Brit. J. vener. Dis. (1968), 44, 103.

SELECTED ASPECTS OF SYPHILIS AND GONORRHOEARESEARCH IN THE UNITED STATES, 1967*t

BY

LESLIE C. NORINSVenereal Disease Research Laboratory, Venereal Disease Program, National Communicable Disease Center,

Bureau of Disease Prevention and Environmental Control, Public Health Service,U.S. Department of Health, Education, and Welfare

Atlanta, Georgia 30333,U.S.A.

In recent years programmes concerned with thecontrol of syphilis and gonorrhoea have dependedlargely on the use of penicillin and on epidemio-logical methods, but many venereologists and otherphysicians concerned with public health problemshave indicated the need for more laboratory researchto provide additional means of controlling andeventually eradicating these diseases. It has thereforeseemed timely to take stock of relevant researchprogrammes being conducted in the United States,selecting for discussion those in which progress isalready being made or is particularly desirable. Itis hoped that this composite description of variousresearches, some of which are still preliminary, willstimulate investigators with similar interests tocontact each other, and will contribute to inter-national research awareness and co-operation.

I. Syphilis ResearchMost people are aware of the decline of syphilis

after the widespread introduction of penicillintherapy, and of the subsequent resurgence of thedisease during a period of public indifference.However, it is not so widely known that the risingincidence of syphilis has not brought about acorresponding increase of research interest in thedisease.Three of the major ways in which present research

might contribute to the control and eradication ofsyphilis are the development of a vaccine, theimprovement of serological tests, and the develop-ment of a skin test.

(A) VaccineSuccess in the elaboration of a syphilis vaccine

has eluded competent workers for several decades(Turner and Hollander, 1957). Nevertheless, the

* Received for publication November 12, 1967.t Trade names are used for identification only and do not represent

an endorsement by the Public Health Service or the U.S. Departmentof Health, Education, and Welfare.

problem is at present being re-examined in severallaboratories. Miller, in Los Angeles, is utilizinggamma-irradiated Treponema pallidum organisms asa type of "attenuated" immunizing agent. His earlystudies, partly in collaboration with Bekker andde Bruijn in Utrecht, showed that the injection ofsuch organisms into rabbits could stimulate theproduction of T. pallidum immobilizing antibodieswithout producing overt lesions at the site ofinoculation. There had been earlier reports that, ofall the antibodies measured in the usual serologicaltests for syphilis, the treponemal-immobilizingantibodies were possibly the best correlated withimmunity.

In his more recent experiments, Miller "vacci-nated" rabbits with intracutaneous injections ofirradiated but motile T. pallidum. Although theresults of the subsequent challenge are not clear-cut,Miller feels that the data are encouraging.The gamma-irradiated treponemes have been

found to cause cardiolipin-reagin reactivity in therabbits vaccinated with them, and a few commentsregarding sero-reactivity and vaccines seem perti-nent.

Because non-treponemal cardiolipin-reagin testsare so widely used as screening tests for the detectionof syphilis, serious problems could arise if wide-spread use were made of a vaccine that triggeredconversion to reactivity in such tests, especially ifthe reaction produced were more than transient.This problem might be circumvented by developinga more sophisticated serological test which coulddifferentiate antibodies to a vaccine from thoseproduced against an active infection. Alternatively,serial testing of sera might reveal stabilization ordecline of the reactivity triggered by vaccine, incontrast to the rising titre indicating active infection.However, a rising titre might also result from avaccine that was replicating and thereby increasingits antigenic mass. Naturally, the administration of

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a vaccine that caused sero-conversion would hamperclinical work and disturb epidemiological surveysthat utilize the present tests on a single-specimenbasis to ascertain the level of treponemal disease ina given population.

