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The isolation and partial characterization of a protective antigen from developing larvae of Ascaris suum
International Journal for Parasitology. Vol. 9. pp. 307-3 Pergamon Press Ltd. 1979. Printed in Great Britain. (0 Austrakm Society for Parasifology.
II.
0020-7519/79/0801-0307$02.00/O
THE ISOLATION AND PARTIAL CHARACTERIZATION OF A PROTECTIVE ANTIGEN FROM DEVELOPING LARVAE OF ASCARIS SUUM BERT
Department
of Pathobiology,
E. STROMBERG
School of Veterinary Medicine HI, University of Pennsylvania, Philadelphia, PA 19104, U.S.A. (Received 9 August 1978)
B. E. 1979. The isolation and partial characterization of a protective antigen from developing larvae of Asearis suum. International Iournal for Parasitology 9: 307-3 I I. An antigen (ACF antigen) obtained by in vitro cultivation of third-stage larvae of Ascaris suum to the fourth stage in defined medium induced significant protection in guinea pigs. The antigen was further characterized by ultrafiltration and gel filtration and was shown to be a single component by gel precipition and immunoelectrophoresis. It induced active cutaneous anaphylaxis in sensitized animals. The ACF antigen is estimated to contain about 79% protein and 22% carbohydrate and to have a molecular weight of approx 67,000.
Abstract-STROMBERG
INDEX KEY WORDS: Ascaris suum; protective product; functional antigen; vaccination.
immunity;
INTRODUCTION
soluble antigens;
MATERIALS
In vitro cultivation of helminth parasites has been used by several investigators to obtain worm immunogens (Silverman, Poynter & Podger, 1962; Soulsby, 1963; Zimmerman & Leland, 1974; and Rothwell & Love, 1974). Such immunogens may induce protection against a homologous challenge infection and may be considered as protective antigens. Perhaps some might be considered as ‘functional antigens’ in that they serve a physiological function in the parasites as we!! as inducing an immune response which significantly affects the parasite (i.e. the exsheathing fluid of Huemonchus contortus is capable of inducing ‘self cure’ in sheep to that infection (Soulsby & Stewart, 1960). Examples of in vitro produced helminth material which are protective are those reported by Silverman et al. (!962), Crandal! & Arean (!965), Guerrero & Silverman (1971) and Rothwell & Love (1974). Stromberg & Soulsby (1977) reported the protection inducing capacity of an immunogen obtained by culturing third-stage larvae of Ascaris suum through the third moult to the fourth stage. The development of a defined low molecular weight culture medium (Stromberg, Khoury & Soulsby, 1977) has allowed further study of this immunogen. This study reports the isolation and partial characterization of the protective antigen produced in vitro by developing larvae of A. suum. 307
Experimental
animals.
excretory-secretory
AND METHODS
Protection was assessed in female outbred Hartly strain guinea pigs (550-600 g). Female New Zealand white rabbits (4 kg) were used to provide third-stage larvae for culture and for producing antisera. A!! animals were purchased from Skippack Farms, Skippack, PA and were fed a commercial diet and water ad lib. Production of culture fluid antigen. Third-stage larvae of A. suum were cultured to the fourth stage in a defined medium as previously described (Stromberg ef al., 1977). Briefly, third-stage larvae recovered from the lungs of rabbits were sterilized and then cultured in medium 199, supplemented with glucose, glycyl-histidyl-lysine, penicillin and streptomycin in an atmosphere of N,:O,:CO, (90:5:5) for 7 days. The larvae were then removed by filtration and the culture medium washed by diafiltration with 0.15 M-NaC! (saline) and concentrated ( x 20) on an Amicon Filter (Mode! 52, Amicon Inc., Lexington, MA) using a YM 10 membrane (IO,000 molecular weight exclusion limit). The fraction with a molecular weight > 10,000 will be referred to as the SD fraction. The effluent was concentrated to a volume equal to that of the SD fraction using a UM2 membrane (2000 molecular weight exclusion limit) and is referred to as the UM fraction (200&!0,000 molecular weight). Assessment of protection. Groups of at least six guinea pigs were immunized with 0.1 ml of the culture fluid antigens (SD, 31 pg and/or UM, 10 pg protein), control culture medium or saline, each emulsified with an equal volume of complete Freund’s adjuvant (CFA) and injected intramuscularly into the muscle of the hind legs.
308
BERTE.
