Archives of Virology
Archives of Virology 61, 141--147 (1979)
© by Springer-Verlag 1979
lnmmno9enieity of Subviral Herpes Simplex Virus Preparations I. Formation of Neutralizin 9 Antibodies in Different Animal Species After Administration of Herpes Simplex Virus Solubilized Antigens By L. KUTINOVX, V. ~LICHTOVX,and V. VONKA Department of Experimental Virology, Institute of Sera and Vaccines, Prague, Czechoslovakia With 3 Figures Accepted February 9, 1979
Summary Production of neutralizing antibodies was followed in guinea pigs, rabbits hamsters and mice immunized with crude antigen extracts (AM) from human diploid cells infected with herpes simplex virus tsTpe 1. The AiM induced relatively high levels of neutralizing antibodies in all four species. The antibodies were predominantly complement-requiring and remained so even after administration of repeated AM doses. With the strains used, the antibody response was predominantly type specific and, surprisingly, the type specificity of sera usually increased after administration of repeated doses of AM. Guinea pigs seemed to be the best responsive animal species. They developed the highest levels of antibodies and complement-nonrequiring antibodies were seen in them earlier than in the other animal species. The dose-response experiments carried out in guinea pigs indicated that after a single dose administration the ratio between complement-requiring and complement-nonrequiring antibodies was dependent on the amount of antigen administered. When AM was given without adjuvant less efficient antibody production was observed than after the administration of the same amount of antigen with adjuvant.
Introduction The development of a subunit type of herpes simplex virus (HSV) vaccine is prefercd to whole virion vaccines for reasons of safety (2, 3, 5, 6). However, a number of problems associated with the development of such vaccine, including its immunogcnicity, remain to be solved. In a previous report (6) we described the production and some properties of HSV neutralization antigens extracted from human diploid cells infected with HSV type 1. In the present study, we investigated the formation of neutralizing antibodies in animals immunized with this preparation. A comparison was made
0304-8608/79/0061/0141/$ 01.40
t42
L. KuTINovX, V. SLIeI~Tov£, and V. V o ~ : A :
b e t w e e n r a b b i t s , h a m s t e r s , m i c e a n d g u i n e a pigs in r e s p e c t of t h e d e v e l o p m e n t of c o m p l e m e n t - r e q u i r i n g (CR +) a n d n o n r e q u i r i n g ( C R - ) n e u t r a l i z i n g a n t i b o d i e s . T h e t y p e s p e c i f i c i t y of t h e a n t i b o d y r e s p o n s e was also e x a m i n e d .
Materials and Methods Cells H u m a n diploid cells (LEP) derived f r o m e m b r y o lungs were c u l t i v a t e d as described (8). I n a few e x p e r i m e n t s rabbit e m b r y o fibroblasts were also used. T h e y were cultivated in the same media as the L E P cells. Virus HSV-1 (strain K O S ) and H S V - 2 (strain 196) were k i n d l y p r o v i d e d b y Dr. J . L. Melnick, B a y l o r College of Medicine, Houston. T h e viruses were g r o w n in L E P cells at a multiplicity of infection ( ~ O I ) of 0.1.-o-0.5 P F U per cell. Eagle's m i n i m a l essential m e d i u m (MEM) s u p p l e m e n t e d w i t h 5 per cent h e a t - i n a c t i v a t e d calf serum, 0.075 per cent NaHCO3 and antibiotics was used as m a i n t e n a n c e m e d i u m .
