Immunogenicity of inactivated purified tissue culture vaccine against hepatitis A (HepAvac) assessed in laboratory rodents L.B. Elbert*, E.A. Lisitzina, Yu.Yu. Kusov and M.S. Balayan Immune response of laboratory rodents (guinea-pigs, CBA and Balb/ c mice, 14:?star and August rats) to inactivated hepatitis A vaccine was quantitatively assessed. Under comparable condttions of experimenL the mice showed the htghest antibody titres and were capable of reacting to the lower doses of immunogen; meanwhile their individual variations m immune response were more pronounced, white rats were the least susceptible to the vaccine, demonstrating the minimal anubody formation; guinea-pigs produced antibody at intermediate levels but the antibody titres were the most homogeneous. The enhancing effect of aluminium hydroxide was observed m guinea-pigs examined at the late postimmumzatton stage. Differences in mTmunogeniclO' of three vaccine lots were essentially similar when these lots were tested as undiluted preparations in guinea-pigs and mice for mean antibody titres and in rowe for 50% m~mune response using serial diluttons of vaccine. All three tests could be routinely employed for vaccine immunogeniclty control. Keywords:Tissue culture vaccine against hepatitis A, laboratory rodents, ~accmatmn schedules, adjuvant
INTRODUCTION During the last decade several variants of inactivated tissue culture vaccine against hepatitis A have been developed and the general principles of procedures for ~ts manufacture and quality controls have been outhned I 9. Since evaluation of protective potency m susceptible primates for each vaccine lot was prohibitively expensave, adequate tests for vaccine antigenic/' ]mmunogemc activity were performed mostly a priori m conventional laboratory animals. The present study was undertaken with the purpose of further evaluation and tmprovement of this vaccine quality control method. This was achieved by a comparative study of humoral immune response (antxbody production) m three species of laboratory rodents, i.e. guinea-pigs, rats and mice, using different immunization schedules MATERIALS
AND METHODS
Vaccine preparation The vaccine was prepared as described earher v 8 from hepatms A vtrus (HAV), grown in continuous Institute of Pohomyehtws and Virus Encephahtldes of Russaan Academy of Medical Sciences, 142782 Moscow reg, Russia. *To whom correspondence should be addressed. (Received 11 December 1991, revised 1 April 1992, accepted 7 April 1992) 0264-410x92.120828-05 ( 1992Butterworth-HememannLtd 828
Vaccine, Vol 10, Issue 12, 1992
heteroploid monkey kidney cell hne 46471° and stored at - 7 0 ° C wtthout sorbent.
Animals Guinea-pigs at the weights of 180-220 g; weeks old with body and Balb/c mice were of 12 14 g.
start of immunization had body Wistar and August rats were 6 8 weights of 100-110 g, and CBA 6 - 7 weeks old with body weights
Timetable of immunization and administration of vaccine 'Standard' schedule consisted of three injections 2 weeks apart (0, 14, 28 days); other schedules, dosage of vaccine, and extempore supplementation of adjuvant (alumlnlum hydroxide 1 1-1.5 mg ml -~ ) are indicated m the Results section. Each vaccine dose was 0 5 ml and was tested in at least five animals; on some occasions 1 0 ml dose was applied The vaccine dose, divided m three equal parts, was introduced to the guinea-pigs intramuscularly in two different legs and subcutaneously. The vaccine was given mtraperitoneally as a smgle dose to the rats and mice.
Antibody Ammal sera were tested for anUbody capable of binding HAV antigen in compeUtive enzyme immunoassay ~1 using partially purified hve HAV ( HAS- 15 strain ) grown m tissue culture. Serum dilution producmg 50% blocking effect was determined as antibody titre.
Hepatttm A vaccme assessed m rodents. L.B Elbert e t al
Mathematical
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Seroconverston m gumea-plgs after vaccination with adjuvant Vaccmatton schemes (a), 0, 14, 28, (b), 0, 14, 76, (c) 0, 35, 65 days
Antibody response of guinea-pigs and Balb/c mice was compared using larger groups (24 and 55) of identically immunized animals (Table 2) revealing that G M T in mice was higher than that m guinea-pigs ( log 2 ! 0.1 versus log 2 6.9 ). Individual variations of titre in mice were more pronounced as well. Dose-effect dependence in guinea-pigs and mice (F~gure 3) shows that the mice were more susceptible to a low concentration of the HAV ~mmunogen Balb,/c m i c e
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rats a n d m i c e
The effect of vaccination was compared in three species of rodents immunized according to the standard schedule, all animals were bled 7 days after the last injection The immunization effect m guinea-pigs and mice appeared to be better than that in rats of both hnes, antibody tltres in individual animals being much more variable in the latter (Table I ).
