MolecularImmunology,Vol. 16. pp. 66S669. Pergamon
TMMUNOGENICITY OF RIFAMYCINS GIOVANNI
MISTRELLO,’ RENATO CRICCH10,2 GIULIANA MARIACARLA MOSCA’ and LUIGLBASSI’
‘Department of Chemotherapy, Natural
Laboratory of Immunology and 2Department
1978; in revisedjorm
S.p.A., 15 March
of Antibotics and 20158 Milano, Italy 1979)
Abstract-The presence of anti-rifamycin antibodies in sera of rabbits injected with a rifamycin-protein conjugate or with some unconjugated rifamycin derivatives, was assayed by equilibrium dialysis. The conjugate was effective in eliciting the production of rifamycin specific antibodies. This capacity also was exhibited by the derivatives P and S/TA. The possible mechanism of the formation of potentially immunogenic rifamycin-protein complexes in the organism is discussed. Competition experiments were performed in order better to clarify the haptenic moiety of rifamycins. We conclude that the antigenic determinant of the rifamycin molecule includes part of the chromophore together with part of the ansa chain.
The rifamycins are a family of antibiotic substances derived from Streptomyces species. Chemical modification of the basic nucleus of rifamycins led to discovery of rifampicin. This is a broad spectrum antibiotic that at the concentration of 10m6M specifically inhibits the bacterial DNA-dependent RNA-polymerase in vitro (Hartman et al., 1967). Rifampicin has been found to be of particular value in the treatment of tuberculosis. Tuberculous patients under intermittent therapy with rifampicin sometimes show side reactions (Nessi et al., 1973). Evidences have been provided that these reactions may be associated with the presence in the serum of antibodies against rifampicin (Boman et al., 1973; Dewdney, 1977). Attempts to immunize experimental animals with rifamycins have often been unsuccessful (Dewdney, 1977). However, Joniau et al. (1976), using a rifamycin-protein conjugate, were able to induce the production of anti-rifamycin antibodies in rabbits. Dukor et al. (1973) provided evidence for delayedhypersensitivity reactions in guinea pigs sensitized with rifampicin in Freund’s complete adjuvant. The present work deals with the use of a rifamycin-protein conjugate to induce the formation of rifamycin specific antibodies in the rabbit. An attempt also was made to characterize the immunogenic capacity of different rifamycins by injecting rabbits with some unconjugated rifamycin derivatives. Correlation of chemical structure with antibody-inducing capacity of unconjugated rifamycins could lead to an understanding of the pathway by which potentially immunogenic rifamycin-macromolecule complexes are generated in the organism. The amount of anti-rifamycin antibodies in the rabbits sera was measured by equilibrium dialysis using [38-14C’3 rifampicin as indicator hapten. In addition, in order to define more clearly the haptenic moiety of ritdmycins, experiments were performed.
The rifamycin-protein conjugate was prepared by reacting 3-formylrifamycin SV with bovine serum albumin (BSA) as already described (Bassi ct al., 1976). After dialysis against phosphate buffered saline (PBS) the conjugate was freed from uncomplexed rifamycin by gel filtration on Sephadex G 25. The rifamycins used as inducing haptens to immunize rabbits are shown in Fig. 1.All rifamycins were dissolved in dimethylformamide (DMF) and diluted with PBS to a final concentration of 0.5?, of DMF. Antisera
Female albino rabbits (2.0-2.5 kg) from our own laboratories were used. The animals were injected subcutaneously in multiple sites with 1 mg of conjugate or 3 mg of the one of the rifamycins in complete Freund’s adjuvant once weekly for five weeks. Three bleedings were done at weekly interval, starting one week after the last injection. One pool of serum from each individual rabbit was used. Gamma:globulin fractions prepared from individual rabbit antisera by sodium sulfate precipitation (Kekwick, 1940) were further purified by DEAE-cellulose chromatography (Fahey & Terry, 1974). Equilibrium
A Dianorm multi-equilibrium dialysis system (20 cells) (Innovativ Medizin, Esslingen, Zurich, Switzerland) was employed (Assandri & Moro, 1977). Dialysis was performed using [38-14C] rifampicin. specific activity 56 mCi/m-mole, as indicator hapten. The radiochemical synthesis was carried out by G. Sartori of Gruppo Lepetit. Solutions were prepared with total rifampicin concentrations ranging from lo-’ to 4.10-” M. The dialysis medium was PBS, pH 7.4. The final solutions contained a trace amount of DMF originally used to dissolve rifampicin. Purified gamma globulins were used at a concentration of 10 mg/ml. Dialysis was for 6 hr at 37°C. Radioactivity measurements were performed with a Packard Tri-Carb model 2650 liquid scintillation spectrometer, utilizing the scintillation cocktail instagel (Packard, Downers, Grove, IL, U.S.A.). Counting had 85”, of efficiency. The method described by Nisonoff and Pressman (1958) was employed for plotting the binding data.
