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Neutralization of bothropic and crotalic venom toxic activities by IgG(T) and IgGa subclasses isolated from immune horse serum
Pergamon PII:
SOO41-OlUl(96)OUl77-X
NEUTRALIZATION OF BOTHROPIC AND CROTALIC VENOM TOXIC ACTIVITIES BY IgG(T) AND IgGa SUBCLASSES ISOLATED FROM IMMUNE HORSE SERUM
1. Fernandes. H. A. ‘Takeham. A. (‘. R. Sanlos, F. Cotmon~. D. Latinne. H. Buzin and 1. Moot. Neutralizatton of hothropic and crotalic venom toxic activities by IgG(T) and IgGa subclasses isolated from itnmunc horse wrum. To.\-icwr~35, 93 1 936. 1997. lgG(T) and IgGa isotypes wet-e isolated from horse hq,perimmune an[i-bothropic and anti-crotalic sera using ;I ccombination ot‘ two affinity chrom;ttographic processes. l&(T) and IgGa isotypes were isolated from theso ser:l b) chrom~ttoFrltphv on protein A-Sepharose followed by separation of the Lwo isot\peb b>, chromatograpll~ on 3 column of anti-lg<;(T)-Sepharose. LO-Ho&T-l. a KIL anti-horse IgC(T) tnonoclonal antibody,, was used. A comparative study 01‘ the efficiency of these isotypes in ncutralwtng the main toxic activities of the l~ot~~ologot~c~CIIOIIIS was carried out. It was found that IgG(T) c\as about three-fold and seven-fold tnorc pl-otec~ive than IgGa for tteu~rali/atiott of the lethal activity of B.,jr,rr/rtr~~ and C’. tl. /cwifrc.t,\ venoms. rcspecti\cly. I@(T) uas also more efYcctive than 1gG;t for the neutraliration of [he hacniorrh;tgic activity induced by .R. j~o.w~~~,l venom. while both isotypes tteutrali;led oquall> well the blood incoagulability induced by this \zenom. The results suggest that I@(T) is the moslt protective isotlpe present in both anti-bothropic and anti-crotalic set-a. followed by IgGu. Owing to their wry low concentration in the scrttni. other IpG isot\ipes arc not likely to be important in neutr;lli/ing the venon~s’ toxic activities. ( IYY7 Elsevier Science Lid
IN l-KoL~CC“rIoN
The present therapeutic prolocol for the treatment of envenomation by snakebite is based primarily on the i.v. adtnintstration of ccluine hyperitnmune antivenom serum. Several problems arc associated with serum therapy. One of lhese is the ditliculty in dctertnining the exact amounl of antivenom required to neutralize Ihe toxic cKects of the venom. which usually leads to adminis~r~ttion of unnecessarily large volun~s of serum. l-he large amount
of useless proteins administered facilitates the induction of immediate adverse reactions. Many attempts have been made to reduce the amount of immunogenic protein injected into patients This has led to the USCof pur;tied (F(ab’),) fragments instead of the whole antibody molecule (Pope, 1919). More recently it has been demonstraM experinient~~lly that purified anti-snake venom antibodies are verl efrective in neutralizing the toxic activities of the venom (Russell (11r/l.. 19x5). In our laboratory wc have been able to separate IgG(T) isotype from horse anti-bothropic serum and to study its role in neutrali;ling the lethality of bothropic venom (Fernandes c/ N/.. 1091. 1094). These experiments were extended in thij study, an efticient and easy method \A;IS devclopcd for isolating the IgGa isotype from hot-w serum. and a comparative study &as conducted on the efficiency of horTe IgG(T) and IgGa antibodies in neutralizing the tnxic activities of bothropic and crotalic venoms.