STROMBERG
days later the animals, while under halothane anesthesia, were challenged with 5000 artificially hatched larvae from infective eggs of A. ~uurn injected into the mesenteric vein. Protection was assessed by comparing the recovery of third-stage larvae from the lungs of experimental vs control animals 6 days after challenge as previously described (Stromberg & Soulsby, 1976). In one experiment two immunizing injections of culture fluid antigen (SD, 31 pg protein) in CFA were given intramuscularly at day 0 and 14; animals were challenged on day 24 and protection was assessed on day 30. Prior to statistical analysis all data on larval recovery were transformed with the arc-sin conversion to obtain normally distributed data. All experimental groups were compared with the controls (culture medium and/or saline) using Student’s t test or the Fisher-Behrens modified t test when there was inequality of the sample variances. A probability value (P) of 0.05 or less was considered to constitute a statistical significance between groups. Producfion of specific nntiseru. Rabbits were immunized by the injection of the two fractions of culture fluid antigen (SD and UM, 41 pg protein) in CFA into the footpads; 30 days later the rabbits were given an intravenous boost of the antigen fractions (41 pg) and were bled 10 days later. Subsequently, these rabbits were boosted as needed by a subcutaneous injection of the antigens and bled 10 days after the boosting dose. Antiserum to an A. suum infection was produced in rabbits by giving three doses of 10,000 embryonated eggs of A. sum per OS at 21 day intervals. The animals were exsanguinated 10 days after the last infection. Anfigen analysis and separation. The protein concentration of the SD fraction was determined by the method of Lowry, Rosebrough, Farr & Randall (1951). The carbohydrate concentration was determined utilizing the Anthrone technique (Kabat & Mayer, 1961). The dry weight of the SD fraction was determined by dialysizing against distilled water and then lyophilizing in a tared weighing vial which was then brought to constant weight inadessicator. Electrophoresis was performed on cellulose acetate in a Beckman Microzone chamber at 250 V for 30 min. The electrophoretogram was stained with
Ten
I.J.P. VOL.
9. 1979
Coomassie Brilliant Blue R 0.5 % in 2% acetic acid. The SD fraction was separated by ascending flow gel filtration chromatography on a colu&m of Sephadex 6200 (2.4 >: 200 cm) using 0.15 M-DhosDhate buffered saline. DH 7.2 as the eluting buffer. ?he effluent was monitoreh’with a U.V. monitor (ISCO, Lincoln, Nebraska) at 280 nm and collected in 10 ml fractions. The molecular weight determinations were performed by Sephadex G75 gel separation (Andrews, 1964). The elution volume of the SD fraction was compared with the elution volumes of known protein markers; bovine serum albumin, ovalbumin, chymotrypsin, ribonuclease and cytochrone c. Ouchterlonv gel double diffusion was performed in agarose (Seakim, Marine Colloids Inc., Rbckland, ME)
in 0.06 M-Tris-barbital buffer, pH 8.8. The wells were cut I cm apart, charged and allowed to develop at room temperature for 48 h. Immunoelectrophoresis was performed in agarose. The antigen was separated by electrophoresing for I h at 175 V and an additional hour at
110 V. Troughs were then charged with antiserum and allowed to develop at room temperature for 48 h. Active cutaneous anaphylaxis was performed by injecting 0.1 cm3 of the SD, UM or the G200 fractions intradermally in the back of guinea pigs which had been immunized previously with seven biweekly subcutaneous injections of eggs of A. sum (Soulsby, 1957). The diameter of the wheal and erythema were measured 30 min after injection. Duplicate animals were injected with 0.5 cm3 of 3 % Evans Blue dye intravenously at the time of the intradermal injection and the area of blueing was measured. The reactions were recorded on a - to 4+ basis according to the size and intensity of the reaction. RESULTS The fractions
protection
inducing
of culture
fluid
capacity
are
of
the
two
presented
in Table 1. Statistically significant resistance was observed when guinea pigs were immunized with 31 pg protein of the SD fraction (> 10,000 MW) and little if any protection was induced with 10 pg protein of the low molecular weight fraction (UM fraction).
TABLE I-MEAN PER CENT RECOVERY* OF THIRD STAGE LARVAE OF Ascuris mum FROM THE LUNGS OF GUINEA PIGS SENSITIZED BY INTRAMUSCULAR INJECTION OF FRACTIONS OF THE EXCRETORY-SECRETORY PRODUCTS OF LARVAE OF A. SUUM DEVELOPING FROM THE THIRD TO THE FOURTH LARVAL STAGE, in Vitro AND CHALLENGED WITH 5000 SECOND STAGE LARVAE OF A. suum BY MESENTERIC VEIN INJECTION Experiment
Group
I 1 2 3 4 II 1 2 3 III 1 2
Treatment
Mean
Diafiltration fractions UM Fraction (6)$ SD Fraction (I I) Medium II (7) Saline (7) G-200 Sephadex fractions Peak 1 (6) Peak 2 (6) Saline (6) SD Antigen administered SD x 2 (6) Saline (7)
*Arc-sin transformed data. tS.E. = Standard error of the mean. iNumbers in parentheses are the number $N.S. = Not significant. IlMedium = Culture medium control.
23.34 17.05 22.11 23.91 16.25 19.52 19.49 twice 10.42 16.42
of animals
i
S.E.I_
P Values
1.85 2.14 1.93 3.95
N.S. 0.05 N.S.
i 1.41 & 2.01 h 1.96
0.05 N.S.
I i
0.025 -
k * + +
0.27 0.45
in the group.
I.J.P. VOL.9. 1979
Isolation of a protective antigen of Ascuris ~urirn
FIG. 1. Immunodiffusion by the Ouchterlony technique. RS: rabbit antiserum to whole culture medium (SD and UM), UM: UM fraction and SD: SD fraction. FIG. 2. lmmunodiffusion by the Ouchterlony technique. Center well contains the SD fraction (SD) and the surrounding wells contain six rabbit antisera to whole culture medium (1 to 6).
