Acta Tropica, 50(1992)353-356
353
© 1992 Elsevier SciencePublishers B.V. All rights reserved 0001-706X/92/$05.00 ACTROP 00190
Short Communication
Antigenic mimicry between piperazine derivatives and Wuchereria bancrofti microfilariae Tahziba Hussain and Balachandran Ravindran Department of lmmunology, Regional Medical Research Centre (I.C.M.R.), Bhubaneswar, India
(Received 17 July 1991; accepted 22 October 1991) Key words: Wucheria bancro~i; Lymphaticfilariasis; Diethylcarbamazine;Antigenicmimicry
Studies performed earlier in our laboratory indicated that antibodies with reactivity to the antifilarial drug, Diethylcarbamazine (DEC) react with microfilariae of Wuchereria bancrofti (Ravindran et al., 1988). The antibodies were raised in rabbits using as hapten, methyl piperazine carboxylic acid (MPCA), an acid hydrolysis product of DEC (Ravindran et al., 1987). The anti-MPCA antibodies not only reacted very strongly with DEC, they also reacted with microfilarial sheath of W. bancrofti. The reactivity to the parasite could be effectively inhibited by preincubation of the antisera with DEC. These observations indicated a possible antigenic similarity between the antifilarial drug DEC and the microfilarial surface of W. bancrofti. In this communication we report the results of our attempts to raise antibodies to two other piperazine derivatives, piperidine carboxylic acid (PCA) and piperidine propionic acid (PPA) with a view to study their reactivity to the parasite. The structures of the various haptens used in the investigation are shown in Fig. 1. MPCA was prepared by acid hydrolysis of DEC using 70% H2SO4 as described elsewhere (Ravindran et al., 1987), PPA (Cat No. 26371-07-3) and PCA (Cat No. 498-94 2) were procured from Aldrich Chemical Co. Inc., U.S.A. The haptens were conjugated with tetanus toxoid (TT) by carbodiimide reaction using 1-ethyl-3(3-dimethylaminoprophyl) carbodiimide (EDCI) and the coupling efficiency was tested by trinitrobenzene sulphonic acid assay (Ravindran et al., 1987). Three rabbits were each immunized with TT-MPCA, TT-PCA or TT-PPA. Two doses at 15 days interval were administered intramuscularly, the first (1.5 mg) with Freund's complete adjuvant (CFA) and the second (0,75 mg) with Freund's incomplete adjuvant (FICA). An intravenous booster of plain antigen (0.6 mg) was administered on the 42nd day. Two control rabbits were injected with tetanus toxoid pretreated with coupling agent EDCI (TT-Ed). Blood for sera were collected from all the rabbits at various time intervals as indicated in results. Indirect immunofluorescent assay (IFA) was performed with the immunized rabbit sera using purified microfilariae of W. bancrofti. ELISA was performed using ultrasonicated microfilarial antigen: Correspondence address." Dr. B. Ravindran, Asst. Director, Dept. Immunology, Regional Medical
Research Centre (I.C.M.R.),Chandrasekharpur,Bhubaneswar751 016, India. [Tel: 55364, 53903, 56191].
354
a.
O /C2H 5 CH3--N _ ~ / N - - C - - N \ C 2 H 5
b.
CH3-- NN__... N--COOH
c. d.
HN
//COOH
~---~N-
CH~CH2-COO H
Fig. 1. Structures of haptens used in this study: (a) diethylcarbamazine (DEC): (b) methyl piperazinc carboxylic acid (MPCA); (c) piperidine carboxylic acid (PCA): (d) piperidine propionic acid (PPA).
briefly, about 140,000 purified microfilariae were taken in 3.0 ml of PBS containing 0.2 mM PMSF and 0.2 mM e-amino caproic acid, glass homogenized and then sonicated (Bronson Sonifer 450) for 8 cycles of 5 rain each at 30% duty cycle with an output of 15 W, centrifuged at 2500 x g for 30 rain at 4-'C and the supernatant containing 300 lag protein/ml was stored at - 2 0 C until further use. Details of purification of microfilariae using polycarbonate (Nucleopore) membranes, performance of IFA and ELISA using appropriate anti-rabbit immunoglobulin-conjugates (Dakopatts, Netherlands) have been described elsewhere (Ravindran et al., 1988). Table 1 shows the results for microfilarial reactivity of all the rabbit sera immunized with different hapten-carrier conjugates. By 30 days post-immunization all the rabbit sera showed anti-microfilarial sheath activity. The titres were variable. The reactivity of anti-hapten antibodies with/to the parasite sheath could be inhibited by preincubating the antisera with free hapten. Cross-reactivity amongst these antibodies is evident in inhibition assays using the three haptens as well as DEC (Table 2). The TABLE 1 lEA
Antibody reactivity of antisera to piperazine derivatives with microfilarial sheath of W.