RI-SLI
TS
When horse hyperimmunc serum wa:, pasml through the protein A-Sepharose column, the bound fraction elutcd with citrate bufltr, pH 6.0. contained I&;(T) + I&a. Pnssage of the l@(T) alnd IgGa fraction iI1 the I.(>-I-lo<;T-I-Sepharose column resulted in the separation of the isotypcs. IgGa N;IS rcco\ered u~th the initial buffer (BBS), whcre:ls the bound l&;(T) was eluted with 0. I5 M tit rate buffer. pH 5.0 (Fig. I ). The k115,,for anti-crotalic IgGa M~S h32..1 (1~ [confidence limits (CL) 05’%. 455.3 7X8.51 a11~1 that for l@(T) WLISX7.2 /lg [<‘I., 95%. 5X.9 120.0). The F:I)~,,for anti-bothropic IgGa w;1s I3 13.6 pp (CL 95%. 964.0 1X41.1) \\hilst that for I@(T) was 401.7 /~g (CL 95’/1,. 36X.7 436.5). As can be xccil. the anti-lethal ;iclir,il\’ of I@(T) was always significantly more elli‘ctiw thn Ig(;a against cithcr crolalic or bothropic venom. The d:rtu \kere analyted hy a probit test (Finney. I97 I ). The elfect 01‘ I&;(T) and I&a on the haemorrhugic activity induced by the bothropic vet~nm is shown in ‘Table I, IyC;(T) U’;IY more ellficicnt than IgGa in the ncutralimtion of the hacmorrhagic activity iuduccd 1-1) H. j~m/.wc/ venom. There \I/;L~ no significant difI’crcncc in the abilit!, 01‘ thcsc iwtypcs to ncutrali/e R. ,j~~~~u~~~w~~ \cnom-induced blood incoagulability (Table I ).
LlIS(~‘LJSSlOh
We have previously SIIOWII that IIIOSI of the anti-lethal activit\, of bothropic hyperimmune horse serLmI is due to the I@.;(T) isotype (Fernandes PI t/l.. 1991 ). In the present communication the role was investigated of IgGa and IgG(T) isotypes present in horse anti-bothropic and anti-crotalic hylwrimmune sew in neutr,zllizing the main toxic activities of the homologous venoms. The results suggest that IgG(T) ncutrali7es some toxic activities of both venoms more efficiently than IgGa. This nlay be due to a either ;I quantitative or ;t qualitatice dilYercnce. For instance, it may be due either to a greater antivenom antibody content in the lgG(T’) isotype or to a higher affinity of IgG(T) antivenom antibodies. or evw to both conditions. It is interesting that hyperilllmuni/ation of horses with snake venoms results in J restricted increase in IgG(T) (Audibert and Sandor. 1972). There are other examples of similar isotype restriction. mainly 10 responses elicited by parasites. For instance. infection of mice with 7i:1,/,rr/lo.voill(l ~~.lcziinducts z considerable increase in the IgG2 isotypc (Takehara c’t (I/., 19XI ) and infection 01‘rats with h’;/~/‘o.v~/.o/l~~,/rr.c /~~~~.~i/io~~.vi.s is associated with increased levels of serum Igt (Olgivic.. 1967). This restriction in the antibody isotypc response in mice and IILIIII~IIS is probably associated with acti\;ation of specific subpopulations of T-helper cells and their pal tern of cytokinc expression. It is possible that
;I similar situation prevails in horses. but at present where are no data on horse lymphocytes and cytokines. 11nmediate adverse reactions are frequently incluccd by SCI-UIII therapy u;ith horse serum, due particularly to its high protein content. Our iresults, showing that IgG(T) is responsible for most antitoxic activities of botl1 bothropic and crotalic vciimis, suggest the possibility of treating bitten patients by injecting the IgG(T) isotypc isol:.lted from anti-snake wnom WI-WI. This procedure could he further improved b\’ isolating l@(T) antivenom antibodies by afiinity chl-omatograph4 and usin,(7the isolated lgG(T) antibodies to protect against the i/l rir,o toxic effects of the \cnon~. Tl1is has already been shown to be feasible in small laboratory animals using only small an~ounts of iIntibodicS. Unfortunately. the current technology has not !et allowed the prcparrltion of the large aniounts of IgG(T)-specific antibodies recluircd to provide protection to II~I;~I~s. HoweLcr. this may be po4sihle in the near futui-c.