When these two fractions were examined with gel precipitation using antiserum prepared against the whole culture medium; there were no precipitin bands formed with the UM fraction and only one band with the SD fraction (Fig. 1). None of the six antisera to the whole culture medium recognized any antigen in the UM fraction, but all showed a single precipitin band when reacted with the SD fraction which demonstrated identity (Fig. 2). One band was observed when antisera to an A. suum infection was diffused against the SD fraction. After freezing and thawing or standing at room temperature for 8 h the SD fraction showed two additional minor bands when reacted with these specific antisera. The results of immunoelectro-
.20
Peak
c
I
2
3
4
5
6
phoresis were identical to the gel double diffusion studies and indicated that the single SD fraction antigen migrated slightly toward the anode during electrophoresis. The SD fraction produced a 4f reaction in active cutaneous anaphylaxis while the UM fraction did not elicit any response. Cellulose acetate electrophoresis also demonstrated a single substance in the SD fraction while nothing was observed in the UM fraction. Because the UM fraction could not induce resistance and did not react with immune sera when tested by electrophoresis, active cutaneous anaphylaxis or immunodiffusion no further studies were carried out on this fraction. The remaining studies were performed on the SD fraction only.
I
Peak
7
8
9
IO II
2
12 13 14 15
Fraction
FIG. 3. Optical density of the elution of the SD fraction from Sephadex G-200 and histograms
active cutaneous anaphylaxis reactions (ACA) for the fractions of the separation.
of the
310
BERT E. STROMBERG
TO isolate this antigen further it was fractionated on Sephadex G200 and produced an elution pattern composed of two peaks illustrated in Fig. 3. These fractions were reacted with the culture fluid specific antiserum in gel diffusion and a precipitin band, which showed identity was formed with fractions 4-8. These fractions were tested for their ability to induce an immediate type hypersensitive reaction in the skin of hyper immune guinea pigs, fractions 4-9 being reactive as indicated by the histograms in Fig. 3. This indicates that the antigenic portion of the SD fraction is found in the first peak; the second peak was identified as medium 199 which had not been completely removed with diafiltration. The two peaks were used to immunize guinea pigs to evaluate their protection inducing capacity and it was found that peak 1 contained all of the protective antigens (Table 1). This was shown by peak 1 inducing significant resistance to a challenge infection, while peak 2 was unable to induce any resistance. When two immunizing doses of the SD fraction were given, there was a stronger resistance to a challenge infection as illustrated in Table I. The dry weight determination was found to be 390 ug/ml. The Lowry technique for the estimation of protein demonstrated 3 10 ug/ml which is 79 “/, of the total SD fraction. The anthrone reaction indicated that there was 88 ug/ml of carbohydrate (22 % of the total). When the SD fraction was chromatographed on G75 with several reference substances, the molecular weight was estimated to be about 67,000. DISCUSSION Immunization against parasitic nematodes with non-viable preparations has generally not been very successful. Several investigators have reported limited success in inducing resistance to an A. suu?n infection with extracts or excretory-secretory products of adults or various developmental stages (Soulsby, 1963; Crandall & Arean, 1965; Leikina, 1965; Guerrero & Silverman, 1969, 1971). Stromberg & Soulsby (1977) found an antigen produced in culture which would induce protection when given intramuscularly in CFA. This protective material was present only in cultures where development had taken place (moult from Ls to L,) and it was postulated that the antigen was involved with the moulting process. Perhaps this antigen is a ‘functional antigen’, i.e. one in which the host responds with a protective response to a substance involved in a vital physiological process of the parasite. The development of a defined culture medium to support the growth and moult of third stage larvae (Stromberg et al., 1977) has made it possible to isolate the parasite produced product which was responsible for protection. The initial fractionation of the culture fluid antigen was on the basis of molecular weight using an Amicon filter. Two
I.J.P. VOL.