Soluble Antigen Mixture Crude antigen m i x t u r e s to be used for i m m u n i z a t i o n were p r e p a r e d as described in detail elsewhere (6). I n brief, L E P cells grown in 1200 ml l~oux bottles were infected with I-ISV-1 at MOI 0.5 P F U per cell and P a r k e r ' s i99 m e d i u m w i t h 2 per cent of calf s e r u m was added to the cultures after 2 hours of virus adsorption. The infected cells were scraped off the glass after 28-hour i n c u b a t i o n at 37 ° C. T h e y were disrupted b y t r e a t m e n t w i t h 0.5 per cent N o n i d e t P-40 (Shell Chemical Co., Ltd., London, E n g l a n d ) in isotonic reticulocyte s t a n d a r d buffer (I~SB), p H 7,4. The e x t r a c t was clarified of cell nuclei and viral nucleoeapsids b y differential eentrifugation. T h e final m i x t u r e of antigens (AM) t h a t h a d n o t s e d i m e n t a t e d after t - h o u r centrifugation at 100,000 × g was extensively dialyzed against R S B for 7 days. Absence of infectious virus was verified in L E P cells; one R o u x bottle (1200 mI) culture was inoculated w i t h 1 ml of AM and k e p t for six days at 37 ° C. Two additional passages were performed. Absence of c y t o p a t h i e changes was considered an indication of absence of a n y s u r v i v i n g virus. 51Cr-Release Inhibition Test (C R I T ) The c o n t e n t of I-ISV solubilized antigens in AMs was d e t e r m i n e d by a c y t o t o x i e i t y inhibition test as described previously (7). T w e n t y c o m p l e m e n t requiring I-ISV-I neutralizing a n t i b o d y units, 30 units of guinea pig c o m p l e m e n t and 4 × 104 cryopreserved t a r g e t H S V - I infected rabbit fibroblast cells were used in CI~IT. T h e least a m o u n t of antigen causing 50 per c e n t inhibition of 51Or release was defined as one CI~IT antigen unit. On the basis of a correlation b e t w e e n the results of C R I T and the blocking neutralization test, it can be concluded t h a t CI~IT measures the c o n t e n t of H S V neutralizing antigens (7). Immunization Procedure Chinchilla rabbits (about 3000 g), guinea pigs (400 g), y o u n g adult Syrian h a m s t e r s and white mice, strain H, (9--11 g) were i m m u n i z e d w i t h three doses of AM and the a n t i b o d y response was tested after e v e r y dose of antigen. The a m o u n t s of neutralizing antigens used for i m m u n i z a t i o n are shown in Table 1. A m i x t u r e of AM with c o m p l e t e F r e u n d a d j u v a n t was administered s u b c u t a n e o u s l y (s.c.) as t h e first dose. J~or t h e second dose, inoculated s.c. four weeks later, the .AM was m i x e d w i t h incomplete F r e u n d a d j u v a n t . The t h i r d dose, w i t h o u t a d j u v a n t , was injected i n t r a p e r i t o n e a l l y (i.p.) after a n o t h e r four-weeks interval. Serial s e r u m samples were w i t h d r a w n f r o m rabbits and gxlinea pigs, sera f r o m h a m s t e r s a n d mice were obtained b y e x s a n g u i n a t i n g 5 - - 6 animals a t each interval. The sera from each individual rabbit, guinea pig and h a m s t e r were k e p t a n d tested separately, while mouse sera were pooled.
Immunogenicity of Subviral HSV-1 Preparations
143
I n a dose-dependent response experiment, each of several AM dilutions with complete F r e u n d a d j u v a n t was administered to a group of guinea pigs. Subsequent doses were administered at the same intervals as in the first experiment. I n a s t u d y on the influence of F r e u n d adjuvant, groups of guinea pigs were immunized simultaneously with AM either mixed or not mixed with adjuvant.
Neutralization Test Sera were inactivated at 56 ° C for 30 minutes and kept at --30 ° C until being used. For the neutralization test they were diluted in two-fold steps starting from i:i0. Freeze-dried pooled guinea pig serum was used as the source of complement. Complement units were determined by the raicrotechnique described by ZXvAl)ovi et al. (i0). Undiluted serum contained 600 complement-fixing units/0.1 ml. Equal amounts of serum dilution, virus suspension (100 TCDs0/0.1 ml) and medium with or without complement (20 units of complement/0.1 ml) were mixed and incubated at 37 ° C in a water bath for I hour. Four tube cultures of LEP cells were inoculated with 0.3 ml of each mixture. The test was read after six days of incubation at 37 ° C. At least 50 per cent reduction of cytopathic effect was considered an indication of antibody presence. Serum antibody titres were determined according to K241~BEI¢ (4). For calculation of geometric mean titres (GMT), sera negative at I:I0 dilution were considered positive at I :5 dilution.