Olgivie. H. M. (I 967) Rcqin-like ;Intihodics in rills inldrd with ~hc nern:ttocle prwitc Nl/‘/“~.\‘/r”‘t~~/~~.~ lwdim~is. ltr~mrrolo~~ If. I I 3,. I 3 I . Ounby. C. L.. Colberg. T. R. rind Odell. G. 1’. (19841 A new method for quantit;tting hwmorrha~ inducrd by r;tttlcsn;tkc vcnotns: ;thility of pol~vnlcnt ttntivcnom IO ncutctlizc hacmorrhagic activity. 7o.vicwt 22. 327-232. Pope. C. G. ( 1939) Tllc don 01‘ proteol>tic cnxymes on ~hc antitoxins and lwotcinx in immune scr;t. II. Hc.tt denalurution alicr parlid enqnic aclitw. /jr. J. c’.v,J. /‘U//I. 20. 20 I 2 12. Russell. F. ti.. Sulliv;m. J. B.. Egcn. N. I%.. Jctsr. W. S.. Mwklantl. I-. S.. Wingcrt. W. t\. and Bar-or. D. (10X.5) Prcpxttion 01’ :I new antivenin by nllinitl shroni;rlc,Er;lphy. .JuI. J. wop. Med. f/jx. 34. l-l I. 150. T;I kchatx H. A.. Pcrini. A.. Silw. tvl. I I. and Xlow. I. ( IOX I I T~:~~~trrros!~,rrcr ( rrrzi: role ol’dio&nt antibody cl;~sscs I?.$ I’wu~irol. 52, I .I? I-16. in protwtion ag;iinst inlixlion in lhe niouw. Takchara. Ii. A.. Fcrn;wJes. I.. C’ormont. I-‘.. Lotinnc. 11.. BGn. II. illd MOI;I. I. t 1095) Ncutr;llizing ability ill' IgGtl') md IgGl rubcl;~sscs ixtklld I'roiii mli-hllml~ic horse scrtiiii. Fir.\r /~rrwntrriorftr/ Cof~.~w.v.\ vu l:irrc~nor~ttrlir,r,s trd rhir 'liw~lmw.\. Ilt~titttl kistciir. Paris. I-rnnw.
SOO41-OlUl(96)OUl77-X
NEUTRALIZATION OF BOTHROPIC AND CROTALIC VENOM TOXIC ACTIVITIES BY IgG(T) AND IgGa SUBCLASSES ISOLATED FROM IMMUNE HORSE SERUM
1. Fernandes. H. A. ‘Takeham. A. (‘. R. Sanlos, F. Cotmon~. D. Latinne. H. Buzin and 1. Moot. Neutralizatton of hothropic and crotalic venom toxic activities by IgG(T) and IgGa subclasses isolated from itnmunc horse wrum. To.\-icwr~35, 93 1 936. 1997. lgG(T) and IgGa isotypes wet-e isolated from horse hq,perimmune an[i-bothropic and anti-crotalic sera using ;I ccombination ot‘ two affinity chrom;ttographic processes. l&(T) and IgGa isotypes were isolated from theso ser:l b) chrom~ttoFrltphv on protein A-Sepharose followed by separation of the Lwo isot\peb b>, chromatograpll~ on 3 column of anti-lg<;(T)-Sepharose. LO-Ho&T-l. a KIL anti-horse IgC(T) tnonoclonal antibody,, was used. A comparative study 01‘ the efficiency of these isotypes in ncutralwtng the main toxic activities of the l~ot~~ologot~c~CIIOIIIS was carried out. It was found that IgG(T) c\as about three-fold and seven-fold tnorc pl-otec~ive than IgGa for tteu~rali/atiott of the lethal activity of B.,jr,rr/rtr~~ and C’. tl. /cwifrc.t,\ venoms. rcspecti\cly. I@(T) uas also more efYcctive than 1gG;t for the neutraliration of [he hacniorrh;tgic activity induced by .R. j~o.w~~~,l venom. while both isotypes tteutrali;led oquall> well the blood incoagulability induced by this \zenom. The results suggest that I@(T) is the moslt protective isotlpe present in both anti-bothropic and anti-crotalic set-a. followed by IgGu. Owing to their wry low concentration in the scrttni. other IpG isot\ipes arc not likely to be important in neutr;lli/ing the venon~s’ toxic activities. ( IYY7 Elsevier Science Lid