9. 1979
fractions, the SD (> 10,000) and the UM (200010,000) were collected and assessed for their ability to induce a protective response in the guinea pig. The SD fraction induced significant protection, while the UM fraction showed no activity. The SD fraction produced a single precipitin band in gel diffusion when reacted with the antisera against whole culture antigen and A. SUNS egg infection. The SD fraction also induced an active cutaneous anaphylactic response in sensitized guinea pigs. The UM fraction did not demonstrate any activity in these tests and it was concluded that it did not contain any parasite products of immunological importance. The SD fraction produced only a single precipitin band with six different antisera raised against the whole culture antigen, thus indicating that there was most likely only one immunogen present. Similarly all samples of the SD fraction demonstrated identity when reacted with antiserum to the antigen in gel diffusion. Therefore, the SD fraction will be designated as the Ascavis culture fluid (ACF) antigen. The fact that only a single immunogen was produced by actively growing and moulting larvae may be due to one of the following reasons: excretions and secretions other than those associated with moulting may be very low molecular weight and therefore not immunogenic, such ES products may be present in amounts too small to be detected by the techniques used or during the moulting process the worm may expend most of its energy in this process and therefore producing little other matabolites at the time. Other antigens obtained from A. suum have been described in some detail. One of the first was an allergen isolated by gel filtration of the perienteric fluid from adult worms by Hogarth-Scott (1967). Ambler, Doe, Gemmell, Roberts & Orr (1972) and Hussain, Strejan & Campbell (1972) have described Ascavis allergen A and allergen ASC 1, respectively from extracts of adult worms. Herzig (1974) has also described an allergen from an extract of adult A. suum. More recently Kuo & YOO (1977) described another allergen from the perienteric fluid of freshly collected worms. Only Hussain et al. (1972) and Hussain, Bradbury & Strejan (1973) described their antigen(s) as being both an allergen and an immunogen (produce precipitating antibody) while the others have only evaluated the ability of the antigen to provoke an allergic reaction. The ACF antigen was able to induce a significant protective immune response as well as t0 provoke an allergic response in sensitized animals. In addition this antigen was able to induce a strong IgE response in guinea pigs with reciprocal titers as high as 50,000 on secondary stimulation when given intraperitoneally (Stromberg, in preparation). The ACF antigen was obtained in fairly pure form directly from culture as was indicated by
I.J.P. VOL. 9. 1979
311
Isolation of a protective antigen of Ascaris swum
Amicon and gel fractionation and electrophoresis. This antigen was similar to Allergen A and Asc 1 of Ambler et al. (1972) and Ambler, Miller, Johnson & Orr (1973) and Hussain et al. (1972) in being primarily protein, however the ACF antigen contained a relatively high proportion of carbohydrate (22%). The carbohydrate content is less than the 45.7% reported for an aqueous extract of A. suum by Kent (1963). Its molecular weight, as estimated by gel filtration, is 67,000 which is considerably higher than that reported for Allergen A or Asc 1, 14,000 and 18,000 respectively (Ambler et al., 1973; Hussain et al., 1973). The high molecular weight may suggest the ACF antigen is a polymer or made of several components and this may explain the occurrence of additional precipitin bands in gel diffusion after the antigen was freezethawed or remained at room temperature for 8 h. These results described an antigen which is associated with a physiological event in the parasite and can induce protective immunity. It can be obtained from defined culture medium which should aid its further analysis. This antigen may help to evaluate more fully the protective immune response in an A. suum infection as well as leading to a better understanding of the cellular and humoral responses to parasite products. author expresses his appreciation to Professor E. J. L. Soulsby for his valuable advice throughout the course of this study. The author would like to thank Miss Antonia Cauobianco. Mrs. Rosetta Goss, Miss Norma Molina, Mrs. Ingrid Tabani and Mr. Derek Muncey for their valuable technical assistance. This work was supported by Research Grant AI 06262.
Acknowledgements-The
HERZIG D. J. 1974. Ascaris sensitivity in the dog-I. Isolation and characterization of an antigen. Journal of Immunological
Methods
5: 219-228.
HOGARTH-SCOTT R. S. 1967. The molecular weight range of nematode allergens. Immunology 13: 535-537. HUSSAIN R., STREJAN G. & CAMPBELLD. H. 1972. Hypersensitivity to Ascaris antigens-VII. Isolation and partial characterization of an antigen. Journal of Immunology
109: 638-647.
HUSSAIN R., BRADBURYS. M. & STREJAN G. 1973. Hypersensitivity to Ascaris antigens-VIII. Characterization of a highly purified allergen. Journal of Immunology
111: 260-268.
KABATE. A. & MAYERM. M. 1961. Kabat and Mayer’s Experimental Immunochemistry. Thomas, Springfield. KENT H. N. 1963. Seminar on immunity to parasitic helminths-V. Antigens. Experimental Parasitology 13: 45-56.
Kuo C. Y. & Yoo T. J. 1977. A new allegren from the perienteric fluid of Ascaris suum with respect to charges. International Archives of Allergy and Applied Immunology
54: 308-3 14.
LEIKINA E. S. 1965. Research on ascariasis immunity and immunodiagnosis. Bulletin of the World Health Organization
32: 699-708.
LOWRY0. H., ROSEBROUGHN.H., FARRA. L. & RANDALL R. J. 1951. Protein measurement with the Folin phenol reagent. Journal of Biochemistry 193: 265-275. ROTHWELLT. L. W. & LOVE R. J. 1974. Vaccination against the nematode Trichostrongylus colubriformisI. Vaccination of guinea-pigs with worm homogenates and soluble products released during in vitro maintenance. International Journal for Parasitology 4: 293-299.
SILVERMANP. H., POYNTERD. & PODGERK. R. 1962. Studies on larval antigen derived by cultivation of some parasitic nematodes in simple media: protection tests in laboratory animals. Journal of Parasitology 48: 562-571.
