Comparison of dimethyl dioctadecyl ammonium bromide, Freund's complete adjuvant and mineral oil for induction of humoral antibodies, cellular immunity and resistance to Newcastle disease

FEMS Immunology and Medical Microbiology 7 (1993) 303-314 © 1993 Federation of European Microbiological Societies 0920-8534/93/$06.00 Published by Elsevier

303

FEMSIM 00355

Comparison of dimethyl dioctadecyl ammonium bromide, Freund's complete adjuvant and mineral oil for induction of humoral antibodies, cellular immunity and resistance to Newcastle disease virus in chickens

David Katz a Itzhak Inbar a, Itzhak Samina b, Ben-Ami Peleg b and Dan E. Heller c

Israel Institute for Biological Research, Ness-Ziona, Israel, b Kimron Veterinary Institute, Bet Dagan, Israel, and c Faculty o f Agriculture, The Hebrew UniL,ersity, RehoL~ot, Israel

(Received 25 May 1993; revision received 20 July 1993; accepted 28 July 1993)

Abstract: Dimethyl dioctadecyl ammonium bromide (DDA), a lipophilic quaternary amine, was evaluated in adult chickens for potentiation of immunological responses to subcutaneously administered inactivated Newcastle disease virus (NDV) vaccines. DDA enhanced humoral and cell-mediated immune (CMI) responses to levels which were significantly higher than those induced by the vaccine alone The haemagglutination inhibition antibody titers induced by DDA were slightly lower than those induced by mineral oil although neutralizing antibody titers seemed to be higher. DDA induced strong CMI (DTH and lymphocyte proliferation) responses, more than those induced by Freund's complete adjuvant and mineral oil. Both DDA and mineral oil induced comparable high levels of protection to challenge doses of 200000 LDs0 per chicken. No toxic effects or local tissue damage were observed in any of the inoculated chickens.

Key words." Immunological adjuvants; Dimethyl dioctadecyl ammonium bromide; Freund's complete adjuvant; Mineral oil; Newcas- tle disease virus; Antibody; Cell-mediated immunity; Protection

Introduction

I m m u n o l o g i c a l ad juvan t s a r e o f m a j o r i m p o r -

t a n c e as a g e n t s for p o t e n t i a t i o n o f t he i m m u n e

Correspondence to." D. Katz, Israel Institute for Biological Research, P.O.B. 19, Ness-Ziona 70450, Israel.

r e s p o n s e to i n a c t i v a t e d vi ra l vacc ines . O u t o f t h e

m a n y e x p e r i m e n t a l ad juvan t s , on ly m i n e r a l salt

ad juvan t s a re l i c ensed fo r h u m a n use and only

two a d d i t i o n a l ad juvan t s , m i n e r a l oi l a n d s a p o n i n

(or its p u r i f i e d de r iva t ive , Q u i l A) , a re l i c e n s e d

fo r v e t e r i n a r y use. H o w e v e r , t h e s e ad juvan t s a r e n o t en t i r e ly w i t h o u t s ide e f fec t s and on ly pa r t i a l ly

e f f ec t ive fo r c e r t a i n v i ra l d i seases [1-6] . T h e q u a -

304

ternary ammonium lipoid compound, dimethyl dioctadecyl ammonium bromide (DDA) is one of the many synthetic materials with adjuvant activity which was discovered and studied as part of the effort for finding new, safe and effective immunostimulators [7-9].

In a previous study [10], we compared, in mice, the immunopotentiating properties of DDA to that of two other well-known adjuvants, Freund's complete adjuvant (FCA) and aluminium hydroxide. The study, in which inactivated Semliki For- est virus (SFV) served as the immunogen, indicated the superiority of DDA in inducing delayed-type hyersensitivity (DTH). DDA enhanced humoral antibodies also but to a similar extent as FCA and aluminium hydroxide. However, it had a typical effect on IgG-isotype switching: it induced a high proportion of IgG2a and IgG2b antibodies and low proportions of IgG1 antibodies. This antibody isotype pattern was similar to the pattern induced by FCA and different from that induced by aluminium hydroxide or by the antigen alone.

In this study, the efficacy of DDA as an adjuvant in chickens for inactivated Newcastle disease virus (NDV) vaccines was tested. This experimental system, which was used before in a short study by Ruke et al. [11], is as close as possible to real field conditions since NDV is also causing a se- vere disease in chickens and is of great concern for the poultry industry all over the world.

There are numerous commercial NDV vaccines, live and inactivated, which perform satis- factorily in the control of the disease in nature. Live attenuated vaccines are administered mainly to young chicks while inactivated vaccines are used mainly for laying hens. Most of the inactivated NDV vaccines are adjuvanted by mineral oil which are effective but do produce local reac- tions at the site of inoculation [12].

The adjuvant effects of DDA, FCA and mineral oil were compared in chickens which were inoculated subcutaneously in combination with inactivated NDV. Immunity was evaluated by determination of antibody response, DTH, lymphocyte proliferation and resistance to challenge with virulent NDV.

