the presence of treponemal antibody usingthe TPI test, or the Fluorescent Treponemal AntibodyAbsorbed test (FTA-ABS).

Inoculation procedure A suspension of T. pallidum(Nichols pathogenic strain) containing approximately108 treponemes per ml. was inoculated into the body of therabbit testis, in a dose of0*5 ml. for each testis. Corticoster-oid was given to the inoculated animals to suppressimmunological response and increase thereby the yieldof treponemes (De Lamater, Saurino, and Urbach, 1952;Turner and Hollander, 1957). This was administeredintramuscularly as cortisone acetate (Cortisul, Roussel

TABLE II Treponemal strains: incubation, and harvest

Treponemal strain

1. T. pallidum

2. Nichols

3. Kazan 2

4. Kazan 4

5. Kazan 5

6. Kazan 8

7. T. refringens

8. T. minutum

9. T. ambigua

10. T. microdentium

Duration of incubation (days)

8-12 (rabbit testicle)

5

4

6

6

6

9

9

8

9

Treponemal harvest

12-4 mg. dry weight/100 rabbits (trep. extract)

1-92 g. wet weight/litre



197 g. wet weight/litre


1 1 g. wet weight/litre

1 72 g. wet weight/litre

134 g. wet weight/litre


copyright. on F


http://sti.bmj.com

/B

r J Vener D


ownloaded from

http://sti.bmj.com/


Laboratories, London), 25 mg. per ml. Daily doses weregiven of 0-2 ml/kg. body weight, starting from the first dayof inoculation until full development of syphilitic orchitis.

Harvesting of treponemes When the testes became red,indurated, and nearly twice the normal size, rabbits werekilled by intravenous injection of 5 ml. pentobarbitalsodium solution (Nembutal, Abbott Laboratories) 60 mg./ml. The testes were removed aseptically via a scrotalincision. In order to increase the surface area and therebyfacilitate elution of the treponemes, the testes were cut intosmall fragments which were placed in an extraction flask towhich was added 5 to 10 ml. of sterile phosphate buffersaline pH 7-2 per testis.The air in the flask was extracted and replaced by 5 per

cent. carbon dioxide and 95 per cent. nitrogen mixture, aprocedure which was repeated three times. The flask wasthen shaken on a mechanical shaker (Griffin flask shaker)for 1 hr at room temperature. Removal of red cells andgross testicular debris was accomplished by centrifugationat 1000 x G for 10 min. Continuity of the strain wasensured by immediate inoculation of a fresh batch ofrabbits.

EXPERIMENT 1

Animals For experiments on the protective effect oftreponemal vaccines against T. pallidum, nine adult New

Zealand white rabbits each weighing 3 to 4 kg. wereimmunized as follows:(1) Five rabbits were immunized with non-pathogenictreponemal strains, Nichols, Kazan 4, Kazan 8, T.minutum, and T. ambigua respectively.(2) Two rabbits with Kazan 2.(3) Two rabbits with Kazan 5.Subsequent challenge procedure was carried out onrabbits which had been immunized with:(i) Nichols non-pathogenic strain (one rabbit).(ii) Kazan 2 (two rabbits).(iii) Kazan 4 and Kazan 5 (one rabbit each).The last four rabbits were reserved for Experiment 3.

Challenge procedure Challenge was carried out in immun-ized rabbits at intervals after immunization as follows:

Nichols (non-pathogenic) 3 wksKazan 2 6 wksKazan 4 and Kazan 5 9 wks

0 5 ml. of T. pallidum suspension (4 x 107-108organisms/ml. in phosphate buffered saline) was injectedintratesticularly into the testis of non-immunized andimmunized rabbits on the same occasion. Both non-immunized and immunized animals were treated withcortisone daily as previously described. See also Table III,where schematic representation of passage, challenge, andimmunization protocols appears.