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RESULTS
After a complete course of immunization following the standard schedule, antibody formation expressed in geometric mean titres ( G M T ) was essentially slmdar in the group of animals that received the vaccine in doses of 0.5 and 1 0 ml. When the vaccine was given without alumimum adjuvant the antibody tltres m serum samples taken 7 days and 1 year after the last injection did not differ significantly. An enhancing effect of alumamum hydroxide was observed in this experiment a year after completing the initial immunization. When the guineapigs were boostered with a fourth vaccine dose further antibody rise took place in the animals which received the vaccine without alumlnlum hydroxide (Figure I ). In the next experiment three schedules of vaccine with alum]mum hydroxide, namely (a) 0, 14, 28 days, (b) 0, 14, 76 days and (c) 0, 35, 65 days, were compared by G M T determination. As shown in Figure 2 a single vaccine dose did not produce detectable antibody for at least 35 days ( schedule (c)). The second injection resulted in a rapid and most pronounced increase in G M T m all three schedules, the tatres reached their maximal level m about 2 weeks, and were slightly higher for scheme (c) with the longest interdose Interval. The third antigen injection led to further antibody increase, but for schedule (a) where the time interval between the second and third injections was the shortest such rise was found to be delayed. Antibody titres after three inoculations were maintained at relatively high levels for at least 135 days (period of observation ).
/+,
a
12
Influence of vaccme dose and adjuvant on seroconverston in gumea-pigs and mice m vaccination scheme O, 14, 28, 395 days Vaccme doses curve 1, 0 5 ml, curve 2, 1 0 ml, curve 3, 1 0 ml + alummJum hydroxide
These animals were immumzed by standard schedule and bled 7 days after the last vaccine injection. Despite the fact that variations in individual antibody titres were greater than those in guinea-pigs the reproducibility of the results of G M T estimation was satisfactory; this has been confirmed in three identical experiments (Figure 4 ). Figure 5 shows that the profiles of antibody titres ( G M T ) during 28 days postvacclnation had the same shape ~rrespectwe of the presence of aluminium hydroxide in
Vaccme, Vol
10, I s s u e 12, 1992
829
Hepattbs A vaccine assessed m rodents L.B E/bert et al Table 1
Comparatwe immunogentctty of HepAvac for dtfferent species of rodents Mtce
Rats
Ammal no
Balb..'c
CBA
August
Wtstar
Guinea-pigs
1 2 3 4 5 GMT(s d )
88 79 93 93 77 8 6(0 8)
91 93 93 93 93 9 3(0 1)
81
INTRODUCTION During the last decade several variants of inactivated tissue culture vaccine against hepatitis A have been developed and the general principles of procedures for ~ts manufacture and quality controls have been outhned I 9. Since evaluation of protective potency m susceptible primates for each vaccine lot was prohibitively expensave, adequate tests for vaccine antigenic/' ]mmunogemc activity were performed mostly a priori m conventional laboratory animals. The present study was undertaken with the purpose of further evaluation and tmprovement of this vaccine quality control method. This was achieved by a comparative study of humoral immune response (antxbody production) m three species of laboratory rodents, i.e. guinea-pigs, rats and mice, using different immunization schedules MATERIALS
AND METHODS
Vaccine preparation The vaccine was prepared as described earher v 8 from hepatms A vtrus (HAV), grown in continuous Institute of Pohomyehtws and Virus Encephahtldes of Russaan Academy of Medical Sciences, 142782 Moscow reg, Russia. *To whom correspondence should be addressed. (Received 11 December 1991, revised 1 April 1992, accepted 7 April 1992) 0264-410x92.120828-05 ( 1992Butterworth-HememannLtd 828
Vaccine, Vol 10, Issue 12, 1992
heteroploid monkey kidney cell hne 46471° and stored at - 7 0 ° C wtthout sorbent.
Animals Guinea-pigs at the weights of 180-220 g; weeks old with body and Balb/c mice were of 12 14 g.
start of immunization had body Wistar and August rats were 6 8 weights of 100-110 g, and CBA 6 - 7 weeks old with body weights
Timetable of immunization and administration of vaccine 'Standard' schedule consisted of three injections 2 weeks apart (0, 14, 28 days); other schedules, dosage of vaccine, and extempore supplementation of adjuvant (alumlnlum hydroxide 1 1-1.5 mg ml -~ ) are indicated m the Results section. Each vaccine dose was 0 5 ml and was tested in at least five animals; on some occasions 1 0 ml dose was applied The vaccine dose, divided m three equal parts, was introduced to the guinea-pigs intramuscularly in two different legs and subcutaneously. The vaccine was given mtraperitoneally as a smgle dose to the rats and mice.