Materials and Methods

Virus NDV (strain Komarov II) was propagated in

Specific Pathogen Free (SPF) 10-day-old, embryonated eggs. Each egg was inoculated into the allantoic cavity with 0.1 ml from a 1:1000 dilution of the seed virus by standard procedures [13]. Eggs containing dead embryosl after 24 h, were discarded. Live embryos were further incubated (37°C) for three days. At the end of the incubation, the eggs were cooled for a few hours at 4°C. The allantoic fluids were harvested individually from each egg and monitored for NDV activity by a qualitative haemagglutination slide test. Haem- agglutinin (HA) positive fluids were pooled, clari- fied by centrifugation (2000 rpm for l0 rain) and stored at -70°C until used. These NDV stocks, thus prepared, were titrated in embryonated eggs by the allantoic route of inoculation. Ten-fold dilutions (10-5-10-10) were prepared in phospate buffered saline (PBS), inoculated into 9-10-day- old embryonated eggs and incubated at 37°C for 72 h. Five eggs were used for each dilution. At the end of the incubation, small samples of the allantoic fluids were withdrawn and tested for HA (slide test). The 50% egg infective dose (EIDs0) was determined by the Reed-Muench method [13].

For inactivation, part of the NDV stock was treated by /3-propiolactone as previously described [10]. The virus was centrifuged in a Beck- man ultracentrifuge at 90 000 × g for 100 rain and resuspended in PBS to one tenth of its original volume. Samples of the concentrated NDV preparations were tested in embryonated eggs for residual live NDV. Inactivation was considered successful when no residual viral infectivity was found. The inactivated antigen preparations were stored in -70°C until used.

A portion of the virus stock was further purified by ultracentrifugation (Beckman, Palo Alto, USA) on a 5%-50% sucrose gradient, in a SW-28 rotor, for 60 rain at 9 0 0 0 0 × g [14]. Fractions, with the majority of HA activity, were pooled and centrifuged in a R-65 rotor, at 100000 × g for 90

min. The pellet was resuspended in PBS to one seventh of the original volume.

N D V haemagglutinins were titrated in 0.05 ml quantities, in a micro-hemagglutination test, performed in round-bottom 96-well microplates, with 0.75% chick red blood cells (ChRBC). The titre (number of H A units per 0.05 ml) was defined as the reciprocal of the last test dilution that still demonstrated haemagglutination [13].

Estimation of the relative purity of viral preparations

The relative purity (RP) of viral preparations, expressed as H A mg-~ protein, was estimated by dividing the number of H A units per ml to the protein concentration (rag ml-~).

Protein concentrations were determined by Bradford's method [15], in comparison to a bovine serum albumin (BSA) standard.

Virus-adjuvant mixtures D D A (Eastman Kodak, USA) was prepared by

suspension of the powder in sterile-pyrogen free physiological saline (Travenol, Israel). A fine ho- mogenous dispersion of the powder was obtained by heating the suspension to 60°C for 15-20 min. After cooling at room tempera ture the D D A suspension was mixed with an equal volume of the inactivated N D V (diluted in saline) and rocked for 20-60 min at room temperature. The concentration of D D A used for each experiment will be recorded below in Results. FCA (Difco, USA) was emulsified with an equal volume of the inactivated virus suspension (in saline) by vigor- ous mixing on a vortex mixer. Emulsions in mineral oil were prepared in a similar way.

Immunization of chickens For determination of humoral antibodies and

resistance to challenge, White Leghorn roosters were reared at the veterinary institute (Bet- Dagan). At the age of 5 weeks they were vaccinated subcutaneously with 0.5 ml of inactivated experimental N D V vaccines or with commercial vaccines. The amount of virus per dose in the experimental vaccine which was prepared from the concentrated stock (RP = 400000 H A ml - l )

305

was 500 H A units which is equivalent to 4.5 x 108 EIDs0 before inactivation. Commercial vaccines contained approximately the same amount of virus per dose.

For determination of delayed-type hypersensitivity White Leghorn roosters were reared at the Faculty of Agriculture in Rehovot, first up to the age of 4 weeks in an electrically heated battery and then in separate cells in regular batteries. For performance of the lymphocyte proliferation assay, roosters were reared up to the age of 5 weeks under sterile conditions in isolator units. Before the beginning of the experiments they were transferred to regular batteries.

All chickens were wing-banded and bled before vaccination. They were kept in pairs and fed on commercial feed and water ad libitum.

Evaluation of humoral immunity Antibody determination by the haemagglutina-

tion inhibition test. The haemaglutination inhibition (HI) test was performed essentially as described elsewhere [16]. Briefly, 0.05 ml of two-fold dilutions of sera in PBS were prepared in U- shaped, 96-well microtitre plates (Dynatech, USA). To each of the wells, 0.05 ml of 4 H A units of N D V were added and incubated for 20 min at RT. Chicken red blood cells (0.75% in PBS) were added, and incubated at 4°C for 60-90 min. The reciprocal of the last serum dilution showing an inhibition of haemagglutination was considered as the HI antibody titre of the serum.

Determination of neutralizing antibodies by the haemadsorption inhibition test. The haemadsorption inhibition (HAdI) test was performed in chick embryo fibroblasts, in microplates as described elsewhere [17]. First, the titre of the virus was determined in HAd units. For titration of HAdI antibodies (neutralizing antibodies) two-fold dilutions of chicken sera were prepared in PBS and incubated for 45 min at 37°C with 100 HAd units of NDV. Cells, at a concentration of 1-1 × 10 6

0.05 ml per well, were added and incubated for 48 h at 37°C. Chicken red blood cells (0.75% in PBS) were added for 1 h at 4°C. The reciprocal of the last serum dilution showing an inhibition of haemadsorption was considered as the HAdI antibody titre of the serum.