TABLE III Experiments in the protective effect of treponemal vaccines and experiments in the attenuation ofT. pallidum

DIAGRAM OF T. pallidum PASSAGE IN RABBITS

NON-IMMMUNIZED RABBITT. pallidum

NON-IMMUNIZED RABBITT. pallidum

NON-IMMUNIZED RABBITT. pallidum

1~NON-IMMUNIZED RABBIT

T. pallidum

IMMUNIZED RABBITNichols non-pathogenic strain

2 IMMUNIZED RABBITSBoth with Kazan 2 strain

2 IMMUNIZED RABBITSKazan 4 and 5, respectively

I2 IMMUNIZED RABBITSKazan 5 and 8, respectively

2 IMMUNIZED RABBITST. minutum and T. ambigua, respectively

NON-IMMUNIZED RABBIT (SYPHILOMA)Treponemal count 2-5 x 108

copyright. on F


http://sti.bmj.com

/B

r J Vener D


ownloaded from

http://sti.bmj.com/

304 British Journal of Venereal Diseases

Results None of the five immunized rabbits gave theusual testicular inflammatory response, a response whichhad been obtained many times previously without failure,in the maintenance of T. pallidum. Usually testicles ofinfected rabbits showed enlargement starting on the fifthday after infection and becoming marked during thesecond week and suitable for harvesting. By contrast, thetesticles of immunized rabbits were not enlarged on theeighth day. By the 12th day the rabbits showed slightenlargement of the testicles. By the 18th day, rabbitsshowed moderate enlargement only, with no induration.These appearances were in marked contrast to those ofnon-immunized rabbits.When treponemal counts were carried out on the

excised testicles, counts in non-immunized rabbits variedfrom 108- 2-5 x 108 organisms/ml. as compared with4 x 107 -1-5 x 108organisms/ml. in immunized rabbits.

It would appear, therefore, that all the immunizingtreponemes, viz. the Nichols non-pathogenic tre-poneme, and Kazan strains 2, 4, and 5 exerted someprotective effect against the challenge dose of T.pallidum, a dose which invariably produced a markedreaction accompanied by high treponemal counts inthe testicles of rabbits used throughout this entirework.

EXPERIMENT 2Animals An extension of the previous experiment wascarried out on two adult male New Zealand white rabbitsweighing 3-4 kg. 7 mths after immunization with T.refringens and T. microdentium.

Control One adult male New Zealand white rabbit (non-immunized) was treated with the T. pallidum challengeprocedure intratesticularly, for comparison with theimmunized animals.

Booster procedure One week before challenge by viableT. pallidum suspension, a single boosting dose of theappropriate non-pathogenic treponeme was given to eachof the two test animals. This dose was divided into twoinjections of 0.5 ml. of 10 per cent. wet weight/volumesuspension, which were given subcutaneously into theright gluteal region and back of the neck.

Challenge procedure This was carried out as describedin the previous experiment, one week after the boostingdose.

Results The control rabbit showed early enlargement ofthe testicles which progressed to marked enlargement bythe 13th day after infection with a treponemal count of2-5 x 108 organisms/ml.By contrast, the rabbit immunized with T. microdentium

showed almost no enlargement of the testicles with atreponemal count of only 6 x 107 organisms/ml., whilethe rabbit immunized with T. refringens showed slow andslight enlargement of the testicles with a treponemal countof 1.5 x 108 organisms/ml.

From this experiment it would appear that bothT. refringens and T. microdentium exert some pro-tective effect against the challenge dose of T. pallidum.

EXPERIMENT 3

Animals Experiments on the attenuation of T. pallidumused four of the adult male New Zealand rabbits reservedfrom Experiment 1, which had been immunized asfollows:

(1) One rabbit with Kazan 5.(2) One rabbit with Kazan 8.(3) One rabbit with T. minutum.(4) One rabbit with T. ambigua.

Passage procedure For intratesticular passage, testicles ofinfected rabbits were pooled and homogenized, and asuspension of T. pallidum was prepared and injected aspreviously described. Rabbits were observed for 18 daysbefore being killed and T. pallidum from their testicleswas passaged.

Test of attenuation Testicles of infected immunizedrabbits were pooled, homogenized, and injected intra-testicularly into one adult male New Zealand whiterabbit which was then observed daily for evidence ofinfection.

Method of investigation In these experiments, T. pallidumsuspensions were obtained from the testicles of a non-immunized rabbit and passed to the testicles, by intra-testicular injection, of two rabbits immunized with tre-ponemal strains Kazan 4 and Kazan 5 respectively (fromExperiment 1).For the second passage, the testicles of the previous

rabbits were pooled and T. pallidum suspension obtainedtherefrom for passage to testicles of two further rabbitsimmunized with Kazan 5 and Kazan 8 strains respectively.

