Evaluation of Tests for Rabies Antibody and Analysis of

Transcription

1 JOURNAL OF CLINICAL MICROBIOLOGY, Mr. 1977, p Copyright C 1977 Americn Society for Microbiology Vol. 5, No. 3 Printed in U.S.A. Evlution of Tests for Rbies Antibody nd Anlysis of Serum Responses After Administrtion of Three Different Types of Rbies Vccines MONICA GRANDIEN Deprtment of Virology, Ntionl Bcteriologicl Lbortory, S Stockholm, Sweden Received for publiction 29 October 1976 Humorl ntibody response to three types of rbies vccines were ssyed by the neutrliztion (NT), the mixed hemdsorption (MH), nd the indirect immunofluorescence (IF) tests. The NT nd MH tests were used to detect ntibodies combining with ntigens t the surfce of virions nd infected cells, wheres the indirect IF test mesured ntibodies minly to the rbies nucleocpsid ntigen. After immuniztion with humn diploid cell vccine, ntibodies were detected by both the NT nd the MH test in the 14th- nd 30th-dy serum smples from ech of eight vccinted persons. There ws good correltion between titers obtined with the two tests in this group of vccinees. Antibodies elicited by duck embryo nd nervous tissue vccines occurred less frequently nd in lower titers. In these groups of vccinees, 5 of 14 nd 5 of 10, respectively, hd ntibodies detectble by the NT test in the 14th- nd 30th-dy ser but were negtive by the MH test. It is suggested tht this ws due to the high levels of immunoglobulin M ntibodies, which re known to be elicited by dily injections of vccine. Since ntibodies of the immunoglobulin M clss re considered to be less importnt for protection ginst rbies, the MH test is recommended for immunity determintions. Compred with the NT test, this test lso offers the dvntge of being techniclly more convenient becuse of its cpcity for testing numerous ser in single run. Antibody titers obtined by the indirect IF test in the humn diploid cell vccine group were reltively low. Titers in the duck embryo nd nervous tissue vccine groups were higher but did not correlte with the results of the NT test. The rbies ntibody responses induced in humns by vccines derived from nervous tissue nd duck embryo hve been thoroughly investigted in the pst. The recently introduced vccine prepred in humn diploid cells is reported to be highly immunogenic (1, 15). The verifiction of n ntibody response fter immuniztion to rbies is of gret importnce, nd is recommended by the World Helth Orgniztion (WHO) Expert Committee on Rbies 1973 (21). Rbies serum ntibody levels hve previously been determined minly by the neutrliztion (NT) test. Until recently, this time-consuming test ws performed in mice, lthough during the lst decde other techniques, exploiting cell culture systems, hve been reported (4, 6, 17, 19). A hemgglutintion inhibition (HI) test (12) nd pssive hemgglutintion test (7) hve been described, which probbly lso mesure ntibodies directed ginst ntigens on the surfce of the virion. Antibodies ginst minly nucleocpsid ntigens hve been ssyed by the indirect immunofluorescence (IF) (11, 14, 22) nd the complement fixtion (13) tests. The technique of mixed hemdsorption (MH) (10) hs been shown to be very sensitive in detecting ntibodies to severl virl nd cell surfce ntigens on monolyer cultures. Estimtion of ntibody levels in ser of rbies-vccinted nimls with this method ws first described by Espmrk et l. (9). The im of this study ws to ssess the MH test for estimtion of humn ntibodies to rbies nd to compre the results with those obtined by the NT nd indirect IF methods. For this purpose the ntibody response fter vccintion with three types of rbies vccine ws studied. MATERIALS AND METHODS Vccintion. Three types of commercilly vilble vccines were used. Recommended dosge schedules were followed. HDCV. The humn diploid cell vccine (HDCV; Institut Merieux, Lyon, Frnce; vccin rbique in- 263