306

Evaluation of cellular immunity Induction and evaluation of delayed-type hyper-

sensitivity. Delayed-type hypersensitivity (DTH) in immunized roosters was measured as described by Hudson and Hay [18]. Groups of roosters, at the age of 7-12 weeks, were inoculated subcutaneously with different compositions of adjuvants and NDV. Ten days later, roosters were inoculated into the right wattle with 101° EIDs0 of NDV and into the left one with saline. Thicken- ing of wattles was measured with calipers (accu- racy = 0.01 cm) 24 h after inoculation or later. At the end of the experiment the width difference between the right and the left wattle for each rooster was calculated.

The lymphocyte proliferation assay. The lymphocyte proliferation assay was performed essentially as described by Timms [19]. Groups of roosters at the age of 5 weeks were first inoculated subcutaneously with combinations of adjuvants and inactivated NDV and boosted later with inactivated NDV alone. The precise sched- ule will be described in the Results section. Hep- arinated blood (5-10 ml) was taken from each bird, and centrifuged at 50 x g for 13 min. Lym- phocytes, derived from the upper layer, were washed in RPMI-1640 supplemented with L- glutamin and antibiotics (1 in 100 dilution of penicillin and streptomycin, Beit-Haemek, Israel), counted and distributed, 2 × 106/well, to microplates (Nunc, Denmark). Peripheral blood lymphocytes from immunized and control chickens were stimulated in vitro in triplicate with: (1) medium alone, (2) inactivated NDV, (3) purified

protein derivative (PPD) and (4) concanavaline A (ConA). NDV and PPD were used at different concentrations, and ConA was used at a concentration of 20 /xg ml 1. Plates were incubated at 39°C, in a gas atmosphere of 5% CO 2 and 80% humidity for 48 or 72 h. Twenty /xl of 1 ~zCi of 3H-thymidine were added to each well. After 18 h, cells were harvested in a cell harvester (Dy- natec, USA) and transferred to vials containing scintillation fluid for radioactivity measurements in a Beckman LS 7800 counter Stimulation indices (SI), representing the degree of thymidine incorporation and lymphocyte proliferation, were calculated by dividing the radioactive counts obtained from wells containing antigens or non- specific stimulators by the counts of the non- stimulated (medium containing) control wells.

Resistance to challenge Chickens, 3 to 4 weeks after immunization,

were transferred for challenge to a special isola- tion unit of the Veterinary Institute. Each chicken received, intramuscularly, 200000 LDs0 of an Is- raeli velogenic strain. Mortality was recorded daily for 3 weeks after infection.

Statistical analysis ELISA or DTH results were expressed as av-

erage values +_ standard error. The statistical significance of differences between means was determined by Student's t-test. In challenge experiments the chi-square test was used for the determination of statistical significance between groups.

Table 1

Titration and characterization of virus preparations

Virus Virus titer preparation EIDs0 ml 1 HA 0.05 ml- 1 HA ml 1

Protein Relative purity mg m l - I (HA mg 1 protein)

Stock from embr. eggs 4.5 × 109 500 10000 ND ND

Virus after BPL inactivated ND 500 10 000 0.8 12 500 Ultracent- rifuged ND 10 000 200 000 0.5 400 000

Purified (gradient) ND 4 000 80 000 0.04 2 000 000

Results

Characterization of virus preparations N D V (Komarov strain) was grown in embry-

ona ted eggs, harvested from the al lantoic fluid and inactivated by /3-propio lac tone . Virus stocks were purif ied and concen t ra ted by ul t racentr i fu-

gat ion and sucrose densi ty-gradient centr ifuga- tion. The virus conten t and relative purity of each of the prepara t ions were tested and summarized

in Table 1.

Effect of adjuvants on resistance to NDV challenge and antibody response to NDV after one single inoculation of vaccines

Exper iments were per formed to study the adjuvant effect of D D A in inactivated N D V vaccines on resistance to N D V chal lenge and on the ant ibody response.

In one experiment , groups of chickens were inocula ted with the inactivated N D V prepara t ion without additives and with prepara t ions conta in- ing minera l oil and different amounts of DDA. Each chicken received 500 H A units per dose which were ob ta ined by di lut ion of the concen- t ra ted stock p repara t ion (RP = 12500 H A m g - 1 ) in saline. In addit ion, one group was inocula ted with the commercial minera l oil-based vaccine of M A B A T and another group was kept un t rea ted . Each group consisted of 10 chickens, except for

307

the last group (the un t rea t ed ) which consisted of 20 chickens. Chickens were bled on the day of vaccinat ion (day 0) and 20 days later for determi- na t ion of HI antibodies. Nine days after the last b leeding they were chal lenged with virulent NDV. No ant ibodies were found in sera from day 0. Results of HI ant ibodies in se rum from blood drawn 20 days after vaccinat ion and of survival

rates after chal lenge are summar ized in Table 2. The adjuvant effect of D D A (groups 1-4) after a

single inocula t ion is, in general , similar to the effect of minera l oil (group 6). The commercial vaccine ( M A B A T ) induced more ant ibodies than

any o ther vaccine. All adjuvant p repara t ions ( D D A or minera l oil) induced statistically significant more ant ibodies than group 5 (NDV without adjuvant). Survival rates were also significantly higher in the groups injected with the adjuvant conta in ing vaccines. The individual survival rates of these groups (70 -90%) were not statistically different.