Antibody Ammal sera were tested for anUbody capable of binding HAV antigen in compeUtive enzyme immunoassay ~1 using partially purified hve HAV ( HAS- 15 strain ) grown m tissue culture. Serum dilution producmg 50% blocking effect was determined as antibody titre.
Hepatttm A vaccme assessed m rodents. L.B Elbert e t al
Mathematical
procedures
14
Numerical data were analysed using Supercalc, Stratgraphics and Harwardgraphics computer programs. "O
oo
8
Guinea-pigs
E
6
Guinea-pigs,
12 3 "~
8
~E
6
4 Jo
/
J
4-
2
I
I
I
I
I
100
200
300
400
500
Days a f t e r f i r s t vaccine mjectlon Figure 1
\
:l--+
2O
I
I
I
I
I
40
60
80
100
120
140
Days a f t e r f i r s t vaccme dose
14
-b
12
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o
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8
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d
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20
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40
60
80
100
120
140
Days a f t e r f i r s t v a c c i n e dose
12
C
10
~o
o
r-
6 4
o
J
2
0
20
40
60
I
I
I
80
100
120
140
Days a f t e r f i r s t vaccine dose Figure 2
Seroconverston m gumea-plgs after vaccination with adjuvant Vaccmatton schemes (a), 0, 14, 28, (b), 0, 14, 76, (c) 0, 35, 65 days
Antibody response of guinea-pigs and Balb/c mice was compared using larger groups (24 and 55) of identically immunized animals (Table 2) revealing that G M T in mice was higher than that m guinea-pigs ( log 2 ! 0.1 versus log 2 6.9 ). Individual variations of titre in mice were more pronounced as well. Dose-effect dependence in guinea-pigs and mice (F~gure 3) shows that the mice were more susceptible to a low concentration of the HAV ~mmunogen Balb,/c m i c e
2 0
2
_~.--~=
14
10
,/
4
rats a n d m i c e
The effect of vaccination was compared in three species of rodents immunized according to the standard schedule, all animals were bled 7 days after the last injection The immunization effect m guinea-pigs and mice appeared to be better than that in rats of both hnes, antibody tltres in individual animals being much more variable in the latter (Table I ).
:+
+
ro
t~ 0"1 0 .--I
/++\/+-+-+,
/ + 4-+ +-+--4-
I0
RESULTS
After a complete course of immunization following the standard schedule, antibody formation expressed in geometric mean titres ( G M T ) was essentially slmdar in the group of animals that received the vaccine in doses of 0.5 and 1 0 ml. When the vaccine was given without alumimum adjuvant the antibody tltres m serum samples taken 7 days and 1 year after the last injection did not differ significantly. An enhancing effect of alumamum hydroxide was observed in this experiment a year after completing the initial immunization. When the guineapigs were boostered with a fourth vaccine dose further antibody rise took place in the animals which received the vaccine without alumlnlum hydroxide (Figure I ). In the next experiment three schedules of vaccine with alum]mum hydroxide, namely (a) 0, 14, 28 days, (b) 0, 14, 76 days and (c) 0, 35, 65 days, were compared by G M T determination. As shown in Figure 2 a single vaccine dose did not produce detectable antibody for at least 35 days ( schedule (c)). The second injection resulted in a rapid and most pronounced increase in G M T m all three schedules, the tatres reached their maximal level m about 2 weeks, and were slightly higher for scheme (c) with the longest interdose Interval. The third antigen injection led to further antibody increase, but for schedule (a) where the time interval between the second and third injections was the shortest such rise was found to be delayed. Antibody titres after three inoculations were maintained at relatively high levels for at least 135 days (period of observation ).
/+,
a
12
Influence of vaccme dose and adjuvant on seroconverston in gumea-pigs and mice m vaccination scheme O, 14, 28, 395 days Vaccme doses curve 1, 0 5 ml, curve 2, 1 0 ml, curve 3, 1 0 ml + alummJum hydroxide
These animals were immumzed by standard schedule and bled 7 days after the last vaccine injection. Despite the fact that variations in individual antibody titres were greater than those in guinea-pigs the reproducibility of the results of G M T estimation was satisfactory; this has been confirmed in three identical experiments (Figure 4 ). Figure 5 shows that the profiles of antibody titres ( G M T ) during 28 days postvacclnation had the same shape ~rrespectwe of the presence of aluminium hydroxide in
Vaccme, Vol
10, I s s u e 12, 1992
829
Hepattbs A vaccine assessed m rodents L.B E/bert et al Table 1
Comparatwe immunogentctty of HepAvac for dtfferent species of rodents Mtce
Rats
Ammal no
Balb..'c
CBA
August
Wtstar
Guinea-pigs
1 2 3 4 5 GMT(s d )
88 79 93 93 77 8 6(0 8)
91 93 93 93 93 9 3(0 1)
81