2 264 GRANDIEN ctiv6 M6rieux) ws prepred in tissue cultures of humn diploid cells (WI-38) nd inctivted with,3- propiolctone. One milliliter of vccine ws given subcutneously on dys 0, 3, 7, nd 21. DEV. Duck embryo vccine (DEV; Lilly Reserch Lbortories, Indinpolis, Ind.) ws given to ptients in dily doses of 1 ml of vccine subcutneously for 14 dys. Neutrl tissue vccine (NTV). Ptients were given different types of killed rbies vccine, produced in dult or suckling nimls. Vccines were given subcutneously in dily doses for 8 to 14 dys. Ser. Humns, vccinted fter suspected exposure to rbies, were exmined serologiclly. Ser were drwn from 21 vccinees before nd on bout dys 14 nd 30 fter the beginning of immuniztion. Virus nd cells. Virus strin. Flury HEP rbies virus ws pssged 16 times in humn diploid cell strin; the clone ws purified in BHK-21/13S cells nd pssged in BHK-21 cells. This strin ws kindly supplied by T. Wiktor nd H. Koprowski of the Wistr Institute, Phildelphi, P. Cell cultures. The BS-C1 grivet monkey cell line ws used throughout the work. Serologicl tests. (i) NT. This test ws performed s described by Debbie et l. (6). Preincubted virusserum mixtures were inoculted into Leighton tubes contining cell monolyers on cover slips. After 5 dys of incubtion, the monolyers were exmined by immunofluorescence for the presence of rbies ntigen. All tests were performed with 30 to 300 tissue culture infective doses (TCID50) of rbies virus. Seril 10-fold dilutions of the virus were mde in the presence of inctivted negtive horse serum to stbilize the virus. Humn ser were tested in fourfold dilutions strting from 1:6.25. Two culture tubes were inoculted with ech virus-serum dilution mixture. The finl serum dilution yielding 50% negtive tubes ws used to express serum titers, which were then converted into interntionl units (21). WHO First Interntionl Stndrd freeze-dried nturl nti-rbies horse serum (1955), contining 86.6 IU/mpoule reconstituted to 1 ml, ws used s reference, nd nti-rbies serum of equine origin (Lederle), contining 1,000 IU/vil (lot no ), ws used s control serum nd included in ll tests. (ii) MH test. Tests were performed in milk dilution bottles with monolyers of cells infected with 5.5 x 106 TCID50 of rbies virus per bottle nd then were incubted for 3 dys t 37 C. At this time no cytopthic chnges could be seen, but immunofluorescence showed cytoplsmic inclusions in ll cells. From filter pper disks soked with undiluted test serum, the ntibodies were llowed to diffuse during 48 h from the top of n gr lyer to the monolyer of either virus-infected or uninfected control cells. After removing the gr lyer, ntibodies combined with the surfce of the virus-infected cells were demonstrted by sheep erythrocytes coted with globulin nd bound to the ntibodies vi n ntiglobulin link (8). Circulr zones of hemdsorption were obtined if ntibodies in test serum hd com- J. CLIN. MICROBIOL. bined with the surfce of the virus-infected cells in the monolyer. The dimeters of the hemdsorption zones were mesured. The dose response curve showed liner reltionship between the log of the serum dilution nd the dimeter of the zone, s hs been shown by others (8). The slope of the curve ws such tht 10- fold reduction of serum or ntibody concentrtion corresponded to 6.5-mm reduction of the dimeter of the hemdsorption zone. In few instnces humn ser showed nonspecific binding of immunoglobulins to the uninfected cell sheet (3 of 20 persons tested). These zones rrely exceeded the size of the pper disk nd were not regulrly reproducible. The rections could be eliminted for both humn nd horse ser by bsorption with uninfected cells. Hemdsorption zones smller thn 8 mm were regrded s nonspecific nd considered