Table 1. Amounts o/ HSV-1 antigens used /or immunization o/ rabbits, guinea pigs,
hamsters and mice CRIT Dose
I~oute of inoculation
1 2 3
s.c. s.c. i.p.
antigen units~
Rabbits
Guinea pigs
Hamsters
Mice
1280 (2) b 1280 (2) 3840 (3)
1280 (2) 1280 (2) 3840 (3)
640 (1) 640 (1) 1920 (1.5)
64 (0.1) 64 (0.1) 192 (0.15)
Per dose a n d animal b Volume in ml inoculated Results
Comparison of Antibody Response in Di//erent Animal Species Titres of a n t i b o d i e s n e u t r a l i z i n g HSV-1 a n d HSV-2 a n d their c o m p l e m e n t r e q u i r e m e n t , as d e t e c t e d in sera of t h e four a n i m a l species e m p l o y e d , a r e given in F i g u r e 1. All these tests were carried o u t in L E P cells. The results o b t a i n e d in r a b b i t s , guinea pigs a n d h a m s t e r s are expressed as GMT. I n mice, t h e d a t a shown refer to s e r u m pools. I t can be seen t h a t h o m o t y p i c n e u t r a l i z i n g a n t i b o d i e s were d e t e c t e d in all a n i m a l species a f t e r one dose of a n t i g e n a n d their titres increased a f t e r t h e second a n d t h i r d doses. A f t e r t h e first dose, r a b b i t s , h a m s t e r s a n d mice p r o d u c e d c o m p l e m e n t - r e q u i r i n g (CR +) a n t i b o d i e s o n l y ; low levels of c o m p l e m e n t i n d e p e n d e n t a n t i b o d i e s were o n l y d e t e c t e d in guinea pigs. C o m p l e m e n t - i n d e p e n d e n t (CR-) h o m o t y p i c a n t i b o d i e s were d e t e c t e d in sera of all species a f t e r t h e second dose of A M ; however, in h a m s t e r s a n d mice t h e t h i r d dose of a n t i g e n was n e c e s s a r y to s t i m u l a t e s y n t h e s i s of C R - a n t i b o d i e s n e u t r a l i z i n g t y p e 2 virus. The highest a n t i b o d y response was o b t a i n e d in guinea pigs. I n mice, which received t h e least a m o u n t of AM, t h e a n t i b o d y t i t r e s were t h e lowest a n d t h e decrease in t i t r e s in t h e
I4zt
L. Ku~INOVX, V. SHeI-tTOVX, and V. VONKA:
course of two m o n t h s following t h e f o u r t h bleeding was t h e m o s t m a r k e d . F o r control purposes some sera were t e s t e d in p a r a l l e l in cultures p r e p a r e d from rabbit, e m b r y o f i b r o b l a s t s ; t h e neutralizing a n t i b o d y titres were c o m p a r a b l e w i t h those d e t e r m i n e d in L E P cells (results unshown). I n a d d i t i o n to these animals, control r a b b i t s were in same w a y i m m u n i z e d with AM p r e p a r e d from uninfected cells. Their sera were f o u n d free of a n y t I S V n e u t r a l i z i n g a c t i v i t y . This is in line with t h e previous d e m o n s t r a t i o n s t h a t AM from uninfected cultures did n o t compete w i t h H S V n e u t r a l i z i n g a n t i b o d i e s (6, 7).