iI ’ 1”
Fig. 1. Structure of rifamycin derivatives. The first class of four compounds have their aromatic chromophore in the hydroquinonic form and can be oxidized to the quinonic form. The second class of two derivatives carries a thiazolic ring condensed on the chromophore and cannot be oxidized to the quinone. Haprcw competition
on a series of rabbit antisera globulin fractions. As shown in Table 1 all of the rabbits immunized with the conjugate had detectable anti-rifamycin antibodies present in the serum. Among the rabbits injected with unconjugated rifamycin derivatives, two of the four rabbits injected with rifamycin P and the two Table
Rifampicin 3-formyl rifamycin Cyclopentyl Rifamycin SV Rifamycin P Rifamycin S;TA 3-formyl rifamycin
CY sv P SiTA C
injected with rifamycin S/TA had antibodies. The results of the equilibrium dialysis studies performed with the sera of rabbits C,, C,, C,, P,, P,, S/TA, and S/TA, are shown in Figs. 2 and 3. The reciprocal bound (b) vs the reciprocal free cfl hapten concentration was plotted. The lines drawn in the figures are theoretical and were derived from the Sips equation (Nisonoff and Pressman, 1958). The experimental values off were raised to the fractional exponents x which are listed in Table 2. The index of heterogeneity x was calculated with the aid of an H.P. 3353 computer as follows: the experimental values of _f were raised to exponents ranging from 1 to 0.5 in steps of 0.01; the exponent giving the smallest value of the sums of the squares of the difference between the experimental and theoretical values of b was taken as 1. The total concentrations of antibody-combining sites and the values of the average intrinsic association constants rabbits
The relative effectiveness of different rifamycins in inhibiting the binding of [38-“C] rifampicin to rabbit antibodies was measured by equilibrium dialysis. Each rifamycin under study was mixed. at concentrations ranging from IO-’ to 10-lM, with [38-‘1C] rifampicin (2.5 x IO-‘Ml. The gamma globulin preparation was added to II concentration of 5 mg;ml. Otherwise conditions were as described in the previous section.
No. of rabbits injected
5 4 4 4 4 2 3
No. of rabbits showing antibodies toward rifampicin
2 2 3
“Rabbits were injected with 1 mg of 3-formyl rifamycin SV-BSA or 3 mg of an unconjugated rifamycin once weekly for five weeks. The animals were bled for 3 times at weekly intervals. starting one week after the last injection. One pool of each individual rabbit sera was used. Salt-precipitated globulin fractions were further purified by DEAE cellulose chromatography and then tested for the presence of anti rifamycin antibodies by equilibrium dialysis using [38-14CJ rifampicin as indicator hapten.
6 3. I u) 5.
(K,) were determined, respectively, from the J intercepts and the slopes of the lines. Assuming a mol. wt of 75.000 per antibody-binding site, the quantity of rifampicin-specific antibodies per ml of serum was estimated. The binding parameters are listed in Table 2. A trifling binding was observed in the case of the negative sera as well as when purified gamma globulins from normal rabbits were used as control preparation. One experiment was performed in the presence and absence of ascorbic acid in order to assess the influence of the possible oxidation of rifampicin from hydroquinone to quinone (Maggi ef al., 1967). No difference was observed. The specific rifampicin antibodies produced in rabbits provided an efficient tool to study the crossreactivity of different rifamycins. The efficiency of Table 2. Binding
Antigen binding sites (M x 10”)
C, C, C, P, P, SITA, SITA, “The gamma
Fig. 3. Binding of [38-t4C] rifampicin to rabbit antirifamycin antibodies. Equilibrium dialysis was performed with purified globulin fractions from sera of rabbits: (&S/TA,, (A)S/TA,, (o)P,, (o)P,. l/b = Reciprocal of the bound hapten; l/f = reciprocal of the free hapten. The lines are theoretical, based upon the Sips distribution. The experimental values of f were raised to the fractional exponents a listed in Table 2.
different rifamycin analogues in inhibiting the binding of [38-14C] rifampicin to rabbit anti-rifamycin antibodies was measured by equilibrium dialysis. The gamma globulin fraction prepared from the serum of rabbit C, was employed in these competition experiments. The haptens used were: rifampicin, 3formylrifamycin SV, cyclopentyl, rifamycin SV and its quinonic form rifamycin S, the chromophore and penicillin. In the case of the first four compounds 1 mg/ml ascorbic acid was added to the dialysis medium. We determined by spectrophotometric analysis that there was no reduction of quinonic or oxidation of hydroquinonic rifamycins under our experimental
data of rabbit
log K, (association constant)
1.6 3.9 3.7 1.4 0.99 0.59 0.74 globulin
Fig. 2. Binding of [38-t4C] rifampicin to rabbit antirifamycin antibodies. Equilibrium dialysis was performed with purified globulin fractions from sera of rabbits: (o)C,, (A)C,, (o)C,. l/b = Reciprocal of the bound hapten; l/j = reciprocal of the free hapten. The lines are theoretical, based upon the Sips distribution. The experimental values offwere raised to the fractional exponent a listed in Table 2.
