Genitourin Med 1988;64:1 18-23

Does lactobacillus vaccine for trichomoniasis, SolcoTrichovac, induce antibody reactive with Trichomonasvaginalis?JOHN F ALDERETE

From the Department of Microbiology, University of Texas Health Science Center, San Antonio, Texas, USA

SUMMARY Solco Trichovac is a vaccine of Lactobacillus acidophilus developed for treatingtrichomoniasis. The efficacy of the vaccine is reportedly due to cross reacting antibody beingproduced in people immunised with the lactobacillus bacteria. Several techniques, including enzymelinked immunosorbent, indirect immunofluorescence, immunoblot, and radioimmunoprecipitationassays were used to assess the extent of antigenic cross reactivity between Lactobacillus acidophilusand Trichomonas vaginalis. Data show a lack of antigenic relatedness between the Solco Trichovaclactobacilli and several strains of T vaginalis. Furthermore, antiserum to L acidophilus failed to inhibittrichomonad cytadherence or host cell killing, as is suggested by the producers ofthe Solco Trichovacvaccine.

Trichomoniasis continues to be a major sexuallytransmitted disease in the world. With the emergenceof resistance to drugs in strains of Trichomonasvaginalis,' the development of a vaccine represents animportant and necessary step towards eradication orat least control of this disease.The use of Lactobacillus acidophilus as a vaccine for

T vaginalis infections has received attention.2'Reports indicate that the successful immunisation ofwomen is due to the generation of antibody thatrecognises determinants common to antigens of bothlactobacillus and the pathogenic human trichomon-ads.56 Data conflict about the extent and nature ofsuch antigenic relatedness in the lactobacillus and Tvaginalis organisms.>' Using several modern tech-niques and biofunctional assays, I obtained dataindicating that no cross reactivity exists between thelactobacillus used in the Solco Trichovac vaccine2and the protozoan responsible for trichomoniasis.

Materials and methods

T VAGINALIS STRAINS, LACTOBACILLI, ANDANTISERAAll the T vaginalis strains had been used in previousstudies.`5 For example, NYH 286 is a common

Address for reprints: Dr John F Alderete, Department ofMicrobiology, UTHSC-SA, 7703 Floyd Curl Drive, San Antonio,Texas 78284-7758, USA

Accepted for publication 16 June 1987

laboratory strain that has been extensively evaluatedby all the assays used in this report. The SolcoTrichovac lactobacillus was a kind gift from DrsValent and Demes of the Institute of Parasitology,Comenius University at Bratislava, Czechoslovakia.Four rabbits were individually injected subcutan-eously and intramuscularly with 1 ml final volumecontaining 7 x 108 lactobacilli prepared in Freund'scomplete adjuvant. At two week intervals thereafterthey were given two booster immunisations with thelactobacillus in Freund's incomplete adjuvant. Theconcentration of antibody in rabbit serum was mon-itored by an enzyme linked immunosorbent assay(ELISA) as described below. Prebleed serum samplesfrom the immunised rabbits served as controls.

IMMUNOLOGICAL ASSAYS TO TEST FORANTIGENIC CROSS REACTIVITYFour assays were used to assess whether antisera to theSolco Trichovac L acidophilus detected T vaginalisantigens. To insure that rabbits indeed producedantibody to lactobacillus, the bacteria in the originalvaccine suspension of 7 x 109 bacteria per ml werediluted 10-fold up to 10 . Volumes of 50 p1 of thevarious dilutions were added to individual wells ofmicrotitre plates and allowed to dry, and 100%ethanol was added as a fixative." Sera from all fourrabbits injected with lactobacillus were thenindividually tested for reactivity with the bacteriacoated microtitre wells. After antibody had been

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No cross reactions between T vaginalis and Solco Trichovac

1r-1

miRI IR IRIa]0c

Antigen:V.Tih

1.y~~~ %_richo ona2s vaginalis

Fig 1 Results ofenzyme linked immunosorbent assay(ELISA) using rabbit antiserum generated against the SolcoTrichovac lactobacillus vaccine (a) and Trichomonasvaginalis strain NYH 286 (b). Abscissa shows seventrichomonal isolates and lactobacilli, which were.fixed tomicrotitre well surfacesfor screening with antiserum.Prebleed normal rabbit serum was used as a control. For thisexperiment, 7 x 105 lactobacilli werefixed to individual wellsofmicrotitre plates. All sera were diluted 1i`.

incubated with antigen for two hours at 4°C, the wellswere washed with phosphate buffered saline (PBS) andincubated for one hour at 37°C with goat anti-rabbitimmunoglobulins conjugated with alkaline phos-phatase. Lactobacillus antisera from all four rabbitsdiluted 10o' and 1'0- yielded maximum reactions when7 x and 7 x 105 lactobacilli were used to coat thepolyvinyl microtitre wells (fig 1 (a)). The antiserum Lacidophilus from the individual rabbits was thenpooled for use in the experiments outlined. Theprocedure for using T vaginalis organisms as the testantigen has been described previously."