10240 5"i20 2560 "1280 64,0 320 "160
A iI
/// / /
""%
80 .~ 4.01
/;, / I /
20"10r~
"~"1280-
=. 640-
C
t?~\xX
320"
1
\
160-
/
80" 4020"10"~7"
/ " t' ~ \ 28
56 63 "123 28 Time o] bteecling['days)
\\
56 63 123
Fig. 1. Neutralizing antibodies in rabbits ( A ) , guinea, pigs ( B ) , hamsters (C) and mice (1)) immunized with solubilized HSV-1 antigens. Arrows indicate the times of antigen administration, o - - - - o complement-requiring ( C R + ) t y p e 1 neutralizing antibody. × × complement-nonrequiring (CR--) type 1 neutrMizing antibody. • -- -- • CR-F type 2 neutrMizing antibody, a - - zxC R - - type 2 neutralizing antibody. A n t i b o d y fibres are expressed as the reciprocal of the highest serum dilution still neutralizing the virus. I n rabbits, guinea pigs and hamsters, titres are expressed as GMTs; in mice, titres determined in serum pools are given F r o m t h e d a t a shown in F i g u r e t, also t h e ratios between CR + a n d C R - a n d between t y p e 1 a n d t y p e 2 n e u t r a l i z i n g a n t i b o d y titres can be d e t e r m i n e d in the sera of t h e different a n i m a l species. I t can be seen t h a t the ratios b e t w e e n t y p e 1 CI~ + a n d CI{ '- a n t i b o d i e s in guinea, pigs, mice a n d r a b b i t s d i d n o t m a r k e d l y v a r y t h r o u g h o u t t h e o b s e r v a t i o n period, i n d i c a t i n g t h a t m o s t of t h e a n t i b o d y f o r m e d r e m a i n e d c o m p l e m e n t - d e p e n d e n t in spite of r e p e a t e d a n t i g e n doses. The same applies to t y p e 2 neutralizing a n t i b o d i e s f o r m e d in guinea pigs ; however, in t h e o t h e r t h r e e a n i m a l species in which t h e CR ÷ : CR r a t i o h a d i n i t i a l l y b e e n tow t h e ratios were higher in the late t h a n in t h e e a r l y serum samples. This implies t h a t
Immunogenieity of Subviral HSV-1 Preparations
145
the increase in heterotypically reactive antibody was mainly at the expense of complement-requiring antibody. In comparing the type 1 and type 2 antibody titres one sees that in all four animal species the respective ratios, i. e. type specificity, increased after the second dose, and in the case of CR- antibody even after the third dose of AM. Dependence o/Antibody Response on A M Dose In the next experiment, guinea pigs were immunized subcutaneously with one dose of 8 different dilutions of AM mixed with complete Freund adjuvant, 5 animals being used per group. The results of this dose-dependent response experiment are shown in Figure 2A. High levels of CR+ antibodies reactive with both type 1 and type 2 virus were observed in sera even after administration of 10 CRIT units of HSV antigens. The CR+ antibody titres detected after administration of 20 C1%IT units were higher than after 10 units; however, a further increase in antigen dose did not result in a further increase in antibody titre. Paradoxically, the type 2 CR + antibody levels were somewhat lower after high than after low antigen doses. On the other hand, the production of CR- antibodies (both type-specific and t)~e-common) was dependent on the antigen dose. Their levels gradually increased with antigen dose until 320 CRIT units; thereafter an increase in antigen dose was not reflected by an increased antibody titre. 20480102405120 2560-
A °'~,.o__,
Archives of Virology 61, 141--147 (1979)
© by Springer-Verlag 1979
lnmmno9enieity of Subviral Herpes Simplex Virus Preparations I. Formation of Neutralizin 9 Antibodies in Different Animal Species After Administration of Herpes Simplex Virus Solubilized Antigens By L. KUTINOVX, V. ~LICHTOVX,and V. VONKA Department of Experimental Virology, Institute of Sera and Vaccines, Prague, Czechoslovakia With 3 Figures Accepted February 9, 1979
Summary Production of neutralizing antibodies was followed in guinea pigs, rabbits hamsters and mice immunized with crude antigen extracts (AM) from human diploid cells infected with herpes simplex virus tsTpe 1. The AiM induced relatively high levels of neutralizing antibodies in all four species. The antibodies were predominantly complement-requiring and remained so even after administration of repeated AM doses. With the strains used, the antibody response was predominantly type specific and, surprisingly, the type specificity of sera usually increased after administration of repeated doses of AM. Guinea pigs seemed to be the best responsive animal species. They developed the highest levels of antibodies and complement-nonrequiring antibodies were seen in them earlier than in the other animal species. The dose-response experiments carried out in guinea pigs indicated that after a single dose administration the ratio between complement-requiring and complement-nonrequiring antibodies was dependent on the amount of antigen administered. When AM was given without adjuvant less efficient antibody production was observed than after the administration of the same amount of antigen with adjuvant.
Introduction The development of a subunit type of herpes simplex virus (HSV) vaccine is prefercd to whole virion vaccines for reasons of safety (2, 3, 5, 6). However, a number of problems associated with the development of such vaccine, including its immunogcnicity, remain to be solved. In a previous report (6) we described the production and some properties of HSV neutralization antigens extracted from human diploid cells infected with HSV type 1. In the present study, we investigated the formation of neutralizing antibodies in animals immunized with this preparation. A comparison was made
0304-8608/79/0061/0141/$ 01.40
t42
L. KuTINovX, V. SLIeI~Tov£, and V. V o ~ : A :
b e t w e e n r a b b i t s , h a m s t e r s , m i c e a n d g u i n e a pigs in r e s p e c t of t h e d e v e l o p m e n t of c o m p l e m e n t - r e q u i r i n g (CR +) a n d n o n r e q u i r i n g ( C R - ) n e u t r a l i z i n g a n t i b o d i e s . T h e t y p e s p e c i f i c i t y of t h e a n t i b o d y r e s p o n s e was also e x a m i n e d .
Materials and Methods Cells H u m a n diploid cells (LEP) derived f r o m e m b r y o lungs were c u l t i v a t e d as described (8). I n a few e x p e r i m e n t s rabbit e m b r y o fibroblasts were also used. T h e y were cultivated in the same media as the L E P cells. Virus HSV-1 (strain K O S ) and H S V - 2 (strain 196) were k i n d l y p r o v i d e d b y Dr. J . L. Melnick, B a y l o r College of Medicine, Houston. T h e viruses were g r o w n in L E P cells at a multiplicity of infection ( ~ O I ) of 0.1.-o-0.5 P F U per cell. Eagle's m i n i m a l essential m e d i u m (MEM) s u p p l e m e n t e d w i t h 5 per cent h e a t - i n a c t i v a t e d calf serum, 0.075 per cent NaHCO3 and antibiotics was used as m a i n t e n a n c e m e d i u m .
Soluble Antigen Mixture Crude antigen m i x t u r e s to be used for i m m u n i z a t i o n were p r e p a r e d as described in detail elsewhere (6). I n brief, L E P cells grown in 1200 ml l~oux bottles were infected with I-ISV-1 at MOI 0.5 P F U per cell and P a r k e r ' s i99 m e d i u m w i t h 2 per cent of calf s e r u m was added to the cultures after 2 hours of virus adsorption. The infected cells were scraped off the glass after 28-hour i n c u b a t i o n at 37 ° C. T h e y were disrupted b y t r e a t m e n t w i t h 0.5 per cent N o n i d e t P-40 (Shell Chemical Co., Ltd., London, E n g l a n d ) in isotonic reticulocyte s t a n d a r d buffer (I~SB), p H 7,4. The e x t r a c t was clarified of cell nuclei and viral nucleoeapsids b y differential eentrifugation. T h e final m i x t u r e of antigens (AM) t h a t h a d n o t s e d i m e n t a t e d after t - h o u r centrifugation at 100,000 × g was extensively dialyzed against R S B for 7 days. Absence of infectious virus was verified in L E P cells; one R o u x bottle (1200 mI) culture was inoculated w i t h 1 ml of AM and k e p t for six days at 37 ° C. Two additional passages were performed. Absence of c y t o p a t h i e changes was considered an indication of absence of a n y s u r v i v i n g virus. 51Cr-Release Inhibition Test (C R I T ) The c o n t e n t of I-ISV solubilized antigens in AMs was d e t e r m i n e d by a c y t o t o x i e i t y inhibition test as described previously (7). T w e n t y c o m p l e m e n t requiring I-ISV-I neutralizing a n t i b o d y units, 30 units of guinea pig c o m p l e m e n t and 4 × 104 cryopreserved t a r g e t H S V - I infected rabbit fibroblast cells were used in CI~IT. T h e least a m o u n t of antigen causing 50 per c e n t inhibition of 51Or release was defined as one CI~IT antigen unit. On the basis of a correlation b e t w e e n the results of C R I T and the blocking neutralization test, it can be concluded t h a t CI~IT measures the c o n t e n t of H S V neutralizing antigens (7). Immunization Procedure Chinchilla rabbits (about 3000 g), guinea pigs (400 g), y o u n g adult Syrian h a m s t e r s and white mice, strain H, (9--11 g) were i m m u n i z e d w i t h three doses of AM and the a n t i b o d y response was tested after e v e r y dose of antigen. The a m o u n t s of neutralizing antigens used for i m m u n i z a t i o n are shown in Table 1. A m i x t u r e of AM with c o m p l e t e F r e u n d a d j u v a n t was administered s u b c u t a n e o u s l y (s.c.) as t h e first dose. J~or t h e second dose, inoculated s.c. four weeks later, the .AM was m i x e d w i t h incomplete F r e u n d a d j u v a n t . The t h i r d dose, w i t h o u t a d j u v a n t , was injected i n t r a p e r i t o n e a l l y (i.p.) after a n o t h e r four-weeks interval. Serial s e r u m samples were w i t h d r a w n f r o m rabbits and gxlinea pigs, sera f r o m h a m s t e r s a n d mice were obtained b y e x s a n g u i n a t i n g 5 - - 6 animals a t each interval. The sera from each individual rabbit, guinea pig and h a m s t e r were k e p t a n d tested separately, while mouse sera were pooled.
Immunogenicity of Subviral HSV-1 Preparations
143
I n a dose-dependent response experiment, each of several AM dilutions with complete F r e u n d a d j u v a n t was administered to a group of guinea pigs. Subsequent doses were administered at the same intervals as in the first experiment. I n a s t u d y on the influence of F r e u n d adjuvant, groups of guinea pigs were immunized simultaneously with AM either mixed or not mixed with adjuvant.
Neutralization Test Sera were inactivated at 56 ° C for 30 minutes and kept at --30 ° C until being used. For the neutralization test they were diluted in two-fold steps starting from i:i0. Freeze-dried pooled guinea pig serum was used as the source of complement. Complement units were determined by the raicrotechnique described by ZXvAl)ovi et al. (i0). Undiluted serum contained 600 complement-fixing units/0.1 ml. Equal amounts of serum dilution, virus suspension (100 TCDs0/0.1 ml) and medium with or without complement (20 units of complement/0.1 ml) were mixed and incubated at 37 ° C in a water bath for I hour. Four tube cultures of LEP cells were inoculated with 0.3 ml of each mixture. The test was read after six days of incubation at 37 ° C. At least 50 per cent reduction of cytopathic effect was considered an indication of antibody presence. Serum antibody titres were determined according to K241~BEI¢ (4). For calculation of geometric mean titres (GMT), sera negative at I:I0 dilution were considered positive at I :5 dilution.
Table 1. Amounts o/ HSV-1 antigens used /or immunization o/ rabbits, guinea pigs,
hamsters and mice CRIT Dose
I~oute of inoculation
1 2 3
s.c. s.c. i.p.
antigen units~
Rabbits
Guinea pigs
Hamsters
Mice
1280 (2) b 1280 (2) 3840 (3)
1280 (2) 1280 (2) 3840 (3)
640 (1) 640 (1) 1920 (1.5)
64 (0.1) 64 (0.1) 192 (0.15)
Per dose a n d animal b Volume in ml inoculated Results
Comparison of Antibody Response in Di//erent Animal Species Titres of a n t i b o d i e s n e u t r a l i z i n g HSV-1 a n d HSV-2 a n d their c o m p l e m e n t r e q u i r e m e n t , as d e t e c t e d in sera of t h e four a n i m a l species e m p l o y e d , a r e given in F i g u r e 1. All these tests were carried o u t in L E P cells. The results o b t a i n e d in r a b b i t s , guinea pigs a n d h a m s t e r s are expressed as GMT. I n mice, t h e d a t a shown refer to s e r u m pools. I t can be seen t h a t h o m o t y p i c n e u t r a l i z i n g a n t i b o d i e s were d e t e c t e d in all a n i m a l species a f t e r one dose of a n t i g e n a n d their titres increased a f t e r t h e second a n d t h i r d doses. A f t e r t h e first dose, r a b b i t s , h a m s t e r s a n d mice p r o d u c e d c o m p l e m e n t - r e q u i r i n g (CR +) a n t i b o d i e s o n l y ; low levels of c o m p l e m e n t i n d e p e n d e n t a n t i b o d i e s were o n l y d e t e c t e d in guinea pigs. C o m p l e m e n t - i n d e p e n d e n t (CR-) h o m o t y p i c a n t i b o d i e s were d e t e c t e d in sera of all species a f t e r t h e second dose of A M ; however, in h a m s t e r s a n d mice t h e t h i r d dose of a n t i g e n was n e c e s s a r y to s t i m u l a t e s y n t h e s i s of C R - a n t i b o d i e s n e u t r a l i z i n g t y p e 2 virus. The highest a n t i b o d y response was o b t a i n e d in guinea pigs. I n mice, which received t h e least a m o u n t of AM, t h e a n t i b o d y t i t r e s were t h e lowest a n d t h e decrease in t i t r e s in t h e
I4zt
L. Ku~INOVX, V. SHeI-tTOVX, and V. VONKA:
course of two m o n t h s following t h e f o u r t h bleeding was t h e m o s t m a r k e d . F o r control purposes some sera were t e s t e d in p a r a l l e l in cultures p r e p a r e d from rabbit, e m b r y o f i b r o b l a s t s ; t h e neutralizing a n t i b o d y titres were c o m p a r a b l e w i t h those d e t e r m i n e d in L E P cells (results unshown). I n a d d i t i o n to these animals, control r a b b i t s were in same w a y i m m u n i z e d with AM p r e p a r e d from uninfected cells. Their sera were f o u n d free of a n y t I S V n e u t r a l i z i n g a c t i v i t y . This is in line with t h e previous d e m o n s t r a t i o n s t h a t AM from uninfected cultures did n o t compete w i t h H S V n e u t r a l i z i n g a n t i b o d i e s (6, 7).
10240 5"i20 2560 "1280 64,0 320 "160
A iI
/// / /
""%
80 .~ 4.01
/;, / I /
20"10r~
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Fig. 1. Neutralizing antibodies in rabbits ( A ) , guinea, pigs ( B ) , hamsters (C) and mice (1)) immunized with solubilized HSV-1 antigens. Arrows indicate the times of antigen administration, o - - - - o complement-requiring ( C R + ) t y p e 1 neutralizing antibody. × × complement-nonrequiring (CR--) type 1 neutrMizing antibody. • -- -- • CR-F type 2 neutrMizing antibody, a - - zxC R - - type 2 neutralizing antibody. A n t i b o d y fibres are expressed as the reciprocal of the highest serum dilution still neutralizing the virus. I n rabbits, guinea pigs and hamsters, titres are expressed as GMTs; in mice, titres determined in serum pools are given F r o m t h e d a t a shown in F i g u r e t, also t h e ratios between CR + a n d C R - a n d between t y p e 1 a n d t y p e 2 n e u t r a l i z i n g a n t i b o d y titres can be d e t e r m i n e d in the sera of t h e different a n i m a l species. I t can be seen t h a t the ratios b e t w e e n t y p e 1 CI~ + a n d CI{ '- a n t i b o d i e s in guinea, pigs, mice a n d r a b b i t s d i d n o t m a r k e d l y v a r y t h r o u g h o u t t h e o b s e r v a t i o n period, i n d i c a t i n g t h a t m o s t of t h e a n t i b o d y f o r m e d r e m a i n e d c o m p l e m e n t - d e p e n d e n t in spite of r e p e a t e d a n t i g e n doses. The same applies to t y p e 2 neutralizing a n t i b o d i e s f o r m e d in guinea pigs ; however, in t h e o t h e r t h r e e a n i m a l species in which t h e CR ÷ : CR r a t i o h a d i n i t i a l l y b e e n tow t h e ratios were higher in the late t h a n in t h e e a r l y serum samples. This implies t h a t
Immunogenieity of Subviral HSV-1 Preparations
145
the increase in heterotypically reactive antibody was mainly at the expense of complement-requiring antibody. In comparing the type 1 and type 2 antibody titres one sees that in all four animal species the respective ratios, i. e. type specificity, increased after the second dose, and in the case of CR- antibody even after the third dose of AM. Dependence o/Antibody Response on A M Dose In the next experiment, guinea pigs were immunized subcutaneously with one dose of 8 different dilutions of AM mixed with complete Freund adjuvant, 5 animals being used per group. The results of this dose-dependent response experiment are shown in Figure 2A. High levels of CR+ antibodies reactive with both type 1 and type 2 virus were observed in sera even after administration of 10 CRIT units of HSV antigens. The CR+ antibody titres detected after administration of 20 C1%IT units were higher than after 10 units; however, a further increase in antigen dose did not result in a further increase in antibody titre. Paradoxically, the type 2 CR + antibody levels were somewhat lower after high than after low antigen doses. On the other hand, the production of CR- antibodies (both type-specific and t)~e-common) was dependent on the antigen dose. Their levels gradually increased with antigen dose until 320 CRIT units; thereafter an increase in antigen dose was not reflected by an increased antibody titre. 20480102405120 2560-
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