Immunizing agent
TT-MPCA
Pre-irnmunization
Intensity of the reaction ~ (days after immunization)
Titre h
15
30
40
50
3+ 3+ 2+
3+ 4+ 2+
3+ 4+ 2+
3+ 3+ 2+
160 160 20
+ +
2+ + 2+
2+ + 2+
+ 2+
40 40 40
3+ 3+ 2+
3+ + +
+ + 2+
40 640 40
TT-PCA
TT-PPA
hancrq/?i
2+ +
TT-Ed
~Sera diluted twenty fold were analysed results of three individual rabbits in each group are shown. bReciprocal of highest serum dilution - - day 30 sera showing reaction with the sheath.
355 TABLE 2 M i n i m u m concentration of haptens (mM) required for inhibition of antibody reactivity to m f in IFA Antibodies to
MPCA PCA PPA
Inhibiting hapten DEC
PCA
PPA
MPCA
0.312 0.625 0.312
l0 5 5
1.25 1.25 0.156
20 40 40
50 p.1 of ten-fold diluted antiserum (pooled from 3 rabbits) were mixed with 50 lal of different concentrations of haptens and the mixture was spotted for 1FA after 90 min incubation at 37°C.
reactivity with microfilarial components could also be demonstrated for all the immunized rabbit sera by ELISA (Fig. 2). The two control rabbits injected with TT-Ed had no detectable anti-parasite activity as shown by IFA or by ELISA. The cross reactivity of anti-MPCA antibodies to microfilariae demonstrated by us earlier (Ravindran et al., 1988) thus does not appear to be unique to that particular hapten since antibodies to other piperazine derivatives also react with microfilariae of W. bancrofti as revealed by the present study. These findings indicate that antiparasite antibodies could be elicited in experimental animals without using microfilariae of W. bancroJ?i, the availability of which is scarce since no routine animal model or in vitro culture systems exist for large scale production of the parasite 1. 6-
1.2
E c 0.8
u
g e o.4
O un 21
353
© 1992 Elsevier SciencePublishers B.V. All rights reserved 0001-706X/92/$05.00 ACTROP 00190
Short Communication
Antigenic mimicry between piperazine derivatives and Wuchereria bancrofti microfilariae Tahziba Hussain and Balachandran Ravindran Department of lmmunology, Regional Medical Research Centre (I.C.M.R.), Bhubaneswar, India
(Received 17 July 1991; accepted 22 October 1991) Key words: Wucheria bancro~i; Lymphaticfilariasis; Diethylcarbamazine;Antigenicmimicry
Studies performed earlier in our laboratory indicated that antibodies with reactivity to the antifilarial drug, Diethylcarbamazine (DEC) react with microfilariae of Wuchereria bancrofti (Ravindran et al., 1988). The antibodies were raised in rabbits using as hapten, methyl piperazine carboxylic acid (MPCA), an acid hydrolysis product of DEC (Ravindran et al., 1987). The anti-MPCA antibodies not only reacted very strongly with DEC, they also reacted with microfilarial sheath of W. bancrofti. The reactivity to the parasite could be effectively inhibited by preincubation of the antisera with DEC. These observations indicated a possible antigenic similarity between the antifilarial drug DEC and the microfilarial surface of W. bancrofti. In this communication we report the results of our attempts to raise antibodies to two other piperazine derivatives, piperidine carboxylic acid (PCA) and piperidine propionic acid (PPA) with a view to study their reactivity to the parasite. The structures of the various haptens used in the investigation are shown in Fig. 1. MPCA was prepared by acid hydrolysis of DEC using 70% H2SO4 as described elsewhere (Ravindran et al., 1987), PPA (Cat No. 26371-07-3) and PCA (Cat No. 498-94 2) were procured from Aldrich Chemical Co. Inc., U.S.A. The haptens were conjugated with tetanus toxoid (TT) by carbodiimide reaction using 1-ethyl-3(3-dimethylaminoprophyl) carbodiimide (EDCI) and the coupling efficiency was tested by trinitrobenzene sulphonic acid assay (Ravindran et al., 1987). Three rabbits were each immunized with TT-MPCA, TT-PCA or TT-PPA. Two doses at 15 days interval were administered intramuscularly, the first (1.5 mg) with Freund's complete adjuvant (CFA) and the second (0,75 mg) with Freund's incomplete adjuvant (FICA). An intravenous booster of plain antigen (0.6 mg) was administered on the 42nd day. Two control rabbits were injected with tetanus toxoid pretreated with coupling agent EDCI (TT-Ed). Blood for sera were collected from all the rabbits at various time intervals as indicated in results. Indirect immunofluorescent assay (IFA) was performed with the immunized rabbit sera using purified microfilariae of W. bancrofti. ELISA was performed using ultrasonicated microfilarial antigen: Correspondence address." Dr. B. Ravindran, Asst. Director, Dept. Immunology, Regional Medical
Research Centre (I.C.M.R.),Chandrasekharpur,Bhubaneswar751 016, India. [Tel: 55364, 53903, 56191].