REFERENCES AMBLERJ., DOE J. E., GEMMELLD. K., ROBERTSJ. A. & ORR T. S. C. 1972. Biological techniques for studying the allergenic components of nematodes-I. Detection of allergenic components in Ascuris suum extracts. Journal of Immunological Methods 1: 317-328. AMBLERJ., MILLER J. N., JOHNSONP. & ORR T. S. C. 1973. Characterisation of an allergen extracted from Ascaris suutn. Determination of molecular weight, isoelectric point, amino acid and carbohydrate content of the native antigen. Immunochemistry 10: 815-820. ANDREWSP. 1964. Estimation of the molecular weights of proteins by Sephadex gel filtration. Biochemical Journal 91: 222-233.
CRANDALLC. A. & AREAN V. M. 1965. The protective effect of viable and nonviable Ascaris suum larvae and egg preparations in mice. American Journal of Tropical Medicine
and Hygiene
14: 765-769,
GUERREROJ. & SILVERMANP. H. 1969. Ascaris suum: Immune reactions in mice--I. Larval metabolic and somatic antigens. Experimental Parasitology 26: 272281.
GUERREROJ. & SILVERMANP. H. 1971. Ascaris suum: Immune reactions in mice-II. Metabolic and somatic antigens of in vitro cultured larvae. Experimental Parasitology 29: 110-I 15.
SOULSBYE. J. L. 1957. Immunization against Ascaris lumbricoides in the guinea pig. Nature (Lond.) 179: 783-784.
SOULSBYE. J. L. 1963. The nature and origin of the functional antigens in helminth infections. Annuls of the New York Academy
of Science 113: 492-509.
SOULSBYE. J. L. & STEWARTD. F. 1960. Serological studies of the self-cure reaction in sheep infected with Haemonchus contortus. Australian cultural Research 11: 595-603.
Journal
of Agri-
STROMBERG B. E. & SOULSBYE. J. L. 1976. The protective effect of developmental stages in an Ascaris suum infection in the guinea pig. Veterinary Parasitology 2: 197-208. STROMBERG B. E. & SOULSBYE. J. L. 1977. Ascaris suum: immunization with soluble antigens in the guinea pig. International
Journal for Parasitology
7: 287-291.
STROMBERG B. E., KHOURYP. B. & SOULSBYE. J. L. 1977. Development of larvae of Ascaris suum from the third to the fourth stage in a chemically defined medium. International
Journal for Parasitology
7: 149-l 51.
ZIMMERMANG. L. & LELANDS. E. 1974. Isolation and partial characterization of exo- and endoantigens of Cooperia punctafa cultured in vitro. Journal of Parasitology
60: 794-803.
II.
0020-7519/79/0801-0307$02.00/O
THE ISOLATION AND PARTIAL CHARACTERIZATION OF A PROTECTIVE ANTIGEN FROM DEVELOPING LARVAE OF ASCARIS SUUM BERT
Department
of Pathobiology,
E. STROMBERG
School of Veterinary Medicine HI, University of Pennsylvania, Philadelphia, PA 19104, U.S.A. (Received 9 August 1978)
B. E. 1979. The isolation and partial characterization of a protective antigen from developing larvae of Asearis suum. International Iournal for Parasitology 9: 307-3 I I. An antigen (ACF antigen) obtained by in vitro cultivation of third-stage larvae of Ascaris suum to the fourth stage in defined medium induced significant protection in guinea pigs. The antigen was further characterized by ultrafiltration and gel filtration and was shown to be a single component by gel precipition and immunoelectrophoresis. It induced active cutaneous anaphylaxis in sensitized animals. The ACF antigen is estimated to contain about 79% protein and 22% carbohydrate and to have a molecular weight of approx 67,000.
Abstract-STROMBERG
INDEX KEY WORDS: Ascaris suum; protective product; functional antigen; vaccination.
immunity;
INTRODUCTION
soluble antigens;
MATERIALS
In vitro cultivation of helminth parasites has been used by several investigators to obtain worm immunogens (Silverman, Poynter & Podger, 1962; Soulsby, 1963; Zimmerman & Leland, 1974; and Rothwell & Love, 1974). Such immunogens may induce protection against a homologous challenge infection and may be considered as protective antigens. Perhaps some might be considered as ‘functional antigens’ in that they serve a physiological function in the parasites as we!! as inducing an immune response which significantly affects the parasite (i.e. the exsheathing fluid of Huemonchus contortus is capable of inducing ‘self cure’ in sheep to that infection (Soulsby & Stewart, 1960). Examples of in vitro produced helminth material which are protective are those reported by Silverman et al. (!962), Crandal! & Arean (!965), Guerrero & Silverman (1971) and Rothwell & Love (1974). Stromberg & Soulsby (1977) reported the protection inducing capacity of an immunogen obtained by culturing third-stage larvae of Ascaris suum through the third moult to the fourth stage. The development of a defined low molecular weight culture medium (Stromberg, Khoury & Soulsby, 1977) has allowed further study of this immunogen. This study reports the isolation and partial characterization of the protective antigen produced in vitro by developing larvae of A. suum. 307
Experimental
animals.
