Zbl. Bakt. Hyg. A 260, 396-402 (1985) Serologic and Immune Responses to Rabies Virus During Different Human Treatments with Tissue Culture and Suckling Mouse Brain Vaccines ANA MARIA DIAZ and VICTOR MANUEL VARELA-DIAZ Pan American Zoonoses Center, Pan American Health OrganizationlWorld Health Organi- zation, 1000 Buenos Aires, Argentina Received October 24, 1984 . Accepted in Revised Form May 10, 1985 Summary The antibody response to rabies virus was studied in twenty volunteers immunized with different schemes of suckling mouse brain and human diploid cell culture rabies vaccines. Throughout the study period, titers in serum neutralization and indirect fluorescent anti- body tests, as well as the class of immunoglobulins with antirabies activity, varied in different individuals with the treatment scheme and the antigenic potency of the vaccine. The results suggest that measurement of the IgG class of antirabies antibodies, and possibly IgA as well, may be a more adequate criterion to assess the imrnunogeniciry of rabies vaccines than the determination of SN titers alone. Zusammenfassung Die Antikorperreakrion auf Tollwutvirus wurde an zwanzig Freiwilligen untersucht, die nach unterschiedlichen Schemata mit Impfstoffen aus Babymaushirn und menschlichen Di- ploidzellkulturen immunisiert worden waren. Wiihrend des gesamten Untersuchungs- zeitraumes schwankten die Titer im Serum-Neutralisations- und FA-Test ebenso wie die Immunoglobulinklassen mit gegen die Tollwut gerichteter Aktivitat bei den einzelnen Perso- nen je nach lmpfschema und antigener Wirksamkeit des lmpfstoffes. Die Ergebnisse deuten an, daB eine Messung der IgG-Tollwut-Antikorper und moglicher- weise auch von IgA geeignetere Kritericn zur Beurteilung der Immunogenitiit von T011- wutimpfstoffen darstellen als die alleinige Bestimmung von Serumneutralisationstitern. Introduction Since the time of Pasteur, post-exposure antirabies treatment consisted of the ad- ministration of a daily vaccine dose during variable numbers of consecutive days. The classical treatment with 14 daily injections followed by two booster doses has been replaced more recently by several abbreviated vaccination schemes, in which the total number of inoculations varies from six to ten doses (5, 8, 16).
Transcript
Zbl. Bakt. Hyg. A 260, 396-402 (1985)
Serologic and Immune Responses to Rabies VirusDuring Different Human Treatments with Tissue Cultureand Suckling Mouse Brain Vaccines
ANA MARIA DIAZ and VICTOR MANUEL VARELA-DIAZ
Pan American Zoonoses Center, Pan American Health OrganizationlWorld Health Organization, 1000 Buenos Aires, Argentina
Received October 24, 1984 . Accepted in Revised Form May 10, 1985
Summary
The antibody response to rabies virus was studied in twenty volunteers immunized withdifferent schemes of suckling mouse brain and human diploid cell culture rabies vaccines.Throughout the study period, titers in serum neutralization and indirect fluorescent antibody tests, as well as the class of immunoglobulins with antirabies activity, varied indifferent individuals with the treatment scheme and the antigenic potency of the vaccine.
The results suggest that measurement of the IgG class of antirabies antibodies, andpossibly IgA as well, may be a more adequate criterion to assess the imrnunogeniciry ofrabies vaccines than the determination of SN titers alone.
Zusammenfassung
Die Antikorperreakrion auf Tollwutvirus wurde an zwanzig Freiwilligen untersucht, dienach unterschiedlichen Schemata mit Impfstoffen aus Babymaushirn und menschlichen Diploidzellkulturen immunisiert worden waren. Wiihrend des gesamten Untersuchungszeitraumes schwankten die Titer im Serum-Neutralisations- und FA-Test ebenso wie dieImmunoglobulinklassen mit gegen die Tollwut gerichteter Aktivitat bei den einzelnen Personen je nach lmpfschema und antigener Wirksamkeit des lmpfstoffes.