In Houston, Knox and Dacres have immunizedrabbits with cultivable Nichols treponemes andbacterial adjuvants, and may have conferred at leastsome resistance to subsequent challenge. They feelthat their method circumvents the problem ofsero-conversion.Another point to be considered is that, even if

treponemal vaccines did not cause conversion of thereagin tests, they might stimulate the appearance oftreponemal antibodies of the type detected in theTreponema pallidum immobilization (TPI) andfluorescent treponemal antibody-absorption (FTA-ABS) tests. In such circumstances, it would not bepossible to use the TPI and FTA-ABS tests asdiagnostic aids if a vaccinated person developedreagin test reactivity for reasons other than syphilis,i.e. a biological false positive reaction. Furthermore,the appearance of treponemal antibodies aftervaccination would complicate screening the popula-tion with an automated test that was specific fortreponemal antibodies; for example, an automatedFTA-ABS test.Another vaccine strategy that aims in a different

way at the modification of T. pallidum is beingconducted at the Venereal Disease ResearchLaboratory (VDRL) in Atlanta. Thomas, Russell,and others are chemically treating T. pallidumorganisms harvested from rabbits in the hope ofenhancing the immunogenicity of these organisms.In the initial series of experiments, the organismshave been coupled through methylation or diazotiza-tion with each other or with bovine serum albumin.At the present time, rabbits have been injected withthese killed and modified T. pallidum organisms, thesero-conversion of these animals in both non-treponemal and treponemal tests is being assessed,and animals are being challenged by intracutaneousinoculation to find out whether the injection ofthese modified organisms has conferred any pro-tection.

Early in the course of this work it became apparentthat chemical modification and detailed studies ofantigenic composition called for large numbers ofpurified T. pallidum. These organisms should befree of the rabbit testicular tissue proteins anddebris that are usually present after conventionalextraction from rabbit testicular syphilomata.Rathlev and Pfau (1965) have accomplished rathersuccessful purification of small numbers of T.pallidum by using density gradient ultracentrifuga-

tion; however, with conventional rotors, it is notpossible to use this method on large volumes of thecrude material.

Accordingly, Russell and Thomas at the VDRLhave undertaken collaborative studies withAndersonand Cline at the Oak Ridge National Laboratory inTennessee to utilize the newer techniques andequipment ofzonal ultracentrifugation to accomplishthe large-scale purification of T. pallidum organisms.By these means they have thus far been able toprocess, in one batch, 13 litres of extract fromrabbit testicular syphilomata, and from this toobtain fractions that are rich in T. pallidum organ-isms, but free, to the limits of present measurement,from rabbit testicular proteins and debris. Althoughthese organisms were no longer infectious, they didretain both their antigenicity, as evidenced by theirability to react with syphilitic human and rabbitsera, and their immunogenicity, as evidenced bytheir ability to incite the formation of anti-trepone-mal antibodies when injected into rabbits. Thiszonal-ultracentrifugation technique will make itpossible for the first time to obtain large quantitiesof purified T. pallidum organisms; fortunately, theprocess does not itself produce too deleterious aneffect upon the treponemes. This ability to obtainlarge numbers of purified organisms will allow betterdefined studies on the antigenic composition ofT. pallidum. It may also provide a plentiful sourceof purified antigen for serological tests using T.pallidum. Also, if rabbit-grown organisms are to beused as a human syphilis vaccine, it would appearadvantageous to have such organisms free of rabbittesticular materials lest the vaccine trigger somekind of anti-testicular autoimmune process whenit is injected into human subjects.Another way of acquiring large numbers of

T. pallidum organisms would be successful cultiva-tion in vitro, a long-elusive goal. Knox, Dacres,Wende, and others, in Houston, are attempting tocultivate the organism in cell culture, and a fewpharmaceutical manufacturers with vaccine experi-ence have expressed interest in similar approaches.I think it fair to say that only a few of the newer celllines that are available to-day have been examinedin attempts to propagate T. pallidum in cell culture.Because anaerobiasis recurs as a persistent themein reports of cultivation attempts in earlier decades,it will no doubt be well to include in the selection ofcell lines various tumour cells or other cells thattend towards anaerobic conditions. However, thistraditional emphasis on anaerobic conditions is madein the face of the observation that T. pallidumthrives in the rabbit testis, a richly oxygenatedtissue. Hardy, in Baltimore, is continuing his studies

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on the growth requirements of both T. pallidum andthe cultivable treponemes.

