144 TRANSACTIONSOF THE ROYAL SOCIETY OF TROPICAL MEDICINE AND HYGIENE, VOL. 71, No. 2,1977.

Host antigens

and parasite

antigens

of murine

Schistosoma

mansoni

0. L. GOLDRING, A. SHER, S. R. SMITHERS AND DIANE J. MCLAREN National

Institute

for Medical

Research,

Summary

An indirect fluorescent antibody technique was used to detect mouse host antigens and parasite antigens on the surface of Schistosoma mansoni. A rabbit anti-mouse rbc antiserum was used to detect host antigens, and serum from mice immune to S. mansoni was used to detect parasite antigens. Schistosomula prepared after penetration of isolated mouse skin did not possess host antigens but bound antibody from immune serum (immune antibody); schistosomula recovered from the lungs of mice five days after infection possessed host antigens and failed to bind immune antibody. In contrast, schistosomula recovered from the skin of normal or immune mice three and 20 hours after cercarial penetration, adult worms, and cryostat sections of adult worms, were positive for host antigens but also bound immune antibody. Strong binding of immune antibody only occurred with the cryostat sections. Anti-schistosome antibody can therefore bind to schistosomes in the presence of host antigen. The lung forms of schistosomula, however, may have different surface properties as there is no evidence that immune antibody binds to these forms. Introduction

A state of concomitant immunity occurs in some experimental animals infected with S. mansoni. Adult worms stimulate immunity to reinfection but are apparently unharmed by the immune response they generate (SMITHERS & TERRY, 1976). The evasion of immunity has been attributed to the presence of host antigens associated with the surface membrane of the parasite (SMITHERS,TERRY & HOCKLEY, 1969; CLEGG, 1974; TERRY & SMITHERS,1975; GOLDRING, CLEGG, SMITHERS&TERRY, 1976). It is believed that host antigens are glycolipids of host origin, are taken up at the surface of the developing schistosomulum and serve to protect the membrane of the worm against the immune response of the host by masking susceptible parasite antigens. Young schistosomula which do not possess host antigens are susceptible to the immune response. Circumstantial evidence to support this hypothesis was recently presented by MCLAREN, CLEGG & SMITHERS(1975) who showed that schistosomula prepared in vitro possessed no host antigen and reacted with immune monkey serum, whereas four-day-old schistosomula recovered from the lungs of normal mice possessed host antigen and failed to bind immune monkey serum. In the present study an indirect fluorescent antibody system was used to investigate (i) whether three-hour and 20-hour schistosomula, recovered from the skin of mice, possesshost antigen and/or bind immune antibody; (ii) whether the immune status of the host influences host antigen expression and (iii) whether adult schistosomes bind immune antibody.

Mill

Hill,

London

NW7

IAA

Materials and methods Parasite and experimental hosts A Puerto Rican strain of S. mansoni was used through-

out this work; its maintenance and the methods of infection and recovery have been described previously (SMITHERS& TERRY, 1965a, b). The animals employed in the study were male outbred Parkes or CBA mice (18-22 g) obtained from our Institute breeding facility. Mice were each immunized by percutaneous exposure to 30-40 cercariae, and were kept for 12-15 weeks before challenge. Adult schistosomes were recovered by portal perfusion from Parkes mice exposed seven weeks previously to lO&150 cercariae each. Schistosomula

Three-hour schistosomula were prepared by allowing cercariae to penetrate isolated abdominal skin from normal Parkes mice (CLEGG & SMITHERS,1972). These schistosomula are referred to as three-hour “in vitro” forms. Three-hour and 20-hour schistosomula were recovered from the skin of normal and immune Parkes mice. The method of CLEGG(1965) was used, except that the skin was not swabbed with alcohol before excision, and Earle’s balanced saline containing 0.5 % lactalbumin hvdrolvsate and 1 ‘X bovine serum albumin (Elac/l% BSA) was used to recover the schistosomula. Five-day-old schistosomula were recovered from the lungs of normal and immune CBA mice by the method of SHER,MACKENZIE& SMITHERS(1974). Schistosomula were recovered in Elac/l ‘A BSA. Schistosomula recovered from either the skin or lungs of mice are referred to as “in viva” forms. Frozen sections of adult worms

10% methanol-free formaldehyde was prepared by dissolving solid paraformaldehyde in distilled water at 65°C and adjusting the pH to 7.1 with IN NaOH. Adult worms were fixed in this solution for 10 minutes at 4°C. The worms were then washed with Hanks’ balanced salt solution (HBSS), snap frozen in liquid nitrogen, and 8pm frozen sections were prepared using a Bright’s cryostat at -25°C. Antisera