The aim of the second exper iment was de- signed to study the effect of the relative purity of the N D V ant igen on the adjuvantici ty of DDA. N D V prepara t ions with different relative puri t ies were ob ta ined by addi t ion of appropr ia te amounts of al lantoic fluid and saline to the concen t ra ted N D V prepara t ion . The final concen t ra t ion of N D V in each p repara t ion was equal (500 H A units per dose). F i f teen groups of chickens were

Table 2

The adjuvant effects of DDA and mineral oil in inactivated NDV vaccines on resistance to virulent NDV challenge and on antibody response in vaccinated chickens

Group n Material Antibodies Survival no. inoculated x HI + SE y S.G. z % S.G. z

1 10 NDV+ DDA (1 mg m1-1) 3.8+0.51 bc 70 a 2 10 NDV + DDA (4 mg ml- 1) 3.9 _+ 0.38 bc 70 a 3 10 NDV+ DDA (8 mg ml - l ) 3.3_+0.47 c 70 a 4 10 NDV + DDA (16 mg ml - 1) 4.1 + 0.52 bc 90 a 5 10 NDV + saline 2.1 + 0.10 d 10 b 6 10 NDV + mineral oil 4.9 +_ 0.45 b 100 a 7 10 MABAT 6.9 _+ 0.60 a 80 a 8 20 Not treated 0.9 + 0.25 e 0 b

x Concentrations of DDA in the vaccine. Y HI values expressed as log 2 +S.E. z S.G. = Significance group. Different groups of HI values (a to e) are statistically different using the t test and the groups of

survival values (a, b) are statistically significant by the chi square test (P < 0.05).

308

used as d e s c r i b e d in T a b l e 3. G r o u p s 1 to 4 w e r e

i n j ec t ed wi th 0.5 m g D D A p e r d o s e (1.0 m g ml - t )

m i x e d wi th 4 N D V p r e p a r a t i o n s , e a c h wi th a

d i f f e r e n t r e l a t ive pur i ty . T h e s a m e 4 d i f f e r e n t

N D V p r e p a r a t i o n s w e r e i n j e c t e d to g r o u p s 5 to 8

bu t w i th a D D A c o n c e n t r a t i o n o f 4.0 m g m l - 1

(2.0 m g p e r dose) ; G r o u p s 9 and 10 w e r e i nocu -

l a t ed wi th only two N D V p r e p a r a t i o n s ( R P =

12500 H A m g -1 and R P = 4 0 0 0 0 0 H A m g -~)

e a c h m i x e d wi th 16.0 m g m l - ~ o f D D A (8.0 m g

p e r dose) . T h e 5 r e m a i n i n g g r o u p s s e r v e d as

con t ro l s : g r o u p 11 was i n j ec t ed wi th t h e c o m m e r -

cial v a c c i n e ( M A B A T ) , g r o u p 12 wi th N D V in

sa l ine ( R P = 400000 H A m g - 1 ) , g r o u p 13 wi th

N D V in a l l an to i c f lu id ( R P = 12500 H A m g -1 )

g r o u p 14 wi th a l l an to i c f lu id w i t h o u t v i rus and

g r o u p 15 was lef t u n t r e a t e d . C h i c k e n s w e r e b l e d

on day 0 and 21 fo r d e t e r m i n a t i o n o f H I a n t i b o d -

ies. O n e w e e k a f t e r t he last b l e e d i n g t h e y w e r e

c h a l l e n g e d wi th v i r u l e n t N D V . N o a n t i b o d i e s

w e r e f o u n d in day 0 sera . R e s u l t s o f H I a n t i b o d -

ies (all g roups ) a n d n e u t r a l i z i n g ( H A d I ) an t i bod -

ies (g roups 9 to 15 only) in day 21 sera , and o f

survival ra tes a f t e r c h a l l e n g e a re s u m m a r i z e d in

T a b l e 3.

T h e ad juvan t i c i ty o f D D A is c o n f i r m e d o n c e

aga in in this e x p e r i m e n t s ince b o t h a n t i b o d y re-

s p o n s e and r e s i s t ance to c h a l l e n g e w e r e signifi-

can t ly h i g h e r in t h e g r o u p s o f ch i ckens wh ich

w e r e v a c c i n a t e d wi th D D A - c o n t a i n i n g vacc ines

( g roups 1 to 10) t h a n in t he ch i ckens wh ich w e r e

i n o c u l a t e d wi th t he i nac t i va t ed vi rus w i t h o u t ad-

j uvan t s (g roups 12 and 13). D D A also i n d u c e d

re la t ive ly h igh levels o f n e u t r a l i z i n g a n t i b o d i e s

( g roups 9 and 10). T h e r e l a t ive pur i ty o f t he

D D A - c o n t a i n i n g v a c c i n e s d id n o t a f fec t signifi-

can t ly t he H I a n t i b o d y r e s p o n s e , t h e n e u t r a l i z i n g

a n t i b o d y leve l ( H A d I ) a n d r e s i s t ance to chal -

l enge .