354
a.
O /C2H 5 CH3--N _ ~ / N - - C - - N \ C 2 H 5
b.
CH3-- NN__... N--COOH
c. d.
HN
//COOH
~---~N-
CH~CH2-COO H
Fig. 1. Structures of haptens used in this study: (a) diethylcarbamazine (DEC): (b) methyl piperazinc carboxylic acid (MPCA); (c) piperidine carboxylic acid (PCA): (d) piperidine propionic acid (PPA).
briefly, about 140,000 purified microfilariae were taken in 3.0 ml of PBS containing 0.2 mM PMSF and 0.2 mM e-amino caproic acid, glass homogenized and then sonicated (Bronson Sonifer 450) for 8 cycles of 5 rain each at 30% duty cycle with an output of 15 W, centrifuged at 2500 x g for 30 rain at 4-'C and the supernatant containing 300 lag protein/ml was stored at - 2 0 C until further use. Details of purification of microfilariae using polycarbonate (Nucleopore) membranes, performance of IFA and ELISA using appropriate anti-rabbit immunoglobulin-conjugates (Dakopatts, Netherlands) have been described elsewhere (Ravindran et al., 1988). Table 1 shows the results for microfilarial reactivity of all the rabbit sera immunized with different hapten-carrier conjugates. By 30 days post-immunization all the rabbit sera showed anti-microfilarial sheath activity. The titres were variable. The reactivity of anti-hapten antibodies with/to the parasite sheath could be inhibited by preincubating the antisera with free hapten. Cross-reactivity amongst these antibodies is evident in inhibition assays using the three haptens as well as DEC (Table 2). The TABLE 1 lEA
Antibody reactivity of antisera to piperazine derivatives with microfilarial sheath of W.
Immunizing agent
TT-MPCA
Pre-irnmunization
Intensity of the reaction ~ (days after immunization)
Titre h
15
30
40
50
3+ 3+ 2+
3+ 4+ 2+
3+ 4+ 2+
3+ 3+ 2+
160 160 20
+ +
2+ + 2+
2+ + 2+
+ 2+
40 40 40
3+ 3+ 2+
3+ + +
+ + 2+
40 640 40
TT-PCA
TT-PPA
hancrq/?i
2+ +
TT-Ed
~Sera diluted twenty fold were analysed results of three individual rabbits in each group are shown. bReciprocal of highest serum dilution - - day 30 sera showing reaction with the sheath.
355 TABLE 2 M i n i m u m concentration of haptens (mM) required for inhibition of antibody reactivity to m f in IFA Antibodies to
MPCA PCA PPA
Inhibiting hapten DEC
PCA
PPA
MPCA
0.312 0.625 0.312
l0 5 5
1.25 1.25 0.156
20 40 40
50 p.1 of ten-fold diluted antiserum (pooled from 3 rabbits) were mixed with 50 lal of different concentrations of haptens and the mixture was spotted for 1FA after 90 min incubation at 37°C.
reactivity with microfilarial components could also be demonstrated for all the immunized rabbit sera by ELISA (Fig. 2). The two control rabbits injected with TT-Ed had no detectable anti-parasite activity as shown by IFA or by ELISA. The cross reactivity of anti-MPCA antibodies to microfilariae demonstrated by us earlier (Ravindran et al., 1988) thus does not appear to be unique to that particular hapten since antibodies to other piperazine derivatives also react with microfilariae of W. bancrofti as revealed by the present study. These findings indicate that antiparasite antibodies could be elicited in experimental animals without using microfilariae of W. bancroJ?i, the availability of which is scarce since no routine animal model or in vitro culture systems exist for large scale production of the parasite 1. 6-
1.2
E c 0.8
u
g e o.4
O un 21