An antiserum raised against mouse host antigens was prepared in rabbits after three intravenous injections of 2 ml of 2% Parkes solution of mouse erythrocytes at two-weekly intervals. The rabbits were bled two weeks after the last injection. Norma1 rabbit serum was used as a control. Serum from mice immune to S. mansoni (immune serum) was prepared from CBA mice 12-15 weeks after exposure to 30-40 cercariae. This serum has been shown by SHER,SMITHERS& MACKENZIE(1975) to confer passive protection against S. mansoni when injected into normal

0. L. GOLDRING

flooded with the appropriate test antiserum and incubated at 37’C for 30 minutes. After decanting the antiserum, each slide was then washed five times with Elac/l % BSA and finally incubated at 20°C for 30 minutes with the appropriate dilution of the relevant fluorescein-labelled antiserum. After incubation, the slides were washed five times with Elac/l ‘x BSA and mounted in glycerine jelly.

mice. Serum from normal CBA mice was used as a control. All sera were heat inactivated for 30 minutes at 56°C and were used undiluted. Fluorescein conjugated IgG of a goat anti-rabbit IgG (FI-GAR) was obtained from Miles Laboratories. In the test system it was used at a final concentration of one in three for frozen sections, and one in ten for whole organisms. The diluent was Elac/l % BSA. Fluorescein conjugated IgG of a horse anti-mouse globulin (FI-HAM) was obtained from Progressive Laboratories Ltd. It was also used in dilutions of one in three and one in ten.

Worms and sections were examined with a Reichert Binolux microscope &ted with a Tiyoda dark held substage condenser. The lamp was a mercury vapour Wotan HB 200 W. The exciter filter was a BG I2/5.2 mm, and the barrier filter an llford 107.

Fluorescent urrtihody technique Livirfg worm

The direct method was used (GOLDRING et cd., 1976). 50-100 schistosomula or 8-10 adult worms in 2in s gin test tubes were washed three times with EIac/l % BSA and the supernatant removed to retain 0.1 ml of fluid over the worms. 100 1~1of the test antiserum was added and the tubes incubated at 37C for 40 minutes. The worms were then washed four times with Elac/l% BSA and finally suspended in 0.1 ml of the medium. To each tube was then added 0.1 ml of the appropriate dilution of the relevant fluorescein-labelled antiserum and the tube was then incubated at 20°C for 30 minutes. After this time the worms were washed four times with Elac/l % BSA and transferred in a minimum volume of Elac/l % BSA on to a microscope slide.

Photogrrrphy

Ektachrome EH I35 colour positive film was used, from which both colour and black and white prints were made. Recording oj’results

Living schistosomula exhibiting surface membrane tluorescence were graded from 4 to T . I Negative schistosomula showed a generalized blue autofluorescence and no yellow-green fluorescence around the outer edges. Positive schistosomula became less blue and more yellow-green from -I- to $ i t . Schistosomula graded - , showed faint yellow-green fluorescence on their outer edges which became greener through the grades 7~$ to 1 1 -. Dead or damaged worms were not assessed for fluorescence.

Sections were dried at room temperature for 30 minutes, Table I

145

et Cd.

Fluorescence of scbistosomula after treatment with anti-host antigen antiserum and immune mouse serum

Age and source of schistosomula 3-h

3-h

3-h

20-h

20-h

5-day

5-day

“in vitro”

“it1 vivo”

“in vivo”

“itI viva”

“in viva”

“it2 viva”

“in viva”

from normal mouse skin

from immune mouse skin

from normal mouse skin

from from from immune immune normal mouse skin mouse lungs mouse lungs

-ve

-ve

-ve

--Ye

Antibody applied to schistosomula Nil -ve Immune mouse serum t lluorescein ~I-T (26)* labelled anti-mouse globulin (FI-HAM) Normal mouse serum i fluorescein -ve (18) labelled anti-mouse globulin (FI-HAM) Fluorescein labelled anti-mouse -ve 17) globulin (FI-HAM) Rabbit anti-mouse rbc antiserum -W 21) I fluorescein labelled anti-rabbit IgG (FI-GAR) Normal rabbit serum I-ttuorescein labelled -ve (17) anti-rabbit IgG (FI-GAR) Fluorescein labelled rabbit 1gG (FI-GAR) -ve (32)

+**(I01

-ve

(28)

-ve

t -ve

t-

(20) (12)

t (20)

t(l5)

-ve -t +

-vc

-ve

-ve(2l)

--e(8)

-i (18)

(6) (5)

-ve

(30)

-ve

(15)

-ve

(15)

--Ye (12)

(16) (10)

-ve

(23)

-ve (42) I- (3)

-ve

(11)

-ve

(II)

t + (32)

t T (9)

t.t(lI)

(1 I)

rll(9)

-ve

(15)

-ve

(23)

-ve

(19)

-ve

(16)

-ve

(7)

-ve

(10)

-ve

(20)

-ve

(17)

-ve

(13)

-ve

(31)

-ve

(14)

-ve

(8)

* Numbers in parentheses indicate the number of worms examined in a particular preparation. ** Degree of Auorescence observed +, + +, + -1.+.