6.14 6.57 6.32 6.93 6.99 6.86 6.87 in the equilibrium
a (index of heterogeneity) 0.93 0.87 0.93 0.95 0.97 0.91 0.93
was 10 mg/ml.
Antibodies present in original serum @g/ml) 98 204 180 80 62 29 31
eliciting an antibody response We had thought that 3-formyl
Fig. 4. Inhibition of binding of [38-‘4C] rifampicin to rabbit by equilibrium dialysis. The measured antibodies concentration of [38-14C] rifampicin was 2.5.10-‘M. The competing haptens were: (0)rifampicin. (V)cyclopentyl, ( x )3-tormyl rifamycin SV, (A) chromophore, (0) rifamycin SV, (m) rifamycin S, (0) penicillin. The purified gamma globulin fraction prepared from serum of rabbit C, was used in the present experiment at the concentration of 5 mg/ml.
Molar extinction coefficients used were: rifampicin, 15390 at 475 nm; 3-formylrifamycin SV, 13790 at 490 nm; cyclopentyl, 15400 at 475 nm; rifamycin SV, 14235 at 445 nm; rifamycin S, 4660 at 525 nm; and chromophore, 16660 at 378 nm. Results of hapten competition experiments are shown in Fig. 4. Rifampicin, 3-formylrifamycin SV, cyclopentyl, rifamycin SV and rifamycin S are about equally potent in inhibiting the binding of [38-14C] rifampicin. The chromophore is clearly a less potent inhibitor while penicillin does not affect the binding of rifampicin to rabbit antibodies. Competition experiments also were performed using the globulin fraction prepared from serum of rabbit P,. Results were essentially similar to those shown in Fig. 4. conditions.
Using a 3-formyl rifamycin SV-BSA conjugate we have been able to induce the formation of low titer, relatively high affinity antibodies against rifamycin in rabbits. Using rifamycin analogues without any carrier protein we failed to induce the production of antibodies except in the case of rifamycin P and its alkyl derivative S/TA. These rifamycins elicited the formation of antibodies at a lower titer than those induced by the conjugate, but with a higher association constant. The effectiveness of rifamycins P and S/TA in
was rather unexpected. rif~imycin SV would be
the more immunogenic rifamycin. This product was suspected to form Shiff bases with the (.-amino groups of lysine and thus give rise to potentially immunogenic hapten-protein complexes. This would explain the formation of the sensitizing antigen in humans since the hydrazone bond of rifampicin is rather unstable in physiological conditions and part of orally ingested rifampicin is recovered in blood as 3-formyl rifamycin SV. In our experimental model, however, rifamycin P and S/TA appear to be more efficient in generating the antigen in the organism. One possible explanation for this may derive from the fact that the first four rifamycins of Fig. I can be readily oxidized to their 1,4 quinonic forms, whereas rifamycins P and S,‘TA cannot. It has been shown that quinonic rifamycins have affinity values for BSA much lower than those of hydroquinonic rifamycins and that of the two hydroquinonic hydroxyl groups that in position 1 is the only one involved in favouring the formation of the rifamycin-albumin complexes (Assandri it al., 1977). It may be possible that oxidation of the first four rifamycins to the guinonic forms reduces their degree of binding to proteins thus limiting the formation of potentially immunogenic haptenprotein complexes. On the other hand, we cannot exclude that the effectiveness of the derivatives P and S/TA in generating the antigen in the organism may be related to the presence of a thiazolic ring in these compounds. Studies with other rifamycins which cannot be oxidized to the quinonic form and do not contain a thiazolic ring are underway. The results of our competition studies confirm previous observations (Joniau rt al., 1976) and point out that the rifamycin nucleus is the basic structure probably responsible for the cross-reactivity of all rifamycins. The quinonic and the hydroquinonic forms of the rifamycin molecule are equally well recognized by the antigen binding site of the antibody, as shown by the inhibitory power exhibited by the rifamycin S which is similar to that of the other rifamycins tested. The fact that thechromophore appears to be a poor inhibitor of the binding of rifamycin to rabbit antibodies suggeststhatthehaptenicconstituentisnotconfinedtothe naphthoquinonic group but is more complex and includes part of the ansachain. Experiments aimed at determining which groups of the ansa chain are involved in the binding of the rifamycins to rabbit antibodies are in progress. In particular the relevance of the hydroxyls in position 21 and 23 will be evaluated. REFERENCES Assandri A. & Moro L. (1977) J. Chromat. 135, 37. Assandri A., Perazzi A. & Berti M. (1977) J. Anrihiot., Tokyo 30, 409. Bassi L.. Di Berardino L., Perna G. & Silvestri L. G. (1976) Am. Rev. Respir. Dis. 114, 1189. Boman G., Nilsson B. S. & Saerens 6. J. (1973) Stand. J. Req. Dis. Suppl. 84, 40. Dewdney J. M. (1977) In The Antigexc (Edited by Sela M. Vol. IV, p. 73. Academic Press. New York. Dukor P., Schumann G. & Dietrich F. M. (1973) Scrmd. J Rrsp. Dis. Suppl. 84, 73. Fahey J. L. & Terry E. W. (1974) In Handbook o
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