negtive. (iii) Indirect IF test. The IF test ws performed ccording to the stndrd method for rbies ntibody determintion (14). Humn ser were tested in seril fourfold dilution series. Antigen substrte slides; () Impression smers of sectioned rbies-infected mouse brins were mde on clen glss slides. (b) Rbies-infected cells on cover slips in Leighton tubes: Monolyers were infected with TCID50 Of ttenuted rbies virus by bsorption for 1 h t 37 C. After 2 dys, foci of infected cells showing intrcytoplsmic inclusions of rbies ntigen were seen. Slides were fixed in nhydrous cetone t -20 C for 1 h nd then stored t -70 C until use. The results of the indirect IF test were obtined by determining the highest serum dilution giving bright-green, distinct, typicl intrcytoplsmic inclusions in the cells. No ttempt ws mde to evlute immunofluorescence t the cell membrne. The sme ntibody titers were obtined for serum smples tested simultneously on the two kinds of ntigen slides. Microscopy. A Zeiss stndrd universl microscope with incident light from n HBO 100-W highpressure mercury lmp ws used with exiter filter BG12 (350 to 450 nm) nd brrier filters 41 nd 50. RESULTS NT test. Tble 1 shows the results of the NT tests. No ntibodies were detectble in the preimmuniztion ser. All ptients who received HDCV hd detectble ntibodies in the 14th-dy serum smple. Individul serum ntibody response to vccintion vried from 0.6 to 9.4 IU/ml (men 3.8) nd from 2.4 to 9.4 IU/ml (men 8.0) in the 30th-dy serum smple. There ws n increse in titer in the second smple for ll individuls except two in whom the titers were equl on dys 14 nd 30. Two ptients vccinted with DEV hd no neutrlizing ntibodies in the lowest dilution (<0.3 IU/ ml) in the 14th-dy serum smple. The positive ser in this group hd low titers (0.6 to 2.4; men 1.0). However, ll ptients hd ntibodies in the serum smples obtined on dy 30 (0.3 to

3 VOL. 5, 1977 TABLE 1. Results of NT Dy of No. ofser of smples with detectble tested (IU/ml ntibodies/totl of serum) no. serum smple HDCV DEV NTV 0 0/8 0/8 0/5 14 8/8 ( ; 6/8 ( ; 5/5 ( ; men 3.8) men 1.0) men 2.4) 30 8/8 ( ; 8/8 ( ; 5/5 ( ; men 8.0) men 1.1) men 6.6) Type of vccine. 4.7; men 1.1). All 5 in the group of ptients vccinted with NTVs hd titers on dy 14 (0.3 to 7.1; men 2.4) s well s on dy 30 (2.4 to 18.9; men 6.6). Thus, compred with DEV, titers were higher fter HDCV on both dys 14 nd 30, wheres those fter NTV were higher only on dy 30. MH test. Tble 2 shows the proportion of positive ser by the MH test in the groups of ptients immunized with the different vccines. All ptients vccinted with HDCV hd ntibodies in the 14th- s well s the 30th-dy serum smples, giving zones with men dimeter of 13 nd 17 mm, respectively. Ser from ptients receiving DEV gve fewer nd smller rection zones. In 14th- nd 30th-dy serum smples, four of eight nd six of eight ser, respectively, gve positive rections. In the group of ptients vccinted with NTV, the MH ntibody response ws lso low: smll zones were found in two nd three of the ptients on the two smpling dys. Indirect IF ntibody test. All preimmuniztion ser were negtive. Figure 1 shows the results with the different vccines. Fourteen dys fter the beginning of immuniztion, more thn hlf of the ptients who received HDCV hd titers of not more thn 6.25, wheres none of the ptients who received DEV nd NTV hd titers below 25. In the 30th-dy serum smple, two ptients in the HDCV group still hd low titers (.6.25). The highest titer (400) ws found in the NTV group. Comprison of results obtined with the different test methods of ser from ptients in the three vccine groups. The correltions between MH nd NT titers for the three vccine groups re shown in Fig. 2. In the HDCV group (Fig. 2A) there is good correltion between the results