excretory-secretory
AND METHODS
Protection was assessed in female outbred Hartly strain guinea pigs (550-600 g). Female New Zealand white rabbits (4 kg) were used to provide third-stage larvae for culture and for producing antisera. A!! animals were purchased from Skippack Farms, Skippack, PA and were fed a commercial diet and water ad lib. Production of culture fluid antigen. Third-stage larvae of A. suum were cultured to the fourth stage in a defined medium as previously described (Stromberg ef al., 1977). Briefly, third-stage larvae recovered from the lungs of rabbits were sterilized and then cultured in medium 199, supplemented with glucose, glycyl-histidyl-lysine, penicillin and streptomycin in an atmosphere of N,:O,:CO, (90:5:5) for 7 days. The larvae were then removed by filtration and the culture medium washed by diafiltration with 0.15 M-NaC! (saline) and concentrated ( x 20) on an Amicon Filter (Mode! 52, Amicon Inc., Lexington, MA) using a YM 10 membrane (IO,000 molecular weight exclusion limit). The fraction with a molecular weight > 10,000 will be referred to as the SD fraction. The effluent was concentrated to a volume equal to that of the SD fraction using a UM2 membrane (2000 molecular weight exclusion limit) and is referred to as the UM fraction (200&!0,000 molecular weight). Assessment of protection. Groups of at least six guinea pigs were immunized with 0.1 ml of the culture fluid antigens (SD, 31 pg and/or UM, 10 pg protein), control culture medium or saline, each emulsified with an equal volume of complete Freund’s adjuvant (CFA) and injected intramuscularly into the muscle of the hind legs.
308
BERTE.
STROMBERG
days later the animals, while under halothane anesthesia, were challenged with 5000 artificially hatched larvae from infective eggs of A. ~uurn injected into the mesenteric vein. Protection was assessed by comparing the recovery of third-stage larvae from the lungs of experimental vs control animals 6 days after challenge as previously described (Stromberg & Soulsby, 1976). In one experiment two immunizing injections of culture fluid antigen (SD, 31 pg protein) in CFA were given intramuscularly at day 0 and 14; animals were challenged on day 24 and protection was assessed on day 30. Prior to statistical analysis all data on larval recovery were transformed with the arc-sin conversion to obtain normally distributed data. All experimental groups were compared with the controls (culture medium and/or saline) using Student’s t test or the Fisher-Behrens modified t test when there was inequality of the sample variances. A probability value (P) of 0.05 or less was considered to constitute a statistical significance between groups. Producfion of specific nntiseru. Rabbits were immunized by the injection of the two fractions of culture fluid antigen (SD and UM, 41 pg protein) in CFA into the footpads; 30 days later the rabbits were given an intravenous boost of the antigen fractions (41 pg) and were bled 10 days later. Subsequently, these rabbits were boosted as needed by a subcutaneous injection of the antigens and bled 10 days after the boosting dose. Antiserum to an A. suum infection was produced in rabbits by giving three doses of 10,000 embryonated eggs of A. sum per OS at 21 day intervals. The animals were exsanguinated 10 days after the last infection. Anfigen analysis and separation. The protein concentration of the SD fraction was determined by the method of Lowry, Rosebrough, Farr & Randall (1951). The carbohydrate concentration was determined utilizing the Anthrone technique (Kabat & Mayer, 1961). The dry weight of the SD fraction was determined by dialysizing against distilled water and then lyophilizing in a tared weighing vial which was then brought to constant weight inadessicator. Electrophoresis was performed on cellulose acetate in a Beckman Microzone chamber at 250 V for 30 min. The electrophoretogram was stained with
Ten
I.J.P. VOL.
9. 1979
Coomassie Brilliant Blue R 0.5 % in 2% acetic acid. The SD fraction was separated by ascending flow gel filtration chromatography on a colu&m of Sephadex 6200 (2.4 >: 200 cm) using 0.15 M-DhosDhate buffered saline. DH 7.2 as the eluting buffer. ?he effluent was monitoreh’with a U.V. monitor (ISCO, Lincoln, Nebraska) at 280 nm and collected in 10 ml fractions. The molecular weight determinations were performed by Sephadex G75 gel separation (Andrews, 1964). The elution volume of the SD fraction was compared with the elution volumes of known protein markers; bovine serum albumin, ovalbumin, chymotrypsin, ribonuclease and cytochrone c. Ouchterlonv gel double diffusion was performed in agarose (Seakim, Marine Colloids Inc., Rbckland, ME)
in 0.06 M-Tris-barbital buffer, pH 8.8. The wells were cut I cm apart, charged and allowed to develop at room temperature for 48 h. Immunoelectrophoresis was performed in agarose. The antigen was separated by electrophoresing for I h at 175 V and an additional hour at
110 V. Troughs were then charged with antiserum and allowed to develop at room temperature for 48 h. Active cutaneous anaphylaxis was performed by injecting 0.1 cm3 of the SD, UM or the G200 fractions intradermally in the back of guinea pigs which had been immunized previously with seven biweekly subcutaneous injections of eggs of A. sum (Soulsby, 1957). The diameter of the wheal and erythema were measured 30 min after injection. Duplicate animals were injected with 0.5 cm3 of 3 % Evans Blue dye intravenously at the time of the intradermal injection and the area of blueing was measured. The reactions were recorded on a - to 4+ basis according to the size and intensity of the reaction. RESULTS The fractions
protection
inducing
of culture
fluid
capacity
are
of
the
two
presented
in Table 1. Statistically significant resistance was observed when guinea pigs were immunized with 31 pg protein of the SD fraction (> 10,000 MW) and little if any protection was induced with 10 pg protein of the low molecular weight fraction (UM fraction).