Die Ergebnisse deuten an, daB eine Messung der IgG-Tollwut-Antikorper und moglicherweise auch von IgA geeignetere Kritericn zur Beurteilung der Immunogenitiit von T011wutimpfstoffen darstellen als die alleinige Bestimmung von Serumneutralisationstitern.
Introduction
Since the time of Pasteur, post-exposure antirabies treatment consisted of the administration of a daily vaccine dose during variable numbers of consecutive days. Theclassical treatment with 14 daily injections followed by two booster doses has beenreplaced more recently by several abbreviated vaccination schemes, in which the totalnumber of inoculations varies from six to ten doses (5, 8, 16).
Antibody Response to Rabies Immunization 397
For the assessment of reduced vaccine schedules prior to their adoption, the WHOExpert Committee on Rabies (18) has recommended the inclusion of serologic studies.Also, the need for information on reliable criteria to correlate antibody levels and theprotective capacity of rabies vaccination in man has been stressed.
The present report describes the antibody response elicited in man during the courseof different rabies vaccine treatments. Data obtained on the serological and immunoglobulin activity in sera from vaccinees was related to the antigenic potency of thevaccine and the degree of sensitization induced by each immunization scheme.
Materials and Methods
Suckling mouse brain (7) and human diploid cell culture (17) rabies vaccines whichfulfilled the requirements of the NIH potency test (3) were used.
Sera were obtained from four groups of five previously unvaccinated volunteers who hadbeen administered one of the following rabies immunization schemes: 14 + 2 == 14 dailydoses of 5MB vaccine (average antigenic value == 1.5) plus two boosters 10 and 20 days afterthe last inoculation (5); 7 + 3 == seven daily doses of the 5MB vaccine (A.V. == 2) followedby three boosters 10,20 and 90 days later (5); 5MB-6 == six doses of 5MB (antigenic value ==1.0) on days 0, 3, 7, 14,30 and 90 (12); HDCV-6 == six doses of a tissue culture vaccine(antigenic value == 5.0) on days 0, 3, 7, 14,30 and 90 (11).
Samples selected for study were collected from the twenty persons 10,21,60,90 and 180days after the initiation of rabies vaccine treatment.
All sera were examined by serum neutralization (SN) and indirect fluorescent antibody(IFA) tests for rabies as described elsewhere (1, 2). Portions of selected sera from thesepersons were treated with 0.1 M 2-mercaptoethanol (ME) as described previously (2) andsubsequently examined by SN.
Immunoglobulin classes with antirabies activity were detected in sera collected 10,21 and180 days after the initiation of the vaccine treatment. This was carried out by IFA usingsmears of rabies virus-infected mouse nervous tissue and rabbit antisera to human IgM. IgGand IgA (4).
Results
The results of SN and IFA tests in sera collected at periodic intervals during immunization of four groups of persons with different rabies vaccination schemes are shown inTable 1. There was a lack of correlation between SN and IFA antibody titers. MaximalSN and IFA titers were observed in HDCY-6 vaccinees and these titers were lower inthe 5MB-6 group throughout the study period.
Findings on the immunoglobulins with antibody activity against rabies virus detected in sera from vaccinated persons are shown in Table 2. Ten days after beginningtreatment, antibody activity to rabies vaccines was detected by IFA in the IgM, IgGand/or IgA classes in variable proportions of sera. At this time, the highest percentageof sera with IgG antibodies was observed in the 5MB-7 + 3 group, while IgM antibodyactivity persisted in HDCY-6 vaccinees until day 21. IgA rabies antibodies were revealed in individuals from all groups at different times, but only in sera from HDCY-6vaccinees were these immunoglobulins detected consistently throughout the studyperiod.