The effect of adjuvants on the cellular immune response

C e l l u l a r i m m u n i t y was t e s t e d in v a c c i n a t e d

ch i ckens by two t e c h n i q u e s , o n e for d e t e r m i n a -

Table 3

The effect of preparations

the relative purity of NDV on antibody response and immunity in chickens vaccinated with DDA-containing

Group n Material Relative Antibodies Survival no. inoculated x purity y HI -+ S.E. z S.G. * HAdI * * % SG *

1 15 NDV+ DDA (1 mg m l - b 12500 2.0_+0.43 c 66 a 2 14 NDV+DDA (1 mg ml -~) 40000 3.3_+0.49 a 78 a 3 14 NDV+DDA (1 mg ml ~) 125000 2.3_+0.45 c 71 a 4 15 NDV + DDA (1 mg ml - t ) 400 000 2.5 _+ 0.52 c 73 a 5 13 NDV+DDA (4 mg ml ~) 12500 3.0+0.35 b 84 a 6 15 NDV+DDA (4 mg ml i) 40000 3.9_+0.38 a 73 a 7 15 NDV+DDA (4 mg ml 1) 125000 2.8_+0.47 b 93 a 8 15 NDV+DDA (4 mg ml - l ) 400000 2.8_+0.46 b 93 a 9 14 NDV+ DDA (16 mg m1-1) 12500 4.1 _+0.61 a 5.2 71 a

10 15 NDV+DDA (16 mg ml i) 400000 5.0_+0.57 a 5.8 73 a 11 30 MABAT 4.5_+0.56 a 2.1 80 a 12 13 NDV + saline 400 000 2.1 _+ 0.71 c 1.5 23 b 13 15 NDV + AI. fluid 12500 1.0_+0.27 d 1.0 13 b 14 10 Allantoic fluid 0.8 -+ 0.25 d 0.5 0 b 15 25 Not treated 0.3 -+ 0.20 e 0.5 8 b

x Concentration of DDA in the vaccine. Y Relative purity of NDV preparation = HA/mg protein. z HI values expressed a s log 2 +S.E. * SG = Significance group. Different groups of HI values (a to c) are statistically different using the t test and the groups of

survival values (a and b) are statistically different by the chi-square test (P < 0.05). ** HAdl = Haemadsorption inhibition test performed on blood pools (only for groups 9 to 15).

309

Table 4

DTH in two groups of 5 chickens vaccinated for two weeks with NDV + FCA and NDV + DDA and tested at 24, 48 and 96 h after inoculation of wattles

Vaccine Hours a DTH b

NDV + FCA 24 0.41 _+ 0.12 48 0.24_+0.11 96 0.07 _+ 0.10

NDV + DDA 24 0.58_+ 0.18 48 0.25 _+ 0.10 96 0.14_+0.12

a Hours after inoculation of wattles at 2 weeks after vaccination.

b Expressed as mean width difference _+ S.E. (mm) between the right wattle which was inoculated with NDV antigen and the left wattle which was injected with saline.

t ion of D T H and the second for de te rmina t ion of lymphocyte proliferat ion.

The effect of adjuvants on the DTH responses in uaccinated chickens. The op t imal t ime for mea- suring D T H in the wattles of vaccinated chickens was de te rmined in two groups of 5 chickens. One group was inocula ted with an emuls ion of inactivated N D V and F C A and the second with a mixture of inact ivated N D V and DDA. Two weeks after vaccinat ion s t imulat ion was performed by inocula t ing the right watt le with inactivated N D V and the left wattle with saline. The widths of the wattles were measured 24, 48 and 96 h after s t imula t ion (Table 4). Significant differences ( P < 0.05) be tween the right and left wattles were observed only 24 h after s t imulat ion

which indicated that the opt imal t ime for measur- ing D T H responses in vaccinated chickens was 24 h after an in t radermal inocula t ion of the st imulat- ing ant igen into the wattles.

In a second exper iment chickens were induced for D T H responses at different t imes after vaccina t ion (7, 14 and 28 days) with mixtures of N D V and DDA. Twenty-four hours after inocula t ion of right wattles with an t igen and left wattles with saline, widths of wattles were measured and the differences be tween the right and left wattles were calculated. A fourth control group of chickens was not vaccinated but was s t imulated by inocula t ion of right (NDV) and left (saline) wattles 14 days after the onset of the experiment . Results, summarized in Table 5, indicate that within the per iod of about 1 to 3 weeks after vaccinat ion, similar D T H responses were ob- ta ined in all 3 groups of vaccinated chickens and that the average width differences induced in the wattles were significantly higher than the one induced in the control group.

The inf luence of adjuvants on the D T H response in vaccinated chickens was compared in 6 groups of chickens as out l ined in Fig. 1. Three groups of chickens were used for compar ison of three adjuvants, minera l oil (in the commercial vaccine of MABAT) , D D A and FCA. The other three groups served as controls, one for the effect

of D D A alone, the second for the effect of inactivated N D V without additives and the third which was inoculated with saline only. Eight days after vaccinat ion, chickens were tested for specific D T H responses to NDV. All ad juvants i n d u c e d

Table 5

DTH at different times after vaccination of chickens with an NDV vaccine containing 4.0 mg ml- 1 DDA. Widths of wattles were measured at 24 h after inoculation with inactivated NDV

Group n Material Days after DTH y S.G. z No. inoculated × vaccination

1 9 NDV + DDA (4 mg ml - 1) 7 1.70 _+ 0.17 a 2 9 NDV + DDA (4 mg ml - 1) 14 1.80 _+ 0.21 a 3 10 NDV+DDA (4 mg ml i) 28 1.60_+0.15 a 4 11 Not treated 14 0.06 _+ 0.02 b

x Concentration of DDA in the vaccine. Y Expressed as mean width difference+ S.E. (mm) between the right wattle which was inoculated with NDV antigen and the left

wattle which was injected with saline. z S.G. = Significance group. Different groups statistically different by the t-test (P < 0.05).