For the third passage, the testicles of the previous tworabbits were pooled, and the T. pallidum suspensionsobtained passed to the testicles of two rabbits immunizedwith T. minutum and T. ambigua respectively.As a crude test of any possible attenuating effect on

T. pallidum of the limited number of passages, the testiclesof the previous two rabbits were pooled and the T.pallidum suspensions passed in a fourth and final passageto a non-infected, non-immunized rabbit.

Results Within 6 days the testicles of the non-immunizedrabbit showed enlargement which progressed to fully-fledged syphilomata in 12 days, with a treponemal countof approximately 2-5 x 108 organisms/ml. This was inmarked contrast to the testicles of the immunized rabbits,none of which showed enlargement or treponemal countscomparable with that of the non-immunized rabbit, orindeed of the rabbits which had been used for themaintenance of the T. pallidum strain throughout thisstudy.

The experiment would appear to show:

(1) Confirmation of the protective effect of immu-nization by Kazan 5.(2) The protective effect of immunization byKazan 8, T. minutum, and T. ambigua.(3) Attenuation of T. pallidum was not apparentafter three passages in immunized rabbits.

copyright. on F


http://sti.bmj.com

/B

r J Vener D


ownloaded from

http://sti.bmj.com/


DiscussionIn the experiments on active immunity describedabove, evidence of immunity or resistance to infectionby T. pallidum was obtained by comparing testicularappearances in non-immune rabbits with those invaccinated rabbits. Clinical appearances in thetesticles of non-immune rabbits infected with T.pallidum had been observed in detail in many rabbitsduring laboratory maintenance of the Nichols strainof T. pallidum. Furthermore, the naked eye appear-ance had also been carefully noted when the testicleswere excised, together with enumeration of livingtreponemes present. The technique employed formaintenance of the T. pallidum strain, which includedcortisone acetate injection, ensured marked testicularenlargement accompanied by numerous treponemes(108 - 25 x 108/ml.). With this technique testicularenlargement reached its maximum in about 10 to 12days, and this cycle remained constant throughout theentire period of this and other work.

Instances of testicular enlargement being less thanmarked, and of treponemes being present in numbersless than 108, were rare and occurred during thesummer months when ambient temperatures werehigh. Where cortisone injections were not used in themaintenance of strains, the period required fortesticular enlargement was longer (3 to 5 weeks).

In this way, therefore, standards for comparisonwere built up over a considerable period of time, sothat any change in the accustomed pattern wasimmediately obvious.

In challenging vaccinated rabbits by intratesticularinjection of T. pallidum, cortisone acetate wasemployed exactly as in the case of the non-vaccinatedcontrols. Thus any prolongation of the periodrequired for testicular enlargement or any otherchanges could be attributed to immunization andnot to the absence of cortisone effect on immuneresponse. Furthermore, it might be suggested thatany such changes would most probably be due tocirculating antibody formed before cortisone injec-tion, the effect of which injection would be to abolishimmune response both cellular and humoral.

In the present work partial resistance to intra-testicular challenge in rabbits has been obtained byimmunization using treponemal strains-Nichols(non-pathogenic), Kazan 2, Kazan 4, Kazan 5,Kazan 8, T. minutum, T. ambigua, T. refringens, andT. microdentium. This is in contrast to the results ofother investigators. Apparent success is attributed tolarge immunizing doses and careful processing at 4°Cwith storage at - 20°C.

In the light of the above results, it is pertinent toexamine the successful results of others. The results,therefore, of three groups of investigators are ofinterest, viz. Tani, Inoue, and Asano (1951), Miller(1967, 1973), Metzger and others (1969), and Metzgerand Smog6r (1969).

Tani and others (1951) studied the effect on pro-tective immunity against T. pallidum in rabbitsimmunized by carbolic acid saline suspensions of T.pallidum. The manner in which these suspensionswere prepared is of interest and was as follows.

Coarsely particulate material was removed fromtesticular suspension by centrifugation at 1000 r.p.m. for5 min. Stock 10 per cent. antiformin solution from the'ice-box' was added to a final concentration of 0-5 to07 per cent. Preparations were shaken by hand, andallowed to stand at ambient temperature for 30 min. Thiswas followed by centrifugation at 3000 r.p.m. for 1 hr, asingle washing of the 'surface of the sediment' by a 'largevolume of saline', re-suspension in 0 5 per cent. carbolicacid in saline, and storage in the 'ice-box'.