obtined with the two methods. All postimmuniztion ser contined ntibodies demonstrble by both methods. Also, ser with reltively low NT titers (0.6 to 2.4 IU/ml) were positive by the MH test. The MH nd NT titers for the DEV nd NTV groups re shown in Fig. 2B nd C. In the two RABIES ANTIBODIES AND VACCINES 265 TABLE 2. Results of the MH Dy of No. of positives/no. of ptients in ech group serum smple HDCV5 DEV NTV 0 0/8 0/8 0/5 14 8/8 (8-16; 4/8 (8-12; 2/5 (11 nd men 13) men 10) 13) 30 8/8 (15-20; 6/8 (9-17; 3/5 (8, 14 men 17) men 12) nd 15) Zone dimeters of positive ser,.8 mm. Rnge of dimeters (milliliters) for positive ser nd men dimeter for positive results. b Type of vccine. ub z Serum smple 1Lth dy Serum smple 30th dy < 6,25 6, HDCV :n Spt 5- DEV 6Pt < 6,25 6, I) 400 <6,25 6, <6,25 6, Serum titer Serum titer FIG. 1. Comprison of serum titers obtined with the IF test on the different groups of vccines. Absciss, indirect IF ntibody titers; ordinte, number of ptients. groups, 5 of 14 ser nd 5 of 10 ser, with ntibodies demonstrble by the NT test, were negtive by the MH test. Becuse of the discrepncy between titers by the two tests, the immunoglobulins of four ser from the 14th-dy serum smple in the NTV group were seprted into sucrose grdients. Between 25 nd 50% of the rbies-specific ntibodies were shown, by the indirect IF test, to be of the immunoglobulin M (IgM) clss. Only one lowtitered serum (8-mm dimeter) of the 15 MH positive ser in the two groups ws negtive by the NT test. DISCUSSION In this study, ntibody responses to three types of rbies vccine were used to evlute tests for rbies ntibodies. The NT test is the

4 266 GRANDIEN A. HDCV B. of DEV zone mm 8 ptients 8 ptients 20- A 20- K0 I1O- 8 1O-, ^^A x<8 x < 8 XXO, A -2; 15- ^ AS C. NTV 5 S ptients 5- o J. CLIN. MICROBIOL A <8 xx 0 A A s <0,3 0,6 2,5 10 IU/ml <0,3 0,6 2,5 10 IU/ml < 0,3 0,6 Z5 10 IU/mt Neutrliztion test FIG. 2. Correltion of results with the NT nd the MH test for ech of the three groups of vccines. Serum smple on dy 0 (x), dy 14 (0), nd dy 30 (A). most logicl test for estimting the protective cpcity of n ntibody response since it mesures ntibodies combining with the surfce of the virus nd hs been shown to correlte with protection ginst virus chllenge. In the pst this hs been the test used lmost exclusively for estimting ntibody responses to rbies vccintion. The use of the test to demonstrte the efficient immune rection of humns to HDCV hs been thoroughly documented (1, 3, 15). Antibodies hve been detected s erly s 7 dys fter the first injection (24). Bhmnyr (3), using the sme immuniztion schedule (1-ml doses dministered t 0, 3, 7, nd 21 dys) nd the sme source of vccine s used in this study, invribly found NT ntibodies by the 21st dy. The neutrlizing-ntibody response fter DEV hs been found by some workers to be poor nd opinion of the efficcy of this vccine vries (20). In report (5) where the neutrlizing cpcity ws mesured for constnt volume of test serum ginst vrying mounts of infectious virus, ntibody (in serum dilution 1:10) ws found to be virtully bsent in 5 of 13 ptients immunized with 14 dily doses of vccine. The NTV type of vccine hs been reported to elicit higher neutrliztion ntibody titers thn the DEV (18). The MH test mesures ntibodies combining with virus-induced ntigen on infected cells. In other test systems the surfce of rbies-infected cells hs likewise been shown to bind rbies virus ntibodies (2, 9, 23). This ntigen should be equivlent to the glycoprotein ntigen present on the exterior of budding virus. Providing ntibodies of the different immune globulin clsses re mesured to the sme extent, there should therefore be correltion between ntibody titers found in NT nd MH tests. The IF test minly shows ntibodies to the intrcytoplsmic rbies nucleocpsid