398 A. M. Dfaz and V. M. Varela-Diaz
Table 1. Serum neutralization (numerator) and indirect fluorescent ant ibody (denominator)titers" in persons immunized with different rabies vaccination schemes
N° of days after vaccination
Vaccination 10 21 60 90 180scheme
5MB-14 + 2 ND 625 1620 419 84ND 240 25 25 25
5MB-7 + 3 37 214 1620 1400 2805 125 125 25 25
5MB-6 5 187 125 1190 845 25 5 5 5
HDCV-6 66 3820 4670 5060 164025 125 25 > 125 125
" Geometric mean titer s
Th e effect of ME-treatment on th e SN titers of selected indiv idu als is shown in Table3. At day 10, most of the SN titers in the HDCV-6 vaccinees were M E-susceptiblewhil e at day 180, SN titers were resistant to ME treatment.
Discussion
The present findings on the anti body response during human ra bies immunizationprovide information to assess the immunogeni city of different vacc ina tion schemes.
SN anti body titers in sera collected 21 days after the initiati on of rab ies tr eatment
Table 2. Percentage of sera with IgM (in parenthesis), IgG and IgA anti body activity revealed by IFA in persons immunized with different rabies vaccination schemes
Immunoglobulin N° of days after vaccinationclass Scheme
U untreated seraM mercaptoethanol-treated seraD difference in titer
with 14 + 2 and 7 + 35MB vaccination schemes (5) were comparable, as were thoseobtained at 60 days. However, the fact that 5N antibody levels were higher at 90 and180 days in the 7 + 3 group suggests that, in the latter scheme, the booster dose givenat day 27 induced a larger antibody response than that administered on day 34 of the14 + 2 scheme. Accordingly, these observations are compatible with a more effectivesensitization of the host immune system by the 7 + 3 than by the 14 + 2 scheme.
In persons treated with the 5MB-6 scheme (11), 5N titers at 21 and 60 days werecomparable. However, the increase in 5N titers which would have been expected to
follow the booster dose given on day 30 was not detectable until day 90. During preexposure rabies immunization with 5MB, similar increments in titer (Ll-fold) werereported after administration of a booster dose (3), but without the time lag observedin the present study. This delay seems difficult to assess but it may be that the antigenicstimulation of the preceding doses (at days 0, 3, 7 and 14) was insufficient to sensitizethe host immune system for the production of higher levels of antibodies to the boosterdose.
A lack of correlation between 5N titers and the antigenic value of rabies vaccinesbecame apparent from the observations made with 5MB immunization schemes. Thus,5N titers observed in the 7 + 3 scheme in this series were comparable to those reportedin a previous study (11) in which the A.V. of the vaccine was 1 instead of 2.
Also, 5N titers in sera collected at days 21 through 180 from HOCV-6 vaccineeswere consistently higher (between 4 and 37 times) than those in persons receiving the5MB-6 scheme. This could be partly attributed to the antigenic value of the vaccineused, which was higher (5.0) for HDCV than for 5MB (1.0) except that 5N titers werelower in the same immunization scheme using other HDCV vaccines whose antigenicvalues were 1.8 and 4.0 (9, 6).
In duck embryo (DEV) vaccinees (2), the concomitant presence of another class ofME-susceptible immunoglobulins such as IgA was postulated to account for a proportion of the 5N titers which were attributed to IgM antibody activity. This explanationis in agreement with the present observations in persons immunized with 5MB andHDCV schemes in that IgM as well as IgA antibodies were identified by IFA in serumsamples (from days 10 and 21) whose 5N titers were not resistant to ME reduction.
400 A.M. Diaz and V.M. Varela-Diaz
The prolongation of the IgM response for up to 225 days recorded after administration of a 14 + 2 DEV scheme was attributed (2) to persistence of viral antigens intissues, as a result of the repeated vaccinations. However, in persons receiving 5MBand HDCV immunization schemes, IgM antirabies antibodies were detected by IFA insome sera on day 10 and, by day 21, only in 20 per cent of HDCV-6 vaccinees. MEtreatment of selected sera from these individuals also showed an absence of IgMactivity by day 180.