146

HOST

AND

PARASITE

ANTIGENS

Living adult worms which were positive showed a green fluorescence associated with the dorsal tubercles, negative worms showed only a blue autofluorescence. Cryostat sections of adult worms showed a bright localized yellow-green positive staining; control sections showed no localized specific fluorescence and appeared dull green in colour. Results The indirect fluorescent antibody technique was used to demonstrate the presence of either host antigens or parasite antigens on the surface of the schistosomula. The specific antisera used were a rabbit anti-mouse rbc antiserum and an antiserum from mice immune to S. munsoni, for the detection of host antigen and parasite antigen respectively. The results are given in Table I. Three-hour “in vitro” schistosomula bound immune antibody but did not bind antibody from an anti-mouse rbc serum, indicating that parasite antigens but not host antigens were exposed to the surface. When “in Go” schistosomula were tested for the presence of host antigens, all forms were found to be positive. There was an increase in the degree of fluorescence as the age of the schistosomula increased from three hours to five days, and the results were similar whether the schistosomula were recovered from normal mice or mice immune to .S. munsoni. On the other hand, the presence of parasite antigens was detected on the surface of three-hour and 20-hour “in viva” forms, but was not detected on five-day “in vivo” forms. With this test system, however, the immune status of the mouse affected the results; approximately 40% of the three-hour “in Go” schistosomula recovered from immune mice, and a small proportion of 20-hour “in viva” forms recovered from similar animals, bound the fluorescentlabelled horse anti-mouse serum directly. Adult worms

Using identical test systems as those used for studying schistosomula, live adult worms were found to be strongly positive for host antigen and weakly positive for parasite

OF MURINE

s.

??lUiZSOni

antigen. The fluorescence obtained with immune antibody (indicating parasite antigens) was associated with the dorsal tubercles of the worm and occurred in scattered patches limited to about 20% of the worm’s surface (Fig. 1). Essentially similar results were obtained with adult worms which had been fixed in 10% paraformaldehyde for two minutes, washed, and were then treated with immune serum. All controls were negative (Fig. 2). Cryostat sections

Treatment of 8 pm cryostat sections of adult worms with either rabbit anti-mouse rbc antiserum or immune serum showed that both sera reacted principally with the surface of the worm, indicating the presence of both host antigens and parasite antigens (Fig. 3). Control sections treated with normal rabbit serum or normal mouse serum showed no specific fluorescence associated with the surface of the worm (Fig. 4). Discussion The indirect fluorescent antibody technique has shown that three-hour “in vitro” schistosomula lacked mouse host antigens but bound antibody from immune serum whilst five-day “in viva” schistosomula, recovered from the lungs of mice, possessed host antigens and did not bind antibody from immune serum. These results corroborate the findings of MCLAREN et al. (1975), who used an antibody-enzyme bridge technique and ultrastructural localization of the enzyme in similar studies on three-hour “in vitro” and four-day “in viva” schistosomula; they suggested that the presence of host antigen serves to protect the surface membrane of the schistosomula against antibody mediated damage. In contrast, our examination of three-hour and 20-hour “in vivo” schistosomula recovered from the skin of mice, demonstrated an ability of these forms to bind immune antibody in the presence of host antigens; although host antigen fluorescence and immune antibody fluorescence was not as strong as that observed on the five-day “in vivo” schistosomula and three-hour “in vitro” schistosomula respectively. These results could indicate that the

Fig. 1. Adult schistosome ( x 930). Tubercles fluorescing with immune mouse serum.

0.

L. GOLDRING

147

Ct d.

Fig. 2. Adult schistosome 1 (x930). No fluorescence of tubercles with normal mouse serum.