310

2.0

1.0

0.0 MABAT NOV+ODA NDV-FCA ODA NDV+SAL SALINE

n=14 n=14 n=14 n=8 n=8 n=5

EXPERIMENTAL GROUPS

Fig. 1. Effect of adjuvants on DT H responses in NDV vaccinated chickens. Three groups of chickens were inoculated with mixtures of NDV and adjuvants ((NDV + FCA, N D V + D D A and NDV in mineral oil (MABAT)). The other three groups which served as controls were inoculated with D D A alone, with NDV without additives and with saline. The number of chickens per group (n) is indicated. Eight days after vaccination, chickens were tested for specific DT H responses to NDV expressed as the width differences of right and left wat t les+S.E. The letters on the columns indicate significance groups which are statistically different using the

t-test (P < 0.05).

DTH responses which were higher than that induced by the virus alone. DDA induced the high- est DTH response, FCA an intermediate response and mineral oil the lowest response (Fig. 1).

The effect of adjuvants on the lymphocyte proliferation assay in vaccinated chickens. The aim of the first experiment was to determine optimal conditions for stimulation of peripheral blood lymphocytes from NDV vaccinated chickens with inactivated NDV antigen preparations. Out of a total of six (5-weeks-old) chickens, 2 were inocu-

lated subcutaneously with saline, 2 with inactivated NDV (500 H A / d o s e ) and 2 with a mixture of NDV and DDA (500 HA of NDV and 2 mg of DDA/dose) . Each chicken received 2 more injections at 14 day intervals. Blood was tested by the lymphocyte proliferation assay at 10 days after each inoculation. Half of the cultures were stimulated after 48 h and the other half after 72 h, for comparison. All lymphocyte preparations were stimulated with ConA (20 Izg ml - ] ) and with 4 concentrations of the purified NDV (250, 500, 1000 and 2000 HA units); results are summarized in Table 6. Since lower SI values were obtained with 250 and 2000 HA units of NDV, they were omitted from the table. Also, only results which were obtained after the third inoculation of the chickens are shown, since no proliferation of lymphocytes was obtained after the first or the second injections. From this experiment it is evident that optimal conditions for stimulation of lymphocytes were obtained in 48 h cultures with both ConA and the NDV antigen. The optimal concentration of the NDV preparation for stimulation was 500 HA units.

In another experiment we compared the effect of 3 adjuvants, DDA, FCA and mineral oil (MABAT) which were incorporated in 3 inactivated NDV vaccines, on the lymphocyte proliferation assay. In addition, two control groups of chickens were included, one of which was inoculated with saline and another which was inoculated with inactivated NDV without adjuvants. The amount of NDV in the FCA- and DDA-containing vaccines was 500 HA units per dose. The

Table 6

Determination of optimal conditions for lymphocyte proliferation assays with peripheral

Chickens Blood assayed In vitro Stimulation index after sensitization with: inoculated at day after incubation

NDV NDV with: 3rd injection time (h) 500 HA 1000 HA

Saline 10 48 1.0 0.9 70 10 72 0.9 0.8 40

NDV + saline 10 48 1.8 1.5 100 10 72 1.3 1.3 30 10 48 12.4 6.2 10 72 3.0 1.8

NDV + D D A

blood from chickens vaccinated with NDV

ConA 20/zg ml i

150 25

concentration of DDA (in the NDV + DDA vaccine) was 4.0 mg m1-1 (2 mg/dose) . The four groups of chickens which were inoculated with NDV received a booster injection 10 days later which consisted of 500 HA units of inactivated virus (without adjuvants). Chickens were bled at the day of booster and 8 days later and tested for HI antibodies and for cellular immunity by the lymphocyte proliferation assay. Lymphocytes were stimulated with PPD (50 /xg ml - ] ) and with 500 HA units of gradient purified NDV. Significant stimulation values were obtained only after the second injection. Results are summarized in Fig. 2A (stimulation with NDV) and Fig. 2B (stimulation with PPD). HI values for each group of chickens are also shown for comparison (Fig. 2A).

311

Discuss ion

The adjuvant activity of DDA is already well established. However, different results were obtained with different experimental systems. In some systems it induced only cell-mediated immunity in the absence of antibodies and in other systems it enhanced antibody production [8,9]:

In the present study, one single inoculation of the inactivated NDV-DDA mixtures in chickens induced enhanced responses of antibodies, cell- mediated immunity and protection to challenge. In one experiment (Table 2), no clear dose effect on HI antibody titres was observed with doses of DDA ranging from 0.5-8.0 mg (1.0-16.0 mg ml-]) . A somewhat higher response, similar to

SI

Sl

12 A

10

8

6

4

2

0

30

20

10

Stimulated wrth NDV

S A L I N E n=3

a ,;

N D V + S A L M A B A T n=2 n=4

EXPERIMENTAL GROUPS

N D V + D D A N D V - F C A n=4 n=4

-$

- 6

- 4

- 2

-0

HI

S A L I N E N D V + S A L M A B A T N D V + D D A N D V - F C A n=3 n=2 n=4 n=4 n=4

EXPERIMENTAL GROUPS

Fig. 2. Effect of adjuvants on stimulation indices (SI) obtained with peripheral blood lymphocytes from NDV vaccinated chickens. The four groups of chickens that were inoculated with NDV received a booster injection ten days later which consisted of 500 H A units of inactivated virus (without adjuvants). Resul ts are from blood taken (from all groups) 8 days after the booster injection. In (A) lymphocyte cultures were st imulated with purified NDV antigen (500 HA), and in (B) with PPD (50 /xg ml-1) . Bars indicate

s tandard errors. HI antibody titres, expressed as Log 2, are also shown (A). Other symbols are as described in Fig. 1.