This more limited IgM response may be accounted for by considering that the higherantigenic potency of these vaccines results in the induction of higher levels of IgGantibodies, which are known to inhibit IgM synthesis. Accordingly, the lower antigenicstimulation produced by DEV would by in agreement with the prolonged IgM responseobserved. This conclusion is also compatible with data obtained in other studies, inthat a persistence of IgM activity was reported during immunization with a HDCVwhich exhibited an A.V. of 1.8 (6). Similarly, the use of an HDCV with an A.V. of 1.9resulted in a delay in the IgM response, while the latter was inhibited with the morepotent (A.V. of 13) vaccine (10).
At day 10, SN titers in HDCV-6 and 5MB 7 + 3 vaccinees were comparable butthose in the latter group were higher after ME-treatment of sera. This observation istherefore compatible with the presence of higher levels of IgG antibodies in 5MB 7 + 3than in HDCV vaccinees. Rabies antibody activity in the IgG immunoglobulin classwas also detected more frequently at this time in 5MB 7 + 3 (l00 per cent) than inHDCV-6 vaccinees (40 per cent). These findings suggest that the 5MB 7 + 3 schememay be more effective than HDCV-6 during the early stages of rabies treatment,particularly if it is considered that the presence of SN antibodies as soon as possibleafter infection is desirable, and that IgG antibodies have a more important role inprotection than IgM immunoglobulins, which do not leave the circulation.
Several of the above findings also seem pertinent to the evaluation of rabies vaccinetreatments. Thus, IFA and SN titers were associated with IgM, IgA and/or IgG antibodies and individual differences in antibody levels occurred in HDCV and 5MB vaccinees. Evidence has also been obtained to suggest that the production of rabies IgGantibodies varies not only with the immunization scheme, but also with the antigenicvalue and the type of vaccine used. Accordingly, it seems reasonable to propose thatmeasurement of the levels of rabies antibody activity in the IgG immunoglobulin classmay be a more adequate parameter to assess the effectiveness and immunogenicity ofrabies vaccines than the currently used determination of SN titers. This conclusion isalso compatible with the lower affinity of IgM antibodies and their limitations forneutralizing virus outside the circulation and with available information on the relationship between antigenic value of vaccines and the induction and duration of protection against rabies (15).
Also, these consideration suggest that the reported failure of some vaccinated individuals to survive challenge with live virus in the presence of variable SN titers (6, 14)may arise from the existence of insufficient levels of IgG rabies antibodies.
In the present study, IgA antibody activity against rabies virus was demonstrated byIFA in sera from variable proportions of individuals, both during the earlv immunization stages and following boosters with 5MB or HDCV vaccines. Information on theIgA response during rabies vaccination is limited, but these findings suggest that antibodies in this immunoglobulin class may play an active role in protection againstinfection through neutralization of the virus in the tissues and/or the circulation.