TABLE I-MEAN PER CENT RECOVERY* OF THIRD STAGE LARVAE OF Ascuris mum FROM THE LUNGS OF GUINEA PIGS SENSITIZED BY INTRAMUSCULAR INJECTION OF FRACTIONS OF THE EXCRETORY-SECRETORY PRODUCTS OF LARVAE OF A. SUUM DEVELOPING FROM THE THIRD TO THE FOURTH LARVAL STAGE, in Vitro AND CHALLENGED WITH 5000 SECOND STAGE LARVAE OF A. suum BY MESENTERIC VEIN INJECTION Experiment
Group
I 1 2 3 4 II 1 2 3 III 1 2
Treatment
Mean
Diafiltration fractions UM Fraction (6)$ SD Fraction (I I) Medium II (7) Saline (7) G-200 Sephadex fractions Peak 1 (6) Peak 2 (6) Saline (6) SD Antigen administered SD x 2 (6) Saline (7)
*Arc-sin transformed data. tS.E. = Standard error of the mean. iNumbers in parentheses are the number $N.S. = Not significant. IlMedium = Culture medium control.
23.34 17.05 22.11 23.91 16.25 19.52 19.49 twice 10.42 16.42
of animals
i
S.E.I_
P Values
1.85 2.14 1.93 3.95
N.S. 0.05 N.S.
i 1.41 & 2.01 h 1.96
0.05 N.S.
I i
0.025 -
k * + +
0.27 0.45
in the group.
I.J.P. VOL.9. 1979
Isolation of a protective antigen of Ascuris ~urirn
FIG. 1. Immunodiffusion by the Ouchterlony technique. RS: rabbit antiserum to whole culture medium (SD and UM), UM: UM fraction and SD: SD fraction. FIG. 2. lmmunodiffusion by the Ouchterlony technique. Center well contains the SD fraction (SD) and the surrounding wells contain six rabbit antisera to whole culture medium (1 to 6).
When these two fractions were examined with gel precipitation using antiserum prepared against the whole culture medium; there were no precipitin bands formed with the UM fraction and only one band with the SD fraction (Fig. 1). None of the six antisera to the whole culture medium recognized any antigen in the UM fraction, but all showed a single precipitin band when reacted with the SD fraction which demonstrated identity (Fig. 2). One band was observed when antisera to an A. suum infection was diffused against the SD fraction. After freezing and thawing or standing at room temperature for 8 h the SD fraction showed two additional minor bands when reacted with these specific antisera. The results of immunoelectro-
.20
Peak
c
I
2
3
4
5
6
phoresis were identical to the gel double diffusion studies and indicated that the single SD fraction antigen migrated slightly toward the anode during electrophoresis. The SD fraction produced a 4f reaction in active cutaneous anaphylaxis while the UM fraction did not elicit any response. Cellulose acetate electrophoresis also demonstrated a single substance in the SD fraction while nothing was observed in the UM fraction. Because the UM fraction could not induce resistance and did not react with immune sera when tested by electrophoresis, active cutaneous anaphylaxis or immunodiffusion no further studies were carried out on this fraction. The remaining studies were performed on the SD fraction only.
I
Peak
7
8
9
IO II
2
12 13 14 15
Fraction
FIG. 3. Optical density of the elution of the SD fraction from Sephadex G-200 and histograms
active cutaneous anaphylaxis reactions (ACA) for the fractions of the separation.
of the
310
BERT E. STROMBERG
TO isolate this antigen further it was fractionated on Sephadex G200 and produced an elution pattern composed of two peaks illustrated in Fig. 3. These fractions were reacted with the culture fluid specific antiserum in gel diffusion and a precipitin band, which showed identity was formed with fractions 4-8. These fractions were tested for their ability to induce an immediate type hypersensitive reaction in the skin of hyper immune guinea pigs, fractions 4-9 being reactive as indicated by the histograms in Fig. 3. This indicates that the antigenic portion of the SD fraction is found in the first peak; the second peak was identified as medium 199 which had not been completely removed with diafiltration. The two peaks were used to immunize guinea pigs to evaluate their protection inducing capacity and it was found that peak 1 contained all of the protective antigens (Table 1). This was shown by peak 1 inducing significant resistance to a challenge infection, while peak 2 was unable to induce any resistance. When two immunizing doses of the SD fraction were given, there was a stronger resistance to a challenge infection as illustrated in Table I. The dry weight determination was found to be 390 ug/ml. The Lowry technique for the estimation of protein demonstrated 3 10 ug/ml which is 79 “/, of the total SD fraction. The anthrone reaction indicated that there was 88 ug/ml of carbohydrate (22 % of the total). When the SD fraction was chromatographed on G75 with several reference substances, the molecular weight was estimated to be about 67,000. DISCUSSION Immunization against parasitic nematodes with non-viable preparations has generally not been very successful. Several investigators have reported limited success in inducing resistance to an A. suu?n infection with extracts or excretory-secretory products of adults or various developmental stages (Soulsby, 1963; Crandall & Arean, 1965; Leikina, 1965; Guerrero & Silverman, 1969, 1971). Stromberg & Soulsby (1977) found an antigen produced in culture which would induce protection when given intramuscularly in CFA. This protective material was present only in cultures where development had taken place (moult from Ls to L,) and it was postulated that the antigen was involved with the moulting process. Perhaps this antigen is a ‘functional antigen’, i.e. one in which the host responds with a protective response to a substance involved in a vital physiological process of the parasite. The development of a defined culture medium to support the growth and moult of third stage larvae (Stromberg et al., 1977) has made it possible to isolate the parasite produced product which was responsible for protection. The initial fractionation of the culture fluid antigen was on the basis of molecular weight using an Amicon filter. Two
I.J.P. VOL.