In experiments in rabbits these investigatorsvaried; (i) Total amount of antigenic material injectedfor immunization, (ii) Period of immunization,(iii) Interval elapsing between termination of immuni-zation and challenge, (iv) Size of challenge dose.Thus the subcutaneous administration of a total

of 1-4 x 108 T. pallidum to eight rabbits over aperiod of 7 days followed by intradermal challengeat four sites on the back of approximately 3 x 106,3 x 105, 3 x 104, and 3 x 103 T, pallidum at eachsite, 10 days after termination of immunizationproduced no evidence of immunity to T. pallidum.By contrast, when the total number of T. pallidum

injected was doubled (2-8 x 108) and the period ofimmunization extended to 21 days in four rabbits,with challenge as described above 9 days after thelast immunizing injection, only six of sixteen intra-dermal inoculation sites produced chancres, com-pared with ten out of twelve areas in three controlrabbits.

In a further experiment, in which the previoustotal immunizing dose was trebled (8-4 x 108) andadministered to nine rabbits over a period of 20 days,challenge doses at three sites of 108, 105, and 104 T.pallidum, 21 days after the last immunizing injectionproduced six rabbits without chancres, of which oneproduced infection on popliteal transfer, and five withno demonstrable infection, which were, therefore,possibly completely immune. All the remaining testrabbits produced chancres. Seven non-immunizedcontrol rabbits produced chancres on all thechallenged sites.

In a further variation of dose and immunizationperiod, 3-6 x 108 T. pallidum were injected over aperiod of 6 days into seven rabbits, which werechallenged 9 days after the last injection as above,with 108, 105, and 104 T. pallidum. Of these rabbits,two produced no chancres, but were infective onpopliteal node transfer. By contrast, all seven controlrabbits produced chancres.

In a second group of eight rabbits immunized asabove but at a point 74 days after termination of

copyright. on F


http://sti.bmj.com

/B

r J Vener D


ownloaded from

http://sti.bmj.com/


immunization, four animals were completely immuneas shown by absence of chancres and negative lymphnode transfer. All six control rabbits producedchancres.These results show that effective immunization in

rabbits required a large total dose of treponemesadministered daily over a period of at least 3 wks.Furthermore, immunity once established lasted for atleast 2j mths. Lack of success on the part of otherinvestigators was attributed by the authors to lowvaccine dosage, in comparison with their own largetotal dosage of antiformin treated material. Althoughthe authors were uncertain as to the exact part playedby antiformin treatment, they believed that it wasimportant and probably exposed underlying antigensessential for the induction of protective immunity.A different approach was employed by Miller

(1967), who used T. pallidum attenuated by gammairradiation. In one set of experiments, rabbitsimmunized intravenously by T. pallidum with a totaldose of 9 7 x 108 over a period of 12 wks werechallenged at the end of this period by intradermalinjection of 500 T. pallidum at each of four sites.None of the animals showed evidence of protectionsince dark-ground positive lesions appeared at thesame time in both test and control animals.

In another set of experiments, the immunizationperiod and the total doses of T. pallidum wereincreased. Thus rabbits were immunized as abovefor 14 wks, rested for 3 wks, and re-immunized for7 wks. At the end of this period of 24 wks when eachrabbit had received a total of 1-6 x 109 T. pallidum,test and control animals were challenged as before.On the basis of delayed incubation period and thedevelopment of atypical cutaneous lesions in com-parison with non-immune control animals, elevenout of seventeen test animals were consideredpartially immune to the challenge dose. Furthermore,two rabbits showed no evidence of infection, as shownby popliteal node and testis transfer to normal non-immune animals.

In a further set of experiments (Miller, 1973), doseand immunization period were increased. Thusrabbits were injected intravenously with a totalamount of 3-71 x 109 T. pallidum, distributed oversixty doses given once to twice weekly during aperiod of 37 wks with rest periods of 14 days at 12and 24 wks. Ten days after completion of immuniza-tion, test and control rabbits were challenged intra-dermally with 1,000 or 100,000 T. patidum. Noneof the test rabbits developed lesions during the94-day period of observation, nor were their trans-ferred popliteal lymph nodes infectious for otherrabbits. By contrast, all control animals developedtypical, dark-ground positive cutaneous lesions.