IN l-KoL~CC“rIoN
The present therapeutic prolocol for the treatment of envenomation by snakebite is based primarily on the i.v. adtnintstration of ccluine hyperitnmune antivenom serum. Several problems arc associated with serum therapy. One of lhese is the ditliculty in dctertnining the exact amounl of antivenom required to neutralize Ihe toxic cKects of the venom. which usually leads to adminis~r~ttion of unnecessarily large volun~s of serum. l-he large amount
of useless proteins administered facilitates the induction of immediate adverse reactions. Many attempts have been made to reduce the amount of immunogenic protein injected into patients This has led to the USCof pur;tied (F(ab’),) fragments instead of the whole antibody molecule (Pope, 1919). More recently it has been demonstraM experinient~~lly that purified anti-snake venom antibodies are verl efrective in neutralizing the toxic activities of the venom (Russell (11r/l.. 19x5). In our laboratory wc have been able to separate IgG(T) isotype from horse anti-bothropic serum and to study its role in neutrali;ling the lethality of bothropic venom (Fernandes c/ N/.. 1091. 1094). These experiments were extended in thij study, an efticient and easy method \A;IS devclopcd for isolating the IgGa isotype from hot-w serum. and a comparative study &as conducted on the efficiency of horTe IgG(T) and IgGa antibodies in neutralizing the tnxic activities of bothropic and crotalic venoms.
RI-SLI
TS
When horse hyperimmunc serum wa:, pasml through the protein A-Sepharose column, the bound fraction elutcd with citrate bufltr, pH 6.0. contained I&;(T) + I&a. Pnssage of the l@(T) alnd IgGa fraction iI1 the I.(>-I-lo<;T-I-Sepharose column resulted in the separation of the isotypcs. IgGa N;IS rcco\ered u~th the initial buffer (BBS), whcre:ls the bound l&;(T) was eluted with 0. I5 M tit rate buffer. pH 5.0 (Fig. I ). The k115,,for anti-crotalic IgGa M~S h32..1 (1~ [confidence limits (CL) 05’%. 455.3 7X8.51 a11~1 that for l@(T) WLISX7.2 /lg [<‘I., 95%. 5X.9 120.0). The F:I)~,,for anti-bothropic IgGa w;1s I3 13.6 pp (CL 95%. 964.0 1X41.1) \\hilst that for I@(T) was 401.7 /~g (CL 95’/1,. 36X.7 436.5). As can be xccil. the anti-lethal ;iclir,il\’ of I@(T) was always significantly more elli‘ctiw thn Ig(;a against cithcr crolalic or bothropic venom. The d:rtu \kere analyted hy a probit test (Finney. I97 I ). The elfect 01‘ I&;(T) and I&a on the haemorrhugic activity induced by the bothropic vet~nm is shown in ‘Table I, IyC;(T) U’;IY more ellficicnt than IgGa in the ncutralimtion of the hacmorrhagic activity iuduccd 1-1) H. j~m/.wc/ venom. There \I/;L~ no significant difI’crcncc in the abilit!, 01‘ thcsc iwtypcs to ncutrali/e R. ,j~~~~u~~~w~~ \cnom-induced blood incoagulability (Table I ).