Indirect immunofluorescence with live T vaginalisparasites was performed using techniques reportedpreviously.9'`'5 In this case, the rabbit antisera tolactobacillus were heat inactivated at 56°C for 15minutes immediately before being added to tri-chomonads. Rabbit antiserum to T vaginalis strain

NYH 28689 was used as a positive control to showfluorescence reactions.

Immunoblot'3 was performed with total tri-chloroacetic acid precipitated proteins of T vaginalisstrains8 and the bovine trichomonad, Tritrichomonasfoetus.'6 Radioimmunoprecipitation assays usingdetergent extracts of both surface radiolabelled9 andintrinsically radiolabelled8 parasites were also perfor-med using methods detailed elsewhere."9 3`5 Ant-iserum` and monoclonal antibodies'"'5 reactive withT vaginalis protein antigens were used as positivecontrols for these assays.

CYTADHERENCE AND CYTOTOXICITY ASSAYSMonolayers of HeLa cells incubated with trichomon-ads labelled with tritiated (3H)thymidine'0 12 were usedto study the effect of antiserum to lactobacillus ontrichomonal cytadherence. Parasites were pretreatedwith up to 50% heat inactivated pooled antiserum tolactobacillus for different times up to one hour. Thesuspension of T vaginalis containing the antiserum tolactobacillus was then added to cell monolayers'2 andincubated in a 5% carbon dioxide humidified atmos-phere for 30 minutes at 37°C. Coverslips were thenextensively washed in PBS before being immersed inscintillation vials containing the cocktail and havingtheir radioactivity measured.A quantitative colorimetric assay using HeLa cells

in monolayer cultures was used to assess the extent ofhost cell killing.'0 For these experiments, differentconcentrations of high titred antiserum to lactobacilliwere included with the mixture of parasite and hostcells throughout the 18 hour incubation.'`

Results

ENZYME LINKED IMMUNOSORBENT ASSAY (ELISA)It was first necessary to show the presence of antibody

Table Antibody titration to two different concentrations ofSolco Trichovac lactobacillifixed to microtitre wells. Numbersare mean (SD) absorbances (at 405 nm) ofduplicate samplesfrom three experiments*

Concentrations oflactobacillit

Dilutions ofserumt 7 x 105/ml 7 x 10J/ml10 0.74(0-01) 1-04(0.01)1o-2 0-64(0-01) 1-12(0-02)10-3 0054 (0.02) 0.94 (0-02)10-4 0.74 (0.02) 0-88 (0-01)0-5 0.16 (0.01) 0-62 (0.02)

*Normal rabbit serum at 10 ', 1o, and 10 5 dilutions gave mean (SD)absorbances of 0.25 (0-01), 0-15 (0), and 0-10 (0), respectively, for7 x 1o0 lactobacilli. Similar low level reactivity was observed fornormal rabbit serum with all concentrations of lactobacilli.tSerum at 14 days after the final booster was used.tOriginal concentration of bacteria was 7 x 109/ml.

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120Dark field Fluorescence

Fig 2 Indirect immunofluorescence and corresponding darkfieldpictures of Trichomonas vaginalis strains treated withantiserum to Solco Trichovac. As a positive control all strainswere incubated with antiserum to strain NYH 286, and a

representativefluorescence ofagglutinated NYH 286parasites is presented in the top panel. Prebleed control serumgave nofluorescence reactions with any of the strains as alsoseen with antiserum to Solco Trichovac.

to lactobacillus in sera of rabbits immunised with theSolco Trichovac vaccine. The table shows the highlevel of recognition of pooled rabbit antisera to theSolco Trichovac lactobacillus by different concentra-tions of L acidophilus as antigen coating microtitrewells. The same antisera, however, failed to give a

reaction greater than that of the control prebleedserum with seven T vaginalis strains (fig 1 (a)).Antiserum to the trichomonal strain NYH 286,however, gave strong ELISA readings with all seven

strains but failed to react with the lactobacilli coatedwells (fig 1 (b)).

AldereteINDIRECT IMMUNOFLUORESCENCEIt was then important to test for any immunofluores-cence of T vaginalis with the pooled antiserum tolactobacillus. Figure 2 shows that, whereas rabbitantiserum to strain NYH 286 gave strong fluorescenceof live organisms, antibody to the Solco Trichovac Lacidophilus gave no fluorescence with parasites ofthreestrains, which indicated no trichomonad surfacerecognition.

IMMUNOBLOT AND RADIOIMMUNOPRECIPITATIONASSAYSFig 3 (a) shows many trichomonad protein antigensimmobilised on nitrocellulose with rabbit antiserum toT vaginalis strain NYH 286. The extent of anytrichomonad protein recognition of duplicate immun-oblots incubated with antiserum to Solco Trichovaclactobacillus (b) equalled the minor reactivityobtained with prebleed control serum (c).

Finally, any cross reaction between antiserum toSolco Trichovac lactobacillus and radiolabelled Tvaginalis antigens was tested by the radioimmuno-precipitation assay (fig 4). Under no circumstanceswere trichomonad proteins labelled with eitherradioiodine ('25I) (surface) or radiosulphate (internal)(5S) precipitated when Staphylococcus aureus bearingprotein A was added to extracts containing antiserumto lactobacillus. Furthermore, antisera from twopeople immunised with the Solco Trichovac vaccine7also failed to show any formation of antigen andantibody immune complexes (data not shown). On theother hand, the use of rabbit antiserum strain NYH28689 or monoclonal antibody C20A3, which is knownto react with a high molecular weight immunogen,'1'5readily resulted in the precipitation of appropriateprotein(s). As expected, no reaction with parasiteproteins was observed with prebleed control serum.

CYTADHERENCE AND CYTOTOXICITY ASSAYSAntiserum to the lactobacillus vaccine may interferewith certain reactions with live parasites,56 such ascytadherence'2 and host cell killing.'0 A report has alsoshown that host cells incubated with lactobacilli wereless vulnerable to parasitism by T vaginalis.'7 Examin-ation for possible inhibition of T vaginalis parasitismand killing of host cells by the antiserum to Lacidophilus was therefore necessary. Pretreatment ofparasites and adding antiserum to lactobacilli duringincubation of trichomonads and host cells did notaffect the extent of cytadherence or host cytotoxicity(data not shown).' 12 Levels of host cell parasitism andkilling by T vaginalis in the presence of antiserum tolactobacilli were equivalent to those seen either with-out the addition of exogenous serum or with prebleedserum.

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No cross reactions between T vaginalis and Solco Trichovac

0C 0)Trichomonas vaginalis

(0

A>V V1 k~.,

I C? I~-G

Rabbit antiserumto NYH 286

Trichomonas vaginalis

,r

e 'aIl1

Rabbit antiserum toSolco Trichovac

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Trichomonas vaginalis

Normal rabbit serum

Fig 3 Immunoblot of various Trichomonas vaginalis strains (NYH 286, RU 375,431, AL2O W, and CDC85) and the bovinetrichomonad, Tritrichomonasfoetus KV-1, with antiserum to Solco Trichovac lactobacillus (b). Antiserum to strain NYH 286(a) andprebleed normal rabbit serum (c) were used as positive and negative controls, respectively, under the sameexperimental conditions.

Discussion

The Solco Trichovac vaccine contains a mixture of"aberrant" L acidophilus,2 and its immunotherapeuticeffect has been ascribed to possible cross reactionsbetween the lactobacillus and surface structures of thepathogenic human trichomonads.256 Thus, antibodywould be produced that would not only interfere withbiofunctional aspects of T vaginalis, such as cytad-herence and contact dependent host cytotoxicity,'012but might neutralise the parasite, as has been des-cribed.'4 It was therefore interesting to assess the

possible nature of cross reactivity between high titredrabbit antiserum to the Solco Trichovac lactobacillusand trichomonad proteins. Detection of commonepitopes in trichomonad molecules and L acidophiluswould give considerable insight into the immunogensof T vaginalis that could be studied further as vaccinecandidates. Alternatively, such antigenic common-ality between the pathogenic human trichomonadsand possibly bacteria of the normal flora'2 6 would berelevant to issues dealing with the ecology of thenormal flora during this infection and after vaccina-tion.

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Alderete

0Rabbit antiserumto Solco Trichovac

--r --

E.tE EF±E3 E

Ofl I,fLjz CO z--C) h

a)Ii I I

I1251 35s "

Fig 4 Radioimmunoprecipitation assay (a) using detergent extracts ofsurface iodinated (`25I) trichomonads and thoseinternally labelled with radiosulphate ("S) incubated with rabbit antiserum to Solco Trichovac lactobacillus. Prebleed normalrabbit serum served as a negative control. Rabbit antiserum to Trichomonas vaginalis strain NYH 286 and a monoclonalantibody (MAb) designated C20A3, which recognises a trichomonad surface immunogen, were used in duplicate preparationsofdetergent extracts labelled with (a) '25I and (b) 5S.