9. 1979
fractions, the SD (> 10,000) and the UM (200010,000) were collected and assessed for their ability to induce a protective response in the guinea pig. The SD fraction induced significant protection, while the UM fraction showed no activity. The SD fraction produced a single precipitin band in gel diffusion when reacted with the antisera against whole culture antigen and A. SUNS egg infection. The SD fraction also induced an active cutaneous anaphylactic response in sensitized guinea pigs. The UM fraction did not demonstrate any activity in these tests and it was concluded that it did not contain any parasite products of immunological importance. The SD fraction produced only a single precipitin band with six different antisera raised against the whole culture antigen, thus indicating that there was most likely only one immunogen present. Similarly all samples of the SD fraction demonstrated identity when reacted with antiserum to the antigen in gel diffusion. Therefore, the SD fraction will be designated as the Ascavis culture fluid (ACF) antigen. The fact that only a single immunogen was produced by actively growing and moulting larvae may be due to one of the following reasons: excretions and secretions other than those associated with moulting may be very low molecular weight and therefore not immunogenic, such ES products may be present in amounts too small to be detected by the techniques used or during the moulting process the worm may expend most of its energy in this process and therefore producing little other matabolites at the time. Other antigens obtained from A. suum have been described in some detail. One of the first was an allergen isolated by gel filtration of the perienteric fluid from adult worms by Hogarth-Scott (1967). Ambler, Doe, Gemmell, Roberts & Orr (1972) and Hussain, Strejan & Campbell (1972) have described Ascavis allergen A and allergen ASC 1, respectively from extracts of adult worms. Herzig (1974) has also described an allergen from an extract of adult A. suum. More recently Kuo & YOO (1977) described another allergen from the perienteric fluid of freshly collected worms. Only Hussain et al. (1972) and Hussain, Bradbury & Strejan (1973) described their antigen(s) as being both an allergen and an immunogen (produce precipitating antibody) while the others have only evaluated the ability of the antigen to provoke an allergic reaction. The ACF antigen was able to induce a significant protective immune response as well as t0 provoke an allergic response in sensitized animals. In addition this antigen was able to induce a strong IgE response in guinea pigs with reciprocal titers as high as 50,000 on secondary stimulation when given intraperitoneally (Stromberg, in preparation). The ACF antigen was obtained in fairly pure form directly from culture as was indicated by
I.J.P. VOL. 9. 1979
311
Isolation of a protective antigen of Ascaris swum
Amicon and gel fractionation and electrophoresis. This antigen was similar to Allergen A and Asc 1 of Ambler et al. (1972) and Ambler, Miller, Johnson & Orr (1973) and Hussain et al. (1972) in being primarily protein, however the ACF antigen contained a relatively high proportion of carbohydrate (22%). The carbohydrate content is less than the 45.7% reported for an aqueous extract of A. suum by Kent (1963). Its molecular weight, as estimated by gel filtration, is 67,000 which is considerably higher than that reported for Allergen A or Asc 1, 14,000 and 18,000 respectively (Ambler et al., 1973; Hussain et al., 1973). The high molecular weight may suggest the ACF antigen is a polymer or made of several components and this may explain the occurrence of additional precipitin bands in gel diffusion after the antigen was freezethawed or remained at room temperature for 8 h. These results described an antigen which is associated with a physiological event in the parasite and can induce protective immunity. It can be obtained from defined culture medium which should aid its further analysis. This antigen may help to evaluate more fully the protective immune response in an A. suum infection as well as leading to a better understanding of the cellular and humoral responses to parasite products. author expresses his appreciation to Professor E. J. L. Soulsby for his valuable advice throughout the course of this study. The author would like to thank Miss Antonia Cauobianco. Mrs. Rosetta Goss, Miss Norma Molina, Mrs. Ingrid Tabani and Mr. Derek Muncey for their valuable technical assistance. This work was supported by Research Grant AI 06262.
Acknowledgements-The
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