Persisting antibody reaction in paragonimiasis after praziquantel treatment is elicited mainly by egg antigens

Abstract

Antibody responses in serum and cerebrospinal fluid (CSF) samples from patients with active and chronic paragonimiasis and in sera from patients on whom follow-up studies were done after praziquantel treatment were analyzed using antigens of Paragonimus westermani prepared from eggs, metacercariae, juveniles of 4- and 7-week old, adult worms and recombinant protein of 28 kDa cruzipain-like cysteine protease (rPw28CCP). The patient sera/CSFs of active and chronic paragonimiasis revealed strong antibody reactions against the crude extracts of 4- and 7-week old juveniles as well as against those from egg and adult. rPw28CCP also showed specific reaction to the sera with active paragonimiasis. After the treatment, levels of specific antibodies in the sera gradually decreased to negative range in most patients. In some cases with persisting high antibody levels, however, the reactions at 27 kDa egg protein were sustained throughout the observation period of 34 months. The reactions at 35 and 32 kDa in adult extract and rPw28CCP disappeared rapidly after the treatment. Persistent antibody reactions even after successful treatment are provoked by continuous antigenic challenge from eggs which were not resolved by treatment.

Fig. 1

Immunoblot findings of crude extracts of the metacercariae, 4- and 7-week old juvenile worms, 16-week old adult worms and egg with mAb (A) or rabbit antiserum immunized with adult Paragonimus extract (B). After transfer-blot of the proteins separated by 7.5-15% SDS-PAGE, culture supernatant (undiluted) or rabbit antiserum (1:5,000 dilution) was probed overnight. The mAb exhibits positive reactions only with the adult and egg extracts (lanes adult and egg) while multiple immunoreactive bands are shown in panel B which was probed with the rabbit anti-serum. mc^*, metacercariae; M_r, molecular mass in kDa.

Fig. 2

A declining pattern of specific antibody levels in follow-up tests against each differently prepared antigens of Paragonimus westermani assessed by ELISA, immunoblot and immunoprecipitation. (A) Typical changes of antibody levels after praziquantel treatment analyzed by ELISA. The patient sera, collected serially after chemotherapy on 2, 8 and 28 months, exhibit the declining pattern regardless of antigenic preparations. Abs. against extracts from metacercariae (○-○), 4-week (□-□), 7-week (▵-▵), adult (★-★) and eggs (▾-▾). (B) Each serum secured from the time of diagnosis, 8 and 28 months after treatment (lanes a-c) is reacted with antigens of metacercaria (panel mc), 4-week (panel 4), 7-week (panel 7) and adult worms (panel 16), eggs (panel egg) and the recombinant protein of 28-kDa cruzipain like cysteine protease (panel rec), respectively. Strong reactions at 35 and 32 kDa in 7-week and adult extracts at the time of diagnosis disappeared rapidly with the time lapse. Weak reaction against 27 kDa of egg protein is observed. The recombinant protein also showed positive reaction at the time of the diagnosis and shows no reactions in the follow-up sera. The sera used in this experiment is the same as in A. M_r, molecular mass in kDa. (C) Another case whose serum antibody titers shows similar declining pattern as in A. Markings are the same as in A. (D) The follow-up sera in C is immunoprecipitated with adult extract and probed with mAb specific to egg protein. Specific antibody reactions at 27 kDa in egg protein (▸) become fainter and disappeared 8 month after treatment.

Fig. 3

Another changing pattern of serum levels of anti-Paragonimus westermani after praziquantel treatment. The specific antibody levels in the patient sera persisted. (A) The patient sera, collected serially 3, 6, 11, 15 and 23 months after treatment show high antibody levels against the egg and adult extracts while those against other antigenic preparations decreased more promptly. Markings are same as in Fig. 2. (B) The follow-up sera of A reacted with different antigens by immunoblot. Each serum collected at the time of diagnosis, 6, 15 and 23 months post-treatment (lanes a-d) is reacted with different crude extracts as shown in Fig. 2B. Strong reactions at 35 and 32 kDa in 7-week and adult extracts disappeared rapidly within 15 months follow-up; reactions against egg protein, at 27 kDa protein sustained throughout the follow-up period of 23 months. The reaction against recombinant protein also disappear quickly. (C) Sera from another patient collected serially 2, 10, 15, 22, 27 and 34 months after successful treatment show high antibody reactions against the egg extract. Markings are the same as described in Fig. 2. M_r, molecular mass in kDa. (D) Immunoprecipitation analysis of the follow-up sera of a case presented in Fig. 3C. Reactions at 94 and 46 kDa faded out within 10 months after treatment (open arrowheads) while the reaction at 27 kDa persisted throughout the observation period of 34 months (closed arrowhead).