In still further experiments, rabbits challengedintradermally with 1,800 T. pallidum 1 yr after thelast immunizing injection showed no cutaneous

lesions, nor were their lymph nodes or testes infectiousfor other rabbits. All non-immunized control rabbits,however, developed cutaneous lesions.Metzger and others (1969) used T. pallidum which

had been rendered non-viable by suspension inphosphate buffer at pH 7-4 at 4°C for 10 days.Rabbits were injected intravenously with a total of8 x 109 T. pallidum thus treated involving fourweekly doses of 1 ml., each containing 1 8 x 108treponemes, for 3 weeks, followed by a further courseof four 2 ml. doses weekly, each containing 3-6 x 108,for 4 wks. Five wks after completing the immuniza-tion, the test and control rabbits were challenged byintradermal injections of 300,000 T. pallidum at eachoffour sites. None ofthe immunized rabbits developedskin lesions, in contrast to non-immune controlrabbits. Of sixteen rabbits immunized, however, sixwere completely immune, while the remaining rabbitswere considered partially immune since their lymphnodes harboured virulent T. pallidum which causedinfection upon transfer to the testes ofnon-immunizedrabbits.A high degree of immunity was also obtained by

intramuscular injection of non-viable T. pallidum,the degree of immunity depending on the number oforganisms injected during the course of immuniza-tion (Metzger and Smog6r, 1969). Thus ten rabbitsimmunized intramuscularly by T. pallidum with atotal dose of 1-2 x 1010 given over a period of 7 wkswere challenged 5 wks after completing the immuniza-tion by intradermal injection at each of four siteswith 300,000 T. pallidum. Of ten rabbits vaccinated,five showed no skin lesions or evidence of infectionon lymph node transfer, two were asymptomatic,but showed evidence of infection on lymph nodetransfer, and three showed cutaneous lesions. Inother experiments, in which rabbits were given dosesof T. pallidum of 6 x 109 and 3 x 109, animalsresponded with lesser degrees of immunity.

In summary, therefore, from the results of Miller,and of Metzger and co-workers, there is little doubtthat immunity against syphilis can be conferred bythe injection of non-viable T. pallidum. Best resultswere obtained by intravenous injection of largenumbers of organisms over many weeks, and to thisextent their results are in agreement with those ofTani and his colleagues. There the similarity ends,for the first mentioned workers were at pains to avoidthe destruction of superficial antigens consideredrelevant to the induction of protective immunity bygamma irradiation or by low temperature short-termstorage, followed by prompt injection, whereasTani believed that such antigens were uncovered byantiformin treatment, and were effectively preservedby 0 5 per cent. carbolic acid. Be that as it may, allthree preparations were effective in large dosage inthe induction of protective immunity.

It should also be noted that in most cases challenge

copyright. on F


http://sti.bmj.com

/B

r J Vener D


ownloaded from

http://sti.bmj.com/


was by large numbers of T. pallidum, but whethersuch numbers are analogous to those likely to be metwith during the course of human sexual behaviour isuncertain. Where numbers are small, intensiveimmunization similar to that in the experimentalrabbit may not be necessary in human beings.Furthermore, it may be that a partial immunitysufficient to confine infecting T. pallidum to theprimary lesion would give rise to a higher degree ofimmunity than that obtainable by vaccination alone.Elucidation of these problems, however, will requiremuch further work.

Successful but less striking results have beenobtained using non-pathogenic treponemes. Gelperin(1951) used the Reiter treponeme suspended in salineor admixed with adjuvant. Two groups of rabbitswere immunized. The animals in the first group were

injected intravenously and intraperitoneally withsaline suspensions of Reiter treponemes three timesa week for 11 wks, each rabbit receiving a total weightof organisms equivalent to 6,075 ,ug. nitrogen duringthat time. Ten days after the last injection the rabbitswere challenged intradermally at four sites on theposterior part of the back with 200 T. pallidum ateach site.

In the case of the second group, treponeme-adjuvant mixtures were injected subcutaneously on

alternate weeks for the same period, each animalreceiving a total weight of organisms equivalent to1,550 ,ug. nitrogen. Fourteen days after the lastinjection, rabbits were challenged as above.