312

the commercial mineral oil vaccine, was obtained with the 8.0 mg /dose in another experiment (Ta- ble 3). In general, on the basis of all experiments (Tables 2 and 3), higher levels of HI antibodies were obtained with the commercial mineral oil vaccine than with most of the DDA-containing preparations. DDA, however, induced high levels of neutralizing antibodies (HAdI) to NDV, higher than those induced by the antigen alone or by the commercial mineral oil vaccine (Table 3). It seems therefore that DDA has a differential influence on the type of antibodies to be produced. Since this finding is based on the results of one experiment, we regard it as a preliminary finding that should be confirmed by more experiments in the future.

In all our experiments (Tables 2 and 3) DDA induced an excellent immunological resistance to a high dose challenge of virulent NDV (200000 LDs0) which was comparable to the resistance induced by the commercial mineral oil vaccine. This resistance also seemed to be dose-indepen- dent, at least within the range of DDA concentrations tested. It is possible that, in order to see a clear dose-dependence, lower doses of DDA should have been used. A hint for this can be found in the experiment of Ruke et al. [11] who obtained a lower survival rate with a dose of 0.1 mg (53%) than with 0.5 mg and 2.5 mg (67 and 73%, respectively).

As already discussed in our previous paper [10] there is no agreement as to the exact mechanism of action of DDA. One group of authors man- tains that the adjuvanticity of DDA is indepen- dent of the presence of antigen [20-23] while others have shown the importance of direct binding of the antigen to DDA or simultaneous addition of the DDA and antigen at the same inoculation site [24-26]. In the present study, DDA vaccines which contained NDV antigen preparations of different purities were compared (Table 3). Assuming that binding of DDA to the antigen is important for its adjuvant activity, we believed that purified antigen preparations will interact more efficiently with DDA than crude preparations and therefore will elicit a better immune response. Surprisingly, the relative purity of the NDV antigen in the DDA containing vaccines

had no effect on the nature or intensity of the immune response. We have no clear-cut explana- tion for this result. It could be that the impurities of the allantoic fluid interfere with the binding of the NDV antigen to DDA but binding is not a prerequisite for its adjuvant activity or, alterna- tively, binding of DDA to the antigen is essential for adjuvanticity yet DDA has the capacity to adsorb the NDV antigen in the presence of impurities.

DDA had a substantial influence on enhancing DTH (Fig. 1) and lymphocyte proliferation (Fig. 2) responses to NDV antigens in chickens which were vaccinated with NDV-DDA mixtures. The degree of responses with DDA-containing vaccines was significantly higher than with vaccines adjuvanted by FCA or mineral oil. These results, which are in agreement with other authors, con- firm once more the efficacy of DDA as an im- munopotentiator of the cellular arm of the immune system. However, this capacity of DDA is most probably not universal since DDA was less effective in enhancing immunity to non-enveloped viruses like encephalomyocarditis virus [27] and foot and mouth disease [28]. In view of the possible mechanisms of action of DDA it seems that immunopotentiation is dependent on the nature of the antigen which presumably dic- tates the correct physicochemical interaction with the adjuvant and the most efficient configuration for presentation to the immune system. It might be very interesting to find the general rule for this interaction. This will enable exploitation of this remarkable adjuvant effect of DDA for a broader spectrum of antigens.

This study has proven the efficacy of DDA as an adjuvant for improving the immune response to inactivated NDV vaccines in chickens. As previously reported for mice [10], we have not observed any undesirable, general or local, side effects in chickens inoculated subcutaneously with the DDA-containing vaccines.

Acknowledgements

We are grateful for the skillful technical assis- tance of Mrs. Dora Shehori from the Kimron

V e t e r i n a r y I n s t i t u t e , B e t D a g a n , a n d o f Mrs .

R i m a W e i m a n f r o m t h e F a c u l t y o f A g r i c u l t u r e ,

R e h o v o t .

References

1 Gregoriadis, G. (1990) Immunological adjuvants: a role for liposomes. Immunol. Today 11, 89-97.

2 Allison, A.C., Byars, N.E. and Waters, R.V. (1986) Im- munological adjuvants: Efficacy and safety considerations. In: Advances in Carriers and Adjuvants for Veterinary Biologics (Nervig, R.M., Gouch, P.M., Kaeberle, M.L. and Whetstone, A.C., Eds.), pp. 91-103. The University Press, Ames, Iowa.

3 Allison, A.C. and Byars, N.E. (1986) An adjuvant formula- tion that selectively elicits the formation of antibodies of protective isotypes and of cell mediated immunity. J. Im- munol. Methods 95, 157-168.

4 Stewart-Tull, D.E.S. (1985) Immunopotentiating conju- gates. Vaccine 3, 40-44.

5 Warren, S.H. and Chedid, L.A. (1988) Future prospects for vaccine adjuvants. CRC Crit. Rev. Immunol. 8, 83-101.

6 Morein, B. and Hoglund, S. (1990) Subunit vaccines against infection by enveloped viruses. In: Advances in Biotechno- logical Processes, Viral Vaccines, (Mizrahi, A., Ed.), pp. 69-90. Wiley-Liss Inc., New York.