LlIS(~‘LJSSlOh
We have previously SIIOWII that IIIOSI of the anti-lethal activit\, of bothropic hyperimmune horse serLmI is due to the I@.;(T) isotype (Fernandes PI t/l.. 1991 ). In the present communication the role was investigated of IgGa and IgG(T) isotypes present in horse anti-bothropic and anti-crotalic hylwrimmune sew in neutr,zllizing the main toxic activities of the homologous venoms. The results suggest that IgG(T) ncutrali7es some toxic activities of both venoms more efficiently than IgGa. This nlay be due to a either ;I quantitative or ;t qualitatice dilYercnce. For instance, it may be due either to a greater antivenom antibody content in the lgG(T’) isotype or to a higher affinity of IgG(T) antivenom antibodies. or evw to both conditions. It is interesting that hyperilllmuni/ation of horses with snake venoms results in J restricted increase in IgG(T) (Audibert and Sandor. 1972). There are other examples of similar isotype restriction. mainly 10 responses elicited by parasites. For instance. infection of mice with 7i:1,/,rr/lo.voill(l ~~.lcziinducts z considerable increase in the IgG2 isotypc (Takehara c’t (I/., 19XI ) and infection 01‘rats with h’;/~/‘o.v~/.o/l~~,/rr.c /~~~~.~i/io~~.vi.s is associated with increased levels of serum Igt (Olgivic.. 1967). This restriction in the antibody isotypc response in mice and IILIIII~IIS is probably associated with acti\;ation of specific subpopulations of T-helper cells and their pal tern of cytokinc expression. It is possible that
;I similar situation prevails in horses. but at present where are no data on horse lymphocytes and cytokines. 11nmediate adverse reactions are frequently incluccd by SCI-UIII therapy u;ith horse serum, due particularly to its high protein content. Our iresults, showing that IgG(T) is responsible for most antitoxic activities of botl1 bothropic and crotalic vciimis, suggest the possibility of treating bitten patients by injecting the IgG(T) isotypc isol:.lted from anti-snake wnom WI-WI. This procedure could he further improved b\’ isolating l@(T) antivenom antibodies by afiinity chl-omatograph4 and usin,(7the isolated lgG(T) antibodies to protect against the i/l rir,o toxic effects of the \cnon~. Tl1is has already been shown to be feasible in small laboratory animals using only small an~ounts of iIntibodicS. Unfortunately. the current technology has not !et allowed the prcparrltion of the large aniounts of IgG(T)-specific antibodies recluircd to provide protection to II~I;~I~s. HoweLcr. this may be po4sihle in the near futui-c.
Olgivie. H. M. (I 967) Rcqin-like ;Intihodics in rills inldrd with ~hc nern:ttocle prwitc Nl/‘/“~.\‘/r”‘t~~/~~.~ lwdim~is. ltr~mrrolo~~ If. I I 3,. I 3 I . Ounby. C. L.. Colberg. T. R. rind Odell. G. 1’. (19841 A new method for quantit;tting hwmorrha~ inducrd by r;tttlcsn;tkc vcnotns: ;thility of pol~vnlcnt ttntivcnom IO ncutctlizc hacmorrhagic activity. 7o.vicwt 22. 327-232. Pope. C. G. ( 1939) Tllc don 01‘ proteol>tic cnxymes on ~hc antitoxins and lwotcinx in immune scr;t. II. Hc.tt denalurution alicr parlid enqnic aclitw. /jr. J. c’.v,J. /‘U//I. 20. 20 I 2 12. Russell. F. ti.. Sulliv;m. J. B.. Egcn. N. I%.. Jctsr. W. S.. Mwklantl. I-. S.. Wingcrt. W. t\. and Bar-or. D. (10X.5) Prcpxttion 01’ :I new antivenin by nllinitl shroni;rlc,Er;lphy. .JuI. J. wop. Med. f/jx. 34. l-l I. 150. T;I kchatx H. A.. Pcrini. A.. Silw. tvl. I I. and Xlow. I. ( IOX I I T~:~~~trrros!~,rrcr ( rrrzi: role ol’dio&nt antibody cl;~sscs I?.$ I’wu~irol. 52, I .I? I-16. in protwtion ag;iinst inlixlion in lhe niouw. Takchara. Ii. A.. Fcrn;wJes. I.. C’ormont. I-‘.. Lotinnc. 11.. BGn. II. illd MOI;I. I. t 1095) Ncutr;llizing ability ill' IgGtl') md IgGl rubcl;~sscs ixtklld I'roiii mli-hllml~ic horse scrtiiii. Fir.\r /~rrwntrriorftr/ Cof~.~w.v.\ vu l:irrc~nor~ttrlir,r,s trd rhir 'liw~lmw.\. Ilt~titttl kistciir. Paris. I-rnnw.