Results showed that, in immunized animals of bothgroups, there was a prolongation of incubation timein cutaneous lesions as compared with those incontrols. Gelperin (1951), however, considered thatthese differences were not statistically significant.In the case of the adjuvant group, however, thecutaneous lesions were significantly smaller thanthose of the non-immune control group. Althoughother investigators commenting on the results ofGelperin (1951) state that his results did not showevidence of immunity, we think that partial immunitywas established in the adjuvant group at least.Knox and others (1967) reported partial protection

in rabbits using mechanically disrupted Nicholsnon-pathogenic treponemes and E. coli lipopoly-saccharide adjuvant. Animals were immunized atweekly intervals for 5 wks by subcutaneous injectionin the skin of the back of 25 mg. of treponemes. Thetotal weight of organisms injected therefore was

125 mg. Two wks after the last immunizing dose,animals were challenged intradermally on the lowerback with a total of 6 x 106 T. pallidum distributedas 106 treponemes in each of two sites, and 2 x 106in each oftwo further sites. Of 23 animals immunized,nine did not develop cutaneous lesions. Significantimmunity was, therefore, provided by the immunizing

procedure against a considerable challenge of virulentT. pallidum.More recently Izzat and others (1970) attempted to

produce immunity against T. pallidum in rabbitsusing the Nichols non-pathogenic treponeme. Threetypes of vaccine were tested:

(i) A suspension of 2 x 106 treponemes per ml. in2 per cent. alum potassium sulphate preservedwith Merthiolate 1:10,000,

(ii) 'Sonicated' treponemes 100 mg./ml., and adju-vant, E. coli lipopolysaccharide 2-4 mg./ml.,

(iii) Lysozyme-treated treponemal suspension, andadjuvant.

The first preparation was injected subcutaneously,weekly, for 10 wks, each injection containing 106treponemes. The total number of organisms givenwas 107.The second preparation was injected sub-

cutaneously, each rabbit receiving a total of 1,600 mg.of sonicated organisms over a period of 16 wks. Twogroups of rabbits were used, one group receivinginjections at weekly, and the other at monthlyintervals.The third preparation was injected subcutaneously,

each rabbit receiving 1,600 mg. of lysozyme-treatedtreponemes, with adjuvant, weekly over a period of53 weeks.

Rabbits receiving the first and second preparationswere challenged with virulent T. pallidum 3 wksafter completion of immunization in the case of thefirst preparation and 2 wks in the case of the second.Challenge doses consisted of 4 x 106 organismsintratesticularly and 5 x 105 organisms intradermallyat two sites on the back. Animals receiving the thirdtype of preparation were challenged with fiftyorganisms per site.

According to the authors, none of the rabbitsshowed any evidence of immunity against virulentT. pallidum. A possible explanation for failure toproduce immunity may lie in the manner of theinitial preparation of treponemal suspensions. Thusit is stated that organisms were washed thirteentimes in physiological saline and stored at - 60°Cuntil used, whereupon they were thawed and washedthree times more in phosphate buffer saline (pH 7-8).It is suggested that the freezing and thawing followedby further washing may have removed materialessential for the induction of protective immunity.By contrast, treponemal preparations used in the

present work were completely processed before beingfrozen. No further washing or processing was carriedout after thawing. Moreover, the greatest care wastaken to avoid denaturation of labile antigenicmaterials during harvesting and preparation, whichwere carried out at 4°C.

Recent work, therefore, by Zeigler, Jones, Jones,

copyright. on F


http://sti.bmj.com

/B

r J Vener D


ownloaded from

http://sti.bmj.com/


and Kubica (1976) is of considerable interest in thatthey have shown the presence of an extra-cellularslime layer on the surface of T. pallidum (Nicholspathogenic strain), a layer which is absent from thenon-pathogenic treponemes, T. refringens biotypeNichols and T. phagedenis biotype reiterii.

Furthermore, it would appear that, when theintegrity of the slime layer and outer envelope arepreserved by means of glutaraldehyde, a singleintraperitoneal injection of T. pallidum suspensionplus adjuvant may provide partial protection againstintratesticular challenge with T. pallidum. Althoughno experimental evidence is produced in their paperthe inference is that non-pathogenic treponemeswhich apparently do not possess this slime layer areunlikely to be effective as vaccines.The results reported here, whereby non-pathogenic

treponemal vaccines provide partial protection inrabbits against intratesticular challenge by T.pallidum, are therefore at variance with those of theseauthors.