ntigen nd to much lesser extent ntibodies to the virl glycoprotein ntigen present in infected cells. In fct, the ntibody mesured in this test my be induced by dministrtion of isolted rbies nucleocpsid ntigen. The discrepncy between indirect IF nd NT results in few individuls receiving duck embryo vccine hs been reported previously (11, 14). Up to 20% of postvccintion ser positive by the indirect IF test were negtive by the NT test (14). These results might be ttributble to the uthors' use of two different ntigens in the test nd to the predominnce of nucleocpsid ntigen in the vccine (5). The extensively documented results reported by others for the NT test re confirmed in the present study. Thus, the HDCV group showed n ntibody response by the NT test for ll the 14th-dy serum smples (0.6 to 9.4 IU/ml; men 3.8). All individuls hd n increse of ntibodies in the 30th-dy serum smple (2.4 to 9.4 IU/ ml; men 8.0) except two who hd unchnged titers in both ser (Tble 1). The neutrliztion ntibody response of the DEV group ws poorest (Tble 1). In two individuls no ntibodies were found in the lowest serum dilution in the 14th-dy smple (<0.3 IU/ml), nd the men titer ws low (1 IU/ml) for the six positive ser. In the 30th-dy serum smple ll individuls hd positive but still rther low vlues (0.3 to 4.7 IU/ml; men 1.1). The NTV group exhibited quite high levels of neutrlizing ntibodies (men 2.4 nd 6.6 IU/ml) for dys 14 nd 30, respectively. All ser in the HDCV group, 14th- nd 30th-dy, hd ntibodies mesurble by the MH test (Tble 2). For this group of vccinees the NT nd the MH tests hd pproximtely the sme degree of sensitivity (Fig. 2A). Furthermore, results from the two tests pper to correlte. Only ntigens exposed on the surfce of the rbies virion induce ntibodies ssocited with protection ginst the virus. Methods for determining ntibodies fter immuniztion with n inctivted vccine should therefore use the glycoprotein of the rbies virus surfce. This is the cse with NT, MH, nd HI tests. Of these, the NT nd MH tests hve been used here. The gret sensitivity to nonspecific inhibition is

5 VOL. 5, 1977 serious drwbck of the HI test nd therefore this test hs not been included in the study. In the postexposure ntibody response, rpid ppernce of ntibodies of the IgG clss is desired, s ntibodies of the IgM clss do not leve the vessels nd thus hve difficulty in reching the loclly introduced virus. Immuniztion with 14 dily doses of vccine hs been shown to prolong IgM ntibody production (11, 16), probbly due to the persistence of the ntigen. In the present study, totl of 10 of 24 ser in the DEV nd NTV groups with neutrlizing ntibodies were negtive by the MH test. The NT test mesures IgG s well s IgM nd IgA ntibodies, wheres the MH test s shown in model tests indicted only IgG ntibodies. The IgM response in the immune rection ws verified by the high proportion (25 to 50%) of rbies IgM ntibodies found by the indirect IF tests in the 14th-dy serum smple in the NTV group. This hs lso been found erlier fter DEV (11). The NT titers in Fig. 2B nd C could thus prtly be ccounted for by IgM ntibodies ppering during n immuniztion schedule with dily injections. These ntibodies re not mesured in the MH test nd low NT titers for these two groups of vccinees might therefore correspond to negtive MH test. The present study shows the usefulness of the MH test for rbies ntibody estimtion fter vccintion. Becuse it mesures ntibodies directed ginst virus surfce ntigens, the test correlted well with the NT test for the HDCV group. The MH test mesured only ntibodies of the IgG clss, nd it ws sensitive nd gve no flse-positive results. This* test lso ws fesible for quntittive ntibody mesurements, ws much less tedious thn the in vitro NT test, nd gve relible result in 2 dys. ACKNOWLEDGMENTS Skillful technicl ssistnce ws given by M. Blomqvist, A. Erlndsson, V. Fernstr6m, B.