7 Gall, D. (1966) The adjuvant activity of aliphatic nitroge- nous bases. Immunology 11,369-386.

8 Hilgers, L.A.T., Zigterman, G.J.W. and Snippe, H. (1989) Immunomodulating properties of amphiphilic agents. In: Autoimmunity and Toxicology (Kammuller, M.E., Bloksma, N. and Seinen, W., Eds.) pp. 293-306. Elsevier Science Publishers B.V. Amsterdam.

9 Katz, D., Kraaijeveld, C.A. and Snippe, H. Synthetic lipoid compounds as antigen-specific immunostimulators for improving the efficacy of killed viral vaccines. In: Adjuvants- Theory and Practical Applications (Stewart-Tull, D.E.S., Ed.), Butterworth-Heinemann (in press).

10 Katz, D., Lehrer, S., Galan, O., Lachmi, B. and Cohen, S. (1991) Adjuvant effect of dimethyl dioctadecyl ammonium bromide, complete Freund's adjuvant and aliminium hydroxide on neutralizing antibody, antibody-isotype and delayed type hypersensitivity responses to Semliki Forest virus in mice. FEMS Microbiol. Immunol. 76, 305-320.

11 Ruke, E.O., Loeffen, A.H.C. and Lutticken, D. (1988) The use of lipid amines as immunopotentiators for viral vaccines. In: Advances of Immunomodulation (Bizzini, B. and Bonmassar, E., Ed.), pp. 433-443. Pithagora Press, Rome.

12 Droual, R., Bickford, A.A., Charlton, B.R. and Kuney, D.R. (1990). Investigation of problems associated with intramuscular breast injection of oil-adjuvant killed vaccines in chickens. Avian Dis. 20, 473-478.

13 Hsiung, G.D. (1982) In: Diagnostic Virology 3rd Edn., pp. 25-41. Yale University Press, New Haven and London.

14 Arora, J.S., Tremblay, P., Bourgault, R. and Boileau, S. (1985) Concentration and purification of influenza virus from allantoic fluid. Anal. Biochem. 144, 189-192.

313

15 Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utiliz- ing the principle of protein dye binding. Anal. Biochem, 72, 248-254.

16 Peleg, B., Soller, M., Ron, N., Brady, T., and Kalmar, E., (1976) Familiar differences in antibody response of broiler chickens to vaccination with attenuated and inactivated Newcastle disease vaccines. Avian Dis. 20, 661-668.

17 Katz, D., Ben-Moshe, H. and Alon, S. (1977) Titration of Newcastle disease virus and its neutralizing antibodies in microplates by a modified hemadsorption and hemadsorption inhibition method. J. Clin. Microbiol. 3, 227-232.

18 Hudson, L. and Hay, F.C. (1976) In: Practical Immunol- ogy, pp. 63-64. Blackwell Scientific Corporation, Oxford, UK.

19 Timms, L.M. (1979) Correlation of the lymphocyte trans- formation, leucocyte migration and the delayed cutaneous hypersensitvity tests in chickens sensitised to Mycobac- terium tuberculosis. Res. Vet. Science 27, 347-353.

20 Gordon, W.C., Prager, M.D. and Carroll, M.C. (1980) The enhancement of humoral and cellular immune responses by dimethyldioctadecylammonium bromide. Cell. Im- munol. 49, 329-340.

21 Hilgers, L.A.T., Snippe, H., Jance, M. and Willers, LM.N. (1984) Immunomodulating properties of two synthetic adjuvants: dependence upon type of antigen, dose and time of administration. Cell. Immunol. 86, 393-401.

22 Smith, R.H. and Ziola, B. (1986) Cyclophosphamide and dimethyl dioctadecyl ammonium bromide immunopotenti- ate the delayed-type hypersensitivity response to inactivated enveloped viruses. Immunology 58, 245-250.

23 Jensen, K.E. (1986) Synthetic adjuvants: avridine and other interferon inducers. In: Advances in Carriers and Adju- vants for Veterinary Biologics (Nervig, R.M., Gouch, P.M., Kaeberle, M.L. and Whetstone, A.C., Ed.), pp. 91-103. The University Press, Ames, Iowa.

24 Dailey, M.O. and Hunter, R.L. (1974) The role of lipid in the induction of bapten-specific delayed hypersensitivity and contact sensitivity. J. Immunol. 4, 1526-1534.

25 Coon, J. and Hunter, R. (1973) Selective induction of delayed hypersensitivity by a lipid conjugated protein antigen which is located in thymus dependent lymphoid tissue. J. Immunol. 110, 183-190.

26 Hopp, T.P. (1984) Immunogenicity of a synthetic HBsAg peptide: enhancement by conjugation to a fatty acid car- rier. Mol. Immunol. 21, 13-16.

27 Kraaijeveld, C.A., Riviere, G., Boutahar-Trouw, K.B., Jansen, J., Harmsen, M. and Snippe, H. (1983) Effect of the adjuvant dimethyl dioctadecyl ammonium bromide on the humoral and cellular immune responses to encephalomyocarditis virus. Antiviral Res. 3, 137-149.

28 Katz, D., Chai, D., Wasserman, A., Reshef, A., Davidson, M. and Yadin, H. (1992) The adjuvant activity of dimethyl dioctadecyl ammonium bromide (DDA) in foot and mouth disease vaccines. The First TAHRP workshop/April 10- 16.

Documents

Comparison of dimethyl dioctadecyl ammonium bromide, Freund's complete adjuvant and mineral oil for induction of humoral antibodies, cellular immunity and resistance to Newcastle disease