Antibody Response to Rabies Immunization 401
References
1. Atanasiu, P., M. Bahmanyar, M. Baltazar, ]. P. Fox, K. Habel, M. Kaplan, R. E.Kissling, A. Komarov, H. Koprowski, P. Lepine, F. Perez-Gallardo and M. Schaeffer:Rabies neutralizing antibody response to different schedules of serum and vaccine inoculations in non-exposed persons. Bull. WId Hlth Org. 14 (1956) 593-611
2. Deand, D.]. and M. K. Abelseth: The fluorescent antibody test. In: Laboratory Techniques in Rabies (M. M. Kaplan and H. Koprowski, eds.), 3rd ed., pp. 73-84. WorldHealth Organization (1976)
3. Diaz; A. M. 0.: Pre-exposure rabies immunization of man with suckling mouse brainvaccine. Amer. J. Epidem. 115 (1982) 274-277
4. Diaz, A. M. and D. M. Myers: Comparison between a modified counter immunoelectrophoresis test and the indirect fluorescent antibody test for detection of antibodies torabies virus in human sera. J. Clin. Microbiol. 14 (1981) 446-448
5. Diaz, A. M. 0., G. Gonzalez Resigno, A. Fernandez Munilla, O. P. Larghi, N. Marchevsky, and}. C. Arrossi: Vacuna antirrabica de cerebro de raton lactante. Esquemasreducidos de inrnunizacion post-exposicion, Rev. Arg. Microbiol. 11 (1979) 42-44
6. Fuenzatida, E., A. M. O. Diaz, and S. Riverson: Vacuna de cerebro de raton lactantesuplementada con adyuvantes: Su aplicacion en bovinos. Rev. Soc. Arg. Microbiol. 10(1978) 47-53
7. Fuenzalida. E. and R. Palacios: Un metodo mejor para preparacion de la vacuna antirrabica, Bol. Inst. Bact. Chile 3 (1955) 3-10
8. Held, J. R., E. Fuenzalida. H. Lopez Adaros, ]. C. Arrossi, N. R. O. Poles, and A.Sciuetti: Inrnunizacion humana con vacuna antirrabica de cerebro de raton lactante. Bol.Ofic. Sanit. Panamer. 72 (1972) 565-575
9. Kuwert, E. K., I. Marcus, and P. G. Hoher: Neutralizing and complement-fixing antibody responses in pre- and post-exposure vaccinees to rabies vaccine produced in human diploid cells. J. BioI. Standard. 4 (1976) 249-262
10. Kuwert, E. K., I. Marcus, O. Thaenhart, C. Barsenbach,]. Werner, N. Scheiermann,and K. G. Nicholson: Variation of dose and antigenic mass of human diploid cell strainvaccine for post-exposure rabies prophylaxis: the 5:5:3 schedule. In: Cell CultureRabies Vaccines and their Protective Effect in Man (E. K. Kuuiert, T. j. Wiktor, and H.Koprowski, eds.), pp. 149-159. International Green Cross, Geneva (1981)
11. Larghi, O. P., A. M. O. Diaz, and j. C. Arrosi: Comparison of human serologicresponse to suckling mouse brain and human diploid cell rabies vaccines. (Submitted forpublication)
12. Rubin, R. H., R. E. Dierks, P. Gough, M. B. Gregg, E. H. Gerlach, and P. K. Sikes:Immunoglobulin response to rabies vaccine in man. Lancet 2 (1971) 625-628
13. Sikes, R. K. and O. P. Larghi: Purified rabies vaccine. Development and comparison ofpotency and safety with two human rabies vaccines. J. Immunol. 99 (1967) 545-553
14. Sikes, R. K., W. F. Cleary, H. Koprowski, and M. M. Kaplan: Effective protection ofmonkeys against death from street virus by post-exposure administration of tissueculture rabies vaccine. Bull. Wid Hlth Org. 45 (1971) 1-11
15. Sikes, R. K., G. V. Peacock, P. Acha, R. [. Arko, and R. Dierks: Rabies vaccines:duration of immunity study in dogs. J. Arner. vet. med. Ass. 159 (1971) 1491-1499
16. Steck, F., G. A. Spengler, R. Hauser, H. Keller, and A. I. Wandeler: IgM and IgGantibody profiles after immunization of man with HDCS-vaccine (Preliminary communication). Developm. BioI. Stand. 40 (1978) 137-139
17. Wiktor, T. j., S. A. Plotkin, and H. Koprowski: Development and clinical trials of thenew human rabies vaccine of tissue culture (human diploid cell) origin. Developm. BioI.Standard. 40 (1978) 3-9
402 A. M. Diaz and V. M. Varela-Diaz
18. Wiktor, T. [: M. V. Fernandez, and H. Koprowski: Cultivation of rabies virus in humandiploid cell strain WI-38. ]. Immunol. 93 (1964) 353-366
19. World Health Organization: Expert Committee on Rabies, 6th Report. Geneva (1973)
Dr. Ana Maria Diaz; Centro Panamericano de Zoonosis, Casilla 3092, Correo Central,1000 Buenos Aires, Argentina