Fig. 3. Adult worms 8 pm section (TS x 115). Positive fluorescence associated principally with the worm surface, obtained with immune mouse serum (similar result obtained with anti-host antigen antiserum).

early acquisition of small amounts of host antigen reduce the capacity of schistosomula to bind immune antibody. Our results also show that recovery of schistosomula from the skin or lungs of immune as opposed to “normal” mice did not influence host antigen uptake, nor did it determine or alter the ability of these schistosomula to bind immune antibody. One difference was observed, however, between schistosomula derived from normal mice and those derived from immune mice. Approximately 40% of the three-hour schistosomula recovered from immune mice reacted weakly with the fluoresceinlabelled horse anti-mouse globulin antiserum, possibly indicating the presence of mouse antibody on the surface of these forms; direct proof of this would require further experimentation. Surprisingly, five-day-old schistosomula

derived from the lungs of immune mice showed no such reaction. Intact whole adult worms, either living or fixed in paraformaldehyde, were strongly positive for host antigens, yet at the same time they bound immune antibody weakly. The ability of adult worms, three-hour and 20-hour “in viva” schistosomula, to bind immune antibody is in direct contrast to the five-day “in viva” schistosomula which were never seen to bind immune antibody. This observation suggests that the surface properties of the lung stage of the schistosome differ from the younger and older forms of the parasite. Examination of cryostat sections of adult worms demonstrated both the presence of host antigens and the ability of the worms to bind immune antibody. Both

HOSTAND PARASITE ANTIGENSOF MURINES.WKZ~SO~

Fig. 4. Adult worms 8 ym section (TS x 115). No fluorescence with’ normal mouse serum (similar result obtained with normal rabbit serum).

reactions were strong and were associated principally with the worm surface. A similar reaction with immune antibody has been observed by VON LICHTENBERG (1967) using cryostat sections of adult worms in situ in infected mouse livers. In the present study the strong binding of antibody from immune serum to cryostat worm sections is in contrast to the weak binding observed with whole adult worms. It is suggested that the freezing process involved in the preparation of the cryostat sections in some way increases the availability of specific parasite surface determinants for binding with antibody from immune serum. It has been suggested that host antigens serve to protect the surface membrane of the schistosome from antibodymediated damage (MCLAREN,CLEGG & SMITHERS, 1975). In this investigation, however, anti-schistosome antibody was found to bind to young “in vivo” schistosomula and adult worms in the presence of host antigens. This finding does not invalidate the hypothesis that host antigens are protective; it may indicate that the presence of host antigens, although not entirely preventing the binding of anti-schistosome antibody, obviates their binding in sufficient quantity or in the correct pattern to cause surface damage.

References

Clegg, J. A. (1965). In vitro cultivation of Schistosoma mansoni. Experimental

Parasitology,

16, 133-147.

Clegg, J. A. (1974). Host antigens and the immune response in schistosomiasis. In: Parasites in the immunized host: mechanisms of survival. Ciba Foundation Symposium, 25 (new series), 161-183. Clega. --_ J. A. & Smithers. S. R. (1972). The effects of immune rhesus monkey serum‘on schistosomula of Schistosoma mansoni during cultivation in vitro. Znternational Journalfor

Parasitology,

antigens by Schistosoma mansoni. Clinical and Experimental Immunology, 26,181-187. McLaren. D. J.. Cleaa. J. A. & Smithers. S. R. (1975). Acquisition of hoiy’antigens by young Schisiosoma mansoni in mice: correlation with failure to bind antibody in vitro. Parasitology, 70,67-75. Sher, F. A., Mackenzie, P. & Smithers, S. R. (1974). Decreased recovery of invading parasites from the lungs as a parameter of acquired immunity to schistosomiasis in the laboratory mouse. Journal of Znfectious Diseases, 130, 626-633.

Sher, F. A., Smithers, S. R. & Mackenzie, P. (1975). Passive transfer of acquired resistance to Schistosoma mansoni in laboratory mice. Parasitology, 70,347-357. Smithers, S. R. & Terry, R. J. (1965a). The infection of laboratory hosts with cercariae of Schistosoma mansoni and the recovery of adult worms. Parasitology, 55, 695-700.

Smithers, S. R. &Terry, R. J. (1965b). Naturally acquired resistance to experimental infections of Schistosoma mansoni in the rhesus monkey (Macaca mulatta). Pardsitology,

55,701-710.

Smithers. S. R. & Terrv. R. J. (1976). The immunoloav of schistosomiasis. 1; Advances ii Parasitology (Ed. Ben Dawes), 7, 41-93. London and New York: Academic Press. Smithers, S. R., Terry, R. J. & Hockley, D. J. (1969). Host antigens in schistosomiasis. Proceedings of the Roval Society. B.. 17L483-494.

Terry, R. J. &‘Smithers, S. R. (1975). Evasion of the immune response by parasites. Symposia ofthe Society for Experimental

Biology, No. 29,435-465.

von Lichtenberg, F. (1967). Mechanism of schistosome immunity. In: Bilharziasis. International Academy of Pathology, Special Monograph (Ed. F. K. Mostofi). Berlin, Heidelberg, New York: Springer-Verlag, pp. 286-300.

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Goldring, 0. L., Clegg, J. A., Smithers, S. R. & Terry, R. J. (1976). Acquisition of human blood group

Accepted for publicution

12th December, 1976.