In summary, there is evidence to show that theinduction of protective immunity against syphilis ispossible by vaccines containing non-viable T.pallidum or certain cultivable non-pathogenic tre-ponemata. Both types of vaccine have until now,required prolonged courses involving high totaldosage, and in view of the difficulties involved in theproduction of sufficient quantities of T. pallidum foruse in vaccines, it is clear that further experimentsmust be undertaken as a matter of urgency toascertain the true effectiveness of vaccines con-taining the comparatively easily cultivable non-pathogenic treponemes, in the induction of protectiveimmunity.

The above work formed part of a Thesis submitted byDr. H. T. Al-Samarrai in 1974 to the Faculty of Medicinein the University of London, in fulfilment of the require-ments for the degree of Doctor of Philosophy.A debt of gratitude is owed to the following Heads of

Department: Professors K. R. Hill, G. B. D. Scott, W.Brumfitt, and K. E. F. Hobbs, and to many friends, toonumerous to mention, on the staff of the Royal FreeHospital, for help, encouragement, and much personalkindness. A similar debt is also owed to Dr. A. E.Wilkinson and his staff at the Venereal Diseases ReferenceLaboratory, The London Hospital.

ReferencesBREwER, J. H. (1940) J. Amer. med. Ass., 115, 598CHRISTIANSEN, A. H. (1963) Acta path. microbiol. scand.,

57, 81DE LAMATER, E. D., SAURINO, V., and URBACH, F.

(1952) Amer. J. Syph., 36, 127DOBELL, C. (1912) Arch. protistenk., 26, 117EAGLE, H., and FLEISCHMAN, R. (1948) J. exp. Med.,

87, 369GELPERIN, A. (1949) Amer. J. Syph., 33, 101- (1951) Ibid., 35, 1

HANSON, A. W. and CANNEFAX, G. R. (1964) J. Bact.,88, 811

IZZAT, N. N., DACRES, W. G., KNOX, J. M., and WENDE,R. (1970) Brit. J. vener. Dis., 46, 451

-, SMITH, E. B., JACKSON, S. W., and KNOX, J. M.(1971) Ibid., 47, 335

JONES, A. M., ZEIGLER, J. A., and JONES, R. H. (1976) Ibid.,52, 9

KNOX, J. M., DAcRES, W. G., SHORT, D. H., andGLICKSMAN, J. M. (1967) WHO/VDT/RES. 67, p. 128

MCLEOD, C. P. (1962) Publ. Hlth Rep. (Wash.), 77, 431- and MAGNUSON, H. J. (1953) Amer. J. Syph., 37, 9

MAGNUSON, H. J., HALBERT, S. P., and ROSENAU, B.(1947) J. Vener. Dis. Inform., 28, 267and ROSENAU, B. J. (1948) Amer. J. Syph., 32, 418

, and CLARK, J. W. (1949) Ibid., 33, 297METZGER, M., MICHALSKA, E., PODWI*4SKA, J., and

SMOG6R, W. (1969) Brit. J. vener. Dis., 45, 299- and SMOG6R, W. (1969) Ibid., 45, 308

MILLER, J. N. (1967)J7. Immunol., 99, 1012(1971) WHO/VDT/RES. 71, p. 234

- (1973) J. Immunol., 110, 1206FAZZAN, F. P., and WHANG, S. J. (1963) Brit. J.

vener. Dis., 39, 199NICHOLS, H. J., and HOUGH, W. H. (1913) J. Amer. med.

Ass., 60, 108NoGUCHI, H. (1912) J. exp. Med., 15, 81SCHAUDINN, F., and HOFFMANN, P. E. (1905), Arb. k.

GesundhAmte, 22, 527SEGUIN, P., and VINZENT, R. (1936) C.R. Soc. Biol.

Paris, 121, 408TANI, T., INOUE, R., and AsANo, 0. (1951) Jap. J. exp.

Med., 4, 71TuRtNER, T. B., and HOLLANDER, D. H. (1957) 'Biology

of the Treponematoses'. WHO, Monograph Ser.,No. 35

- and NELSON, R. A. (1950) Trans. Ass. Amer. Phys.,63, 112

WARING, G. W., Jr., and FLEMING, W. L. (1951) Amer.3J.Syph., 35, 568

ZEIGLER, J. A., JONES, A. M., JoNES, R. H., and KUBICA,K. M. (1976) Brit.3'. vener. Dis., 52, 1

copyright. on F


http://sti.bmj.com

/B

r J Vener D


ownloaded from

http://sti.bmj.com/

immunity in syphilis studies in active immunity · immunityin syphilis studies in active immunity...

Documents