In other experiments at the VDRL, Cannefax andHanson have begun attempts to produce a hybridtreponeme that is both virulent and cultivable.The general approach has been to try to achievegenetic transformation of the virulent organismsto a cultivable state, or vice versa, through the useof nucleic acids from treponemes having thedesired properties. Cannefax and associates havebeen utilizing an in vivo transformation system,modelled on an early report by Zaffiro (1962) thatReiter organisms persisted in the rabbit testes in thepresence of T. pallidum infection. A genetic trans-formation in vivo between the cultivable Reitertreponemes and the virulent T. pallidum would makean interesting parallel to the classic demonstrationof the transformation in vivo of pneumonococci fromavirulence to virulence. To date, none of these initialattempts at transformation have proved successful.

In other experiments aimed towards a vaccine,Knox and Dacres are attempting to immunizerabbits with the cultivable Nichols strain oftreponeme, sometimes injected with adjuvants,especially a lipopolysaccharide from Gram-negativebacteria. Although they feel their preliminaryresults are encouraging, the situation is not yetclear-cut.

(B) Serological TestsIn the area of improving serological tests for

syphilis (STS), we at the VDRL have three maingoals: to find a test that will recognize syphilis verysoon after invasion by spirochaetes; to differentiateearly from late syphilis and treated from inadequatelytreated or untreated syphilis; and to automateserological tests for syphilis.

(i) Our main strategy for developing a serumtest for incubating syphilis is based on the observa-tion that in many experimental antigen-antibodysystems there is a prompt recognition of foreignantigen by the body. Failure to detect this presumedrecognition would then result from not using thecorrect antigen, or from using an assay system thatis insensitive to the interaction of our antigen withthe early antibody. Of course, one might alwaysargue that there is no early recognition of T.pallidum, but this suggestion is not heuristicallyuseful. Tringali (1965) in Italy and Julian andcolleagues at the VDRL have found that, inexperimental rabbit syphilis, the earliest non-treponemal test reactivity is localized to the IgMantibodies; later in the infection the activity includesor is restricted to the IgG antibodies. Unfortunately,both Tringali and Julian have found that serum

obtained from human subjects with primary syphilisdoes not show that the cardiolipin-reagin activity isconfined to the IgM molecules; instead, it is presentin both IgM and IgG. Thus, at least superficially,the human situation does not follow the simplerabbit model. However, it is necessary to keep inmind the fact that what is clinically "primary" or"early" syphilis may, immunologically, represent ahost response that is already well advanced. Toexamine this point, one needs serum from patientsat the very earliest time that the non-treponemaltests become reactive. This will present practicaldifficulties, since in many cases the patient may notyet have developed the clinical symptoms that willbring him to the attention of a physician.However, Kuhn and colleagues may shed valuable

light upon this point by their studies of experimentalsyphilis infection in chimpanzees maintained at theVDRL. Although larger subhuman primates wereused for experimental syphilis by some of the veryearliest investigators, it was not possible at that timeto maintain them in a state of good health and undercontrolled conditions. Kuhn has demonstrated thatthe chimpanzee responds to experimental infectionwith T. pallidum in much the same way as does thehuman; early lesions develop, as do reactive non-treponemal and treponemal serological tests. Thus,sequential serum samples from the chimpanzees canbe used to examine the very important intervalbetween the time of infection and the developmentof reactivity in the present conventional STS. Totry to show that reactivity is present in early serumsamples that appear to lack reactivity in the currentconventional tests, newer treponemal antigens arebeing used. These are being prepared from thelarger supply of purified T. pallidum that is nowavailable through the zonal-ultracentrifugationtechniques described earlier.An unexpected observation in the course of these

studies was that a significant proportion of "normal"chimpanzees give reactive STS, both in the non-treponemal screening test and in the confirmatorytreponemal tests, such as the TPI and FTA-ABS.The nature of the organism (if indeed there is anorganism) that causes this serological reactivity, andthe possible relevance of such an organism to asyphilis vaccine are subjects of considerable interest.Certainly it is relevant to inquire whether or notthere is an endemic treponematosis, perhaps some-thing like yaws, among chimpanzees in their nativehabitat.

(ii) The studies regarding the differentiation ofadequately treated from untreated syphilis are stillin the preliminary stages. In a collaborative study

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with Atwood and Miller in New York, we haverecently found that reactivity in the TPI and FTA-ABS tests persists for 13 years or longer in patientstreated in the later stages of syphilis. Thus, it willnot be possible to use either of these two treponemaltests, as performed at present, as an index of thera-peutic response.At this point a few remarks are appropriate on the

use of immunofluorescent techniques as a means ofidentifying T. pallidum and other spiral organisms.The early interest in identification by immuno-fluorescent techniques was based on the value of theprocedure for finding T. pallidum organisms inmaterials from clinical lesions [the fluorescentantibody darkfield (FADF), Kellogg and Deacon,1965) and rapid immunofluorescence staining (RIS)procedures (Kellogg and Deacon, 1964)] and on itsusefulness in identifying T. pallidum in tissues ofexperimentally infected animals (Yobs, Brown, andHunter, 1964). The early applications of thesetechniques were fairly straightforward, but interest inthem was considerably heightened after the stimu-lating reports by Collart (1964) that T. pallidumorganisms might persist in rabbits and humans evenafter penicillin therapy of syphilis infection. Morerecently, wider interest in similar questions has beenprovoked by the reports of Smith and Israel (1967a,1967b) and Goldman and Girard (1967) that spiralorganisms, which are sometimes motile, can bedemonstrated in the aqueous humour and cerebro-spinal fluid of treated syphilitics. Both Smith andGoldman have strongly suggested that this spiralorganism is T. pallidum.

For the investigation of these phenomena itseemed desirable to produce a fluorescein-conju-gated anti-T. pallidum antiserum of rather well-defined properties and specificities. This project hasrecently been completed by Mothershed, whoutilized sequential bleedings from syphilitic rabbits.After conjugation with fluorescein, the immuneglobulins from each bleeding were examined forreactivity against a variety of spiral organisms.After ascertaining which of the sequential bleedingscontained the greatest reactivity against T. pallidum(in these experiments it was the 6-week bleeding),the fluorescein-conjugated immune globulins fromthat bleeding were made more specific for T.pallidum by a combination of various absorptionsand dilutions. A detailed description of this conju-gate and a standardized technique for its use areavailable from the VDRL. It is hoped that thisconjugate will also be available commercially in thenear future. For the present, we prefer to interpretwith caution the results obtained through the use ofthis conjugate, since it is always possible that it

might stain some unusual but nonpathogenic spiralorganisms with which it has not been specificallyabsorbed.

(iii) Another area of current research activityis the automation of serological tests for syphilis.This would offer several advantages in certaincircumstances:

(a) Help cope with the estimated 38 million STSbeing performed at present in the United States;

(b) Help alleviate the shortage of trained serologists;(c) Encourage hospitals to re-institute routine STS

on all patients;(d) Allow STS to be included in automated multi-

phasic screening programmes, including those forblood-typing;

(e) Improve reproducibility through reducing varia-tions inherent in manual tests.

Two of the various systems under developmentmay be described as examples. An automatedcardiolipin-reagin test was worked out in a collabora-tive study by McGrew at the VDRL and DuCros atthe Technicon Instruments Corporation (Ardsley,N.Y.). The instrument is a Technicon AutoAnalyzermachine and the antigen is rapid plasma reagin(RPR) card test antigen obtained from Hynson,Westcott, and Dunning, Inc. (Baltimore, Md.).This antigen-a modified VDRL antigen suspensioncontaining choline chloride-also contains specially-prepared carbon particles as a visualization agent.In brief, serum or plasma samples are tested at therate of 100 per hour. Reactions are seen grossly asblack agglutinates deposited on a moving whitefilter-paper strip. Although with a little experiencea serologist can easily judge the degree of reactivityon the tape, an instrumented assessment, perhapswith a densitometer, will probably be a necessity forlarge-scale use of this test. Both in trials at theVDRL, and in the field, the results of the automatedtest so far approximately parallel the performanceof the manual VDRL slide and RPR (Circle) cardtests.

In other studies, the Space-General Corporation(El Monte, Calif.) has under development a systemaimed towards the automated performance of theFTA-ABS test. If it were possible to automate aspecific and sensitive treponemal test at a relativelylow cost, then it might be possible to use it forscreening purposes in preference to reagin tests andthus circumvent the problem of false positivereactors. VDRL staff members and Dr James N.Miller are among the serologists who have co-operated on various aspects of this system.

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(C) Skin TestsAn immediate hypersensitivity skin test, even if it

were presumptive and not definitive, would be veryuseful for certain purposes, such as screening in theclinic and in field studies. Ideally, such a test wouldbe reactive even during the incubation stage ofsyphilis, so that it might also be useful in epidemio-logical investigations. Recent basic immunologicalstudies have shown that there are two kinds ofantibodies, gamma-i and gamma-2, that can stimu-late the skin for immediate hypersensitivity reactions.However, of these, only the gamma-I antibodies cansensitize homologous skin, and yet these samegamma-i antibodies do not fix complement in testsin vitro (Ovary, 1965). With respect to a syphilisskin test, this property of gamma-i antibodies hasthe important implication that skin-test reactivityin syphilis need not correlate with the humoralreactivity demonstrated in the conventional tests invitro. Thus, although the present serological testsdo not become reactive until some time afterinfection, skin tests ofthe immediate hypersensitivitytype may follow a different pattern. At the VDRLthis new knowledge about skin-sensitizing anti-bodies is being applied in studies in guinea-pigs,rabbits, monkeys, and chimpanzees by Cohen,Bullard, Logan, and Kuhn. One important aspect isthat emphasis is being given to assay in the homolo-gous system (guinea-pig antibodies assayed inguinea-pigs; rabbit antibodies in rabbits; andhuman, chimpanzee, and monkey antibodies inchimpanzees and monkeys). At this time, research iscentred on exploring various antigenic extracts ofT. pallidum and other treponemes to discoverpromising skin-test materials.

II. Gonorrhoea ResearchTo the best of my knowledge, most of the basic

research on gonorrhoea, except for antibiotic trials,which is being conducted in the Urnted States istaking place at the VDRL. At the present time, ourchief aim is the development of a serological screen-ing test, especially one that will detect the asympto-matic female carrier. The use of the selectiveculture media developed by Thayer and Martin(1966) indicates that there are many such carriers,but the acquisition of specimens is probably toocumbersome for mass screening. However, approxi-mately 38 million blood specimens are already beingexamined for syphilis each year in the UnitedStates, and virtually all of these could also bescreened with a serological test for gonorrhoea withthe expenditure of relatively little extra effortoutside the laboratory. I look forward, in fact, to theuse of an automated system that will divide a blood

sample into two aliquots, and perform on one ofthese a serum test for syphilis and on the other aserum test for gonorrhoea.The serological response to the gonococcus is at

present being investigated at the VDRL. Cohen andNorins (1966), Cohen (1967), and Cohen and others(in press) have found that humans possess "natural"antibodies to the gonococcus in all three majorclasses of immunoglobulins: IgG, IgM, andIgA. The "natural" IgG antibodies in the sera ofnormal subjects were more reactive with heat-stablethan with heat-labile gonococcal antigens, contrastingwith the IgG antibodies in the sera of infectedpersons, which were more reactive with heat-labilesurface antigens. Moreover, the immune IgGantibodies were more resistant to heating than werethe "natural" IgG antibodies. In other studies onexperimental gonorrhoea in male volunteers (Cohenand others, in press), the sequential humoralresponse to gonococcal antigens was studied. A fewpatients showed an increase of IgA or IgM anti-bodies to heat-labile gonococcal antigens but nineof ten patients showed an increased IgG titre tothem. However, seven of ten patients showedan increased titre of IgA antibodies to heat-stablegonococcal antigens, but there was no increase ofIgG or IgM antibodies. It was also found that theIgG antibodies that were present 14 days afterinoculation showed a greater heat stability than didthe IgG antibodies present just before inoculation.The organisms which were infectious in these

studies had maintained their virulence during 35months of selective passage in vitro. Experimentalstudies by Kellogg, Peacock, Deacon, Brown, andPirkle (1963) had shown that there were fourdistinct types of colonial morphology among thegonococci, which could be maintained and carriedforward in culture by selective passage. Two of theseknown as Type 1 and Type 2, were virulent for malevolunteers, while the two others, Type 3 and Type 4,were avirulent. If cultures of gonococci werecarried forward in the laboratory by randomunselected passages, then the avirulent types ofgonococci came to predominate. (Many "laboratory"strains which have been passaged randomly foryears may now be comprised of the avirulentcolonial types of gonococci.) The ability to recognizevirulent gonococci has an important bearing on thedevelopment of antigens for a serological test.At the present time experiments are in progress

(partly in co-operation with Dr Edgar Ribi andassociates at the Rocky Mountain Laboratory of theNational Institutes of Health) to characterize cellwall and other antigens of the gonococcus. However,despite the fact that these organisms can infect

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human subjects, it has not so far proved possible toproduce experimental infection in chimpanzeesmaintained at this laboratory.

DiscussionThe research aspects of syphilis and gonorrhoea

that have been mentioned do not, of course, includeall work going on at present in the United States.On the other hand, it is important to realize that inthe United States there are only about five placeswhere research on syphilis is being conducted; andapparently there are only one or two places whereany volume of research concerns gonorrhoea.Moreover, in these relatively few places, I know ofonly one or two instances in which a younger

investigator has recently entered the field on anykind of a long-term basis. I have the impression thatthe situation is not very different in other countries.This research situation stands out rather sharplyagainst the magnitude of the problem. In the UnitedStates, for the fiscal year 1967, there were 120,000reported cases of syphilis and 400,000 of gonorrhoea.If one takes into account the under-reporting ofcases, the contrast is even greater.

Perhaps one way in which an increase in researchmay come about is through an increase in collabora-tive projects between those having the newest basictools and approaches for research in infectiousdisease and immunology and those with experienceand knowledge concerning T. pallidum and thegonococcus.

Certainly the relatively small amount of researchtaking place makes it even more important for variousinvestigators to keep in touch, and emphasizes thecontinuing value of international communication insyphilis and gonorrhoea research.

REFERENCESCohen, I. R. (1967).J7. Bact., 94, 141.-, Kellogg, D. S., and Norins, L. C. "The Serum

Antibody Response in Experimental HumanGonorrhoea: Immunoglobulins G, A, and M."In press.and Norins, L. C. (1966). Science, 152, 1257.

Collart, P. (1964). In "Proc. World Forum on Syphilisand Other Treponematoses, Washington, 1962",U.S. Public Health Service Publication No. 997.Government Printing Office, Washington, D.C.

Goldman, J. N., and Girard, K. F. (1967). Arch.Ophthal. (Chicago), 78, 47-50.

Kellogg, D. S., Jr., Cohen, I. R., Norins, L. C., Schroeter,A. L., and Reising, G. "Neisseria gonorrhoeae. II.Colonial variation and pathogenicity during 35months in vitro." In press.

-~ -~(1965). "Bact. Proc. Amer. Soc. Microbiol.,Abstracts 65th Annual Meeting", p. 60, M128.and Deacon, W. E. (1964). Proc. Soc. exp. Biol.

(N.Y.), 115, 963., Peacock, W. L., Jr., Deacon, W. E., Brown, L.,and Pirkle, C. I. (1963). . Bact., 85, 1274.

Ovary, Z. (1965). Fed. Proc., 24, 94.Rathiev, T., and Pfau, C. J. (1965). Scand. J. clin. Lab.

Invest., 17, 130.Smith, J. L., and Israel, C. W. (1967a). Arch. Ophthal.

(Chicago), 77, 474.-~ -~(1967b). J. Amer. med. Ass., 199, 980.

Thayer, J. D., and Martin, J. E., Jr. (1966). Publ. HlthRep. (Wash.), 81, 559.

Tringali, G. (1965). Riv. Ist. sieroter. ital., 40, 243.Turner, T. B., and Hollander, D. H. (1957). "Biology

of the Treponematoses." WHO Monograph SeriesNo. 35. World Health Organization, Geneva.

Yobs, A. R., Brown, L., and Hunter, E. F. (1964).Arch. Path., 77, 220.

Zaffiro, P. (1962). Riv. Ist. sieroter. ital., 37, 614.

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