Several immunological assays, including highly sen-sitive and specific immunoblot and radioimmuno-precipitation techniques, as well as two biofunctionalassays, were used in this study. No evidence wasobtained to indicate any cross reactivity betweenantigenic epitopes of T vaginalis and the Solco Tri-chovac lactobacillus. These data suggest strongly thatany protection in man claimed by the producers of theSolco Trichovac vaccine2 is not due to direct antibodyinterference or neutralisation of the parasite.56 Our

results and those of others7 suggest that non-specificimmune mechanisms are more probably responsiblefor any dimunition or resolution of trichomoniasis orits symptoms in patients. Clearly, much more researchappears to be warranted before allowing large scalehuman vaccination with bacteria originally derivedfrom the urogenital and vaginal tract of women.

Finally, the inability ofantiserum to lactobacillus toinhibit host cytadherence and cytotoxicity is especiallynoteworthy in the light of a report showing that

a

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No cross reactions between T vaginalis and Solco Trichovacpretreatment of cell monolayers with lactobacillidecreased trichomonal parasitism levels.'` Theseresults now appear to be attributable to steric con-

siderations imposed on the host cell surface whenintact bacteria bind to the cells.

This study was supported by Public Health Service grants Al-18768 and AI-22380 from the National Institute of Allergyand Infectious Diseases. I am the recipient of ResearchCareer Development Award K04-AI-00584. I acknowledgethe assistance of Lorraine Kasmala, Edmund C Metcalfe,and Guillermo E Garza in performing the variousexperiments, and thank Ms Diana Hinojosa for typing thismanuscript.

References

I Meingassner JG, Mieth H, Czok R, Lindmark DG, Muller M.Assay conditions and the demonstration of nitroimidazoleresistance in Tritrichomonas foetus. Antimicrob AgentsChemother 1978;13:1-3.

2 Symposium on trichomoniasis. Basle, 20 October 1981. GynakolRundsch 1983;23 suppl:1-88.

3 Pavic R, Stojkovic L. Vaccination with Solco Trichovac. Immun-ological aspects ofa new approach for therapy and prophylaxisof trichomoniasis in women. Gynakol Rundsch 1983;23 suppl2:27-38.

4 Milovanovic R, Grcic R, Stojkovic L. Serological study with SolcoTrichovac, a vaccine against Trichomonas vaginalis infection inwomen. Gynakol Rundsch 1983;23: suppl 2:39-45.

5 Stojkovic L. New evidence elucidating the mechanism ofaction ofgynatren/Solco-Trichovac. Gynakol Rundsch 1984;24 suppi3:29-37.

6 Bonilla-Musoles F, Sanches-Pena JM, Pellicer A, Guevara F,Ziegler WJ. A vaccine against vaginal trichomoniasis inhumans. A temporary solution of the problem? RevistaEspaniola de Obstetricia y Ginecologia 1983;42:777-89.

7 Gombosova A, Demes P, Valent M. Immunotherapeutic effect ofthe lactobacillus vaccine, Solco Trichovac, in trichomoniasis isnot mediated by antibodies cross reacting with Trichomonasvaginalis. Genitourin Med 1986;62:107-10.

8 Alderete JF. Antigen analysis of several pathogenic strains ofTrichomonas vaginalis. Infect Immun 1983;39:1041-7.

9 Alderete JF. Identification of immunogenic and antibody-bindingmembrane proteins of pathogenic Trichomonas vaginalis. InfectImmun 1983;40:284-91.

10 Alderete JF, Pearlman E. Pathogenic Trichomonas vaginaliscytoxicity to cell culture monolayers. British Journal of VenerealDiseases 1984;60:99-105.

11 Alderete JF. Enzyme linked immunosorbent assay for detectingantibody to Trichomonas vaginalis: use of whole cells andaqueous extract as antigen. British Journal of Venereal Diseases1984;60:164-70.

12 Alderete JF, Garza GE. Specific nature of Trichomonas vaginalisparasitism of host cell surfaces. Infect Immun 1985;50:701-8.

13 Alderete JF, Suprun-Brown L, Kasmala L. Monoclonal antibodyto a major surface glycoprotein immunogen differentiatesisolates and sub-populations of Trichomonas vaginalis. InfectImmun 1986;S2:70-5.

14 Alderete JF, Kasmala L. Monoclonal antibody to a majorglycoprotein immunogen mediates differential complement-independent lysis of Trichomonas vaginalis. Infect Immun1986;53:697-9.

15 Alderete JF, Deme;s P, Gombosovi A, et al. Phenotypes andprotein epitope phenotypic variation among fresh isolates ofTrichomonas vaginalis. Infect Immun 1987;SS: 1037-41.

16 Steinbuchel A, Muller M. Anaerobic pyruvate metabolism ofTrichomonas foetus and Trichomonas vaginalis hydrogen-osomes. Mol Biochem Parasitol 1986;20:57-65.

17 Martinotti MG, Martinetto P, Savoia D. Adherence of Tri-chomonas vaginalis to cell culture monolayers. Eur J ClinMicrobiol 1986;5:320-3.

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