-I. Crlsson, nd L. Svensson. LITERATURE CITED 1. Aoki, F. Y., D. A. J. Tyrrell, L. E. Hill, nd G. S. Turner Immunogenicity nd cceptbility of humn diploid-cell culture rbies vccine in volunteers. Lncet 1: Atnsiu, P., P. Tsing, P. Perrin, nd S. Fvre Les IgG nti-glycoproteines rbiques mrquees pr l peroxidse et l'isothiocynte de fluoresceine. Resultts. Ann. Microbiol. (Inst. Psteur) 126B: Bhmnyr, M Results of ntibody profiles in mn vccinted with the HDCV vccine with vrious schedules, p In Regmey, Hennesen, nd Perkins (ed.), Int. symp. on rbies (II), Lyon. Symp. Series Immunobiol. Stnd., vol. 21. S. Krger, Bsel. 4. Cho, H. C., nd P. Fenje Rbies neutrlizing ntibody determintion in tissue culture by direct fluorescent ntibody technique. J. Biol. Stnd. 3: RABIES ANTIBODIES AND VACCINES Crick, J., nd F. Brown Efficcy of rbies vccine prepred from virus grown in duck embryo. Lncet 1: Debbie, J. G., J. A. Andrulonis, nd M. K. Abelseth Rbies ntibody determintion by immunofluorescence in tissue culture. Infect. Immun. 5: Dierks, R. E., nd P. M. Gough Pssive hemgglutintion test for rbies ntibodies in rbies, p In Kpln nd Koprowski (ed.), Lbortory techniques, 3rd ed., vol. 23. WHO monogrph ser. WHO, Genev. 8. Espmrk, J. A., nd A. Fgreus Identifiction of the species of origin of cells by mixed hemdsorption: mixed ntiglobulin rection pplied to monolyer cell cultures. J. Immunol. 94: Espmrk, J. A., A. Fgreus, nd J. Jonsson Mixed hemdsorption: mixed ntiglobulin rection in cell cultures for virologicl nd immunologicl studies. Symp. Ser. Immunobiol. Stnd. 4: Fgreus, A., nd J. A. Espmrk Use of "mixed hemdsorption" method in virus infected tissue cultures. Nture (London) 190: Grndien, M., nd J. A. Espmrk Follow-up of ntibody responses fter post-exposure rbies immuniztion, p In Regmey, Hennesson, nd Perkins (ed.), Interntionl Symposium on Rbies (II), Lyon. Symp. Ser. Immunobiol. Stnd., vol. 21, S. Krger, Bsel. 12. Hlonen, P. E., F. A. Murphy, B. N. Fields, nd D. M. Reese Hemgglutinin of rbies nd some other bullet-shped viruses. Proc. Soc. Exp. Biol. Med. N. Y. 127: Kuwert, E The complement fixtion test, p In Kpln nd Koprowski (ed.), Lbortory techniques in rbies, 3rd ed., vol. 23. WHO monogrph series WHO Genev. 14. Lrsh, S. E Indirect fluorescent ntibody nd serum neutrliztion response in pre-exposure prophylxis ginst rbies. Ann. Intern. Med. 63: Plotkin, S. A., T. J. Wiktor, H. Koprowski, E. E. Rosnoff, nd H. Tint Immuniztion schedules for the new humn diploid cell vccine ginst rbies. Am. J. Epidemiol. 103: Rubin, R. H., R. E. Dierks, P. Gough, M. B. Gregg, E. H. Gerlch, nd R. K. Sikes Immunoglobulin response to rbies vccine in mn. Lncet ii: Sedwick, W. D., nd T. J. Wiktor Reproducible plquing system for rbies, lymphocytic choriomeningitis, nd other ribonucleic cid viruses in BHK-21/ 13S grose suspensions. J. Virol. 1: Sikes, K. R., nd 0. P. Lrghi Purified rbies vccine. Development nd comprison of potency nd sfety with two humn rbies vccines. J. Immunol. 99: Smith, J. S., P. A. Yger, nd G. M. Ber A rpid tissue culture test for determining rbies neutrlizing ntibody, p In (Kpln nd Koprowski (ed.), Lbortory techniques in rbies, 3rd ed., vol. 23. WHO monogr. WHO, Genev. 20. Turner, G. S Rbies vccines. Br. Med. Bull. 25: WHO Expert Committee on Rbies, 6th report WHO technicl report series no World Helth Orgniztion, Genev. 22. Wiktor, T. J., E. Gyorgy, H. D. Schlumberger, F. Sokol, nd H. Koprowski Antigenic properties of rbies virus components. J. Immunol. 110: Wiktor, T. J., E. Kuwert, nd H. Koprowski Immune lysis of rbies virus-infected cells. J. Immunol. 101: Wiktor, T. J., S. A. Plotkin, nd D. W. Grell Humn cell culture rbies vccine. J. Am. Med. Assoc. 224: