adjuvant effects of dimethyl dioctadecyl ammonium bromide, complete freund's adjuvant and...

FEMS Microbiology Immunology 76 (1991) 305-320 © 1991 Federation of European Microbiological Societies 0920-8534/91/$03.50 Published by Elsevier

305

FEMSIM 00177

Adjuvant effects of dimethyl dioctadecyl ammonium bromide, complete Freund's adjuvant and aluminium hydroxide

on neutralizing antibody, antibody-isotype and delayed-type hypersensitivity responses to Semliki Forest virus in mice

David Katz, Shoshana Lehrer, Orna Galan, Bat-El Lachmi and Shoshana Cohen

Department of Virology, Israel Institute for Biological Research, Ness-Ziona, Israel

Received 20 April 1991 Revision received 10 June 1991

Accepted 20 June 1991

Key words: Immunological adjuvant; Dimethyl dioctadecyl ammonium bromide; Complete Freund's adjuvant; Aluminium hydroxide; Semliki Forest virus; Antibody-isotype; Delayed-type hypersensitivity

1. SUMMARY

Outbred mice were inoculated subcutaneously with inactivated Semliki Forest virus (SFV) in saline and combinations of the virus with complete Freund's adjuvant (CFA) aluminium hydroxide (Al) and dimethyl dioctadecyl ammonium bromide (DDA). The immune response was evaluated for delayed-type hypersensitivity, for total ELISA antibodies and antibody-isotypes and for neutralizing antibodies. DDA was the most effi- cient adjuvant in inducing DTH, CFA the second and A1 induced a DTH response that was only slightly higher (statistically not significant) than

Correspondence to: D. Katz, Department of Virology, Israel Institute for Biological Research, P.O. Box 19, Ness-Ziona, 70450, Israel.

that induced by the inactivated virus without adjuvants. All adjuvants enhanced the production of ELISA antibodies to similar levels. However, the levels of neutralizing antibodies induced were low in mice which were inoculated with the inactivated SFV alone or mixtures of the virus with Ai. DDA induced high levels of neutralizing antibodies and CFA induced intermediate levels. The pattern of antibody-isotypes induced by DDA and CFA was different from the pattern induced by the inactivated virus or by the virus mixed with Al: DDA and CFA induced low amounts of IgG 1 antibodies and relatively higher amounts of IgGza and IgG2u antibodies while the inactivated virus and the mixture of the virus with A1 induced higher proportions of IgG~ antibodies. In sera from convalescent mice the majority of antibody activity resided in the IgG2a and IgG2b im- munoglobulin subclasses, while IgG 1 antibodies were undetectable.

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2. I NTR ODUC TION

Performance of viral vaccines composed of inactivated whole viruses or non-replicating antigens (polypeptides, synthetic peptides, etc.) can be highly improved by immunological adjuvants. Different immunological adjuvants may have different effects on cellular and humoral immunological responses [1-3]. Cell-mediated immunity is recognized as important and sometimes essential for protection against certain viral diseases. For other diseases, humoral responses seem to be sufficient [4,5]. The distribution of antibodies within the various classes and subclasses of im- munoglobulins (isotypes) is influenced by the structure of the antigen and its presentation to the immune system with or without adjuvants [5-10]. In mice, antibodies of the complement binding isotypes IgGza and IgG2b seem to be more valuable than IgG~ or IgG 3 for combating infectious agents [3,4,6-8]. However, the combination of a variety of immunological mechanisms might be necessary for the ultimate elimination of viral pathogens and recovery from infection [4,11,12]. An ideal adjuvant should therefore induce a broad spectrum of immunological responses.

Many immunological adjuvants enhance immunity experimentally but may also cause unac- ceptable side-effects [1,5,11,13,14]. So far, only mineral salt adjuvants are licensed and widely used in human vaccines. In the veterinary field only two additional adjuvants are licensed: mineral oil and saponin. However, even these three adjuvants are not ideal since they still cause un- wanted side-effects a n d / o r may be only partially effective for certain viral diseases [1,5,6,13-15]. Efforts are therefore made to fractionate and purify biological immunostimulators or to search for chemical compounds that possess beneficial adjuvant properties with minimal toxic effects [1,2,5,6,11,13-15]. In an investigation for the evaluation of adjuvant activities of aliphatic nitroge- nous bases several substances possessed im- munostimulating effects and amongst them was a quaternary ammonium lipoid compound, dimethyl dioctadecyl ammonium chloride (DDA) [16]. The adjuvanticity of the bromide derivative of this

substance was extensively tested in a variety of laboratory animals in combination with proteins and haptens [16-18], red blood cells [19-21] tu- mour cells [22] and viruses [23-27]. DDA was also tried in humans in conjunction with tetanus toxoid vaccines [28,29].

In this paper, like Kraaijeveld et al. [23], we have used inactivated Semliki Forest virus (SFV) as the immunogen. However, we have chosen to work with outbred mice (rather than inbred) which were inoculated subcutaneously (rather than intradermally), to simulate conditions of practical situations in which vaccines are actually used. Also, in our experimental system, the efficacy of DDA, as an adjuvant, was compared to two other well-known adjuvants, complete Freund's adjuvant (CFA) and aluminium hydroxide (AI). In addition to the effect of the adjuvants on delayed-type hypersensitivity and total antibody responses, the effect of these adjuvants on the isotype distribution within the anti-SFV antibodies was studied. The pattern of antibody-isotypes obtained after vaccination with the inactivated SFV with or without adjuvants was compared with the pattern of antibody-isotypes in mice that recovered from an experimental SFV infection.

3. MATERIALS AND METHODS

3.1. Virus

SFV strain (B26146-ATCC) was propagated in BHK-21 cells, in Dulbecco modified Eagle's medium (DMEM) supplemented with 5% foetal calf serum (FCS) and 70/xg of gentamycin ml - ~, and clarified by low-speed centrifugation. Stocks of viruses thus prepared were pooled and titrated in 3- to 4- week-old mice for the determination of the 50% lethal dose (LDs0) and in tissue cultures (BHK-21) for the determination of titre in plaque-forming units (pfu) [12]. The titres obtained were 3 × 10 s LDs0 ml-1 and 5 × 10 9 pfu m1-1, respectively. Portions of these live virus preparations were stored at - 7 0 °C until used.

For inactivation, part of the SFV pooled stock was treated by /3-propiolactone (Sigma) as fol-

lows: 20.0 ml of 1.0 M Tris buffer (pH 8.0) were added to 80.0 ml of SFV suspension for pH adjustment. One mi of /3-propiolactone, diluted 1:10 in cold distilled water, was slowly added to 99.0 ml of virus suspension. The mixture was left overnight in the cold room (4°C), and cen- trifuged for 75 min at 28000 rpm (68400 x g) in a refrigerated Beckman ultracentrifuge (rotor '30'). Pellets were resuspended in a total of 8.0 ml PBS (10-fold less than the starting volume) by soaking for a few hours in the buffer and sonication for 1 min in a water-bath sonicator. The inactivated SFV preparat ion was kept at - 2 0 °C until used.

By an indirect ELISA (see 3.2 below) before and after inactivation and ultracentrifugation procedures, the final antigen concentration of the inactivated virus stocks was only four times higher than the antigen concentration in the source stock and not 10 times as expected. These results indicate that about 60% of the original antigenic mass was lost during the preparat ion of the inactivated vaccine. The final concentration in the inactivated SFV vaccine stock was therefore esti- mated to be equivalent to 2 × 10 l° pfu ml-1.

Absence of residual live virus in the inactivated virus preparat ion was confirmed by intra- cerebral inoculation of 16 newborn and eight adult (6-week-old) mice.

3.2. Assay for SFV antigens by ELISA

SFV antigens were detected by an indirect 'sandwich' type of ELISA. Ninety-six-well, flat- bot tom, polystyrene plates (Dynatech) were coated with 0.2 ml per well of a 1 : 10 000 dilution of rabbit anti-SFV serum in carbonate buffer (50 mM) containing sodium azide (0.02% w/v) , pH 9.6, by overnight incubation at room temperature. After three washes in PBS containing 0.02% (w/v) sodium azide and 0.05% (w/v) Yween 20 (PBS + Az + T), the wells were blocked with 0.25% (w/v) gelatin in PBS + Az for 1 h at room temperature . The coated plates were then dried and kept in sealed plastic bags at - 7 0 °C until used.

For use, 0.15 ml of serial viral dilutions in PBS + Az + T were incubated in the wells at 37 °C for 1 h. Two or more wells were incubated

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with PBS + Az + T alone to serve as controls. The wells were washed three times with PBS + Az + T and incubated with 0.15 ml of a mouse- anti-SFV ascitic fluid (1:400 dilution) at 3 7 ° C for 1 h. After additional three washing cycles, 0.15 ml of a 1 : 500 dilution of rabbit-anti-mouse IgG conjugated to alkaline phosphatase (Sigma) was added to each well for further incubation at 37 °C for 1 h. Colour development was obtained after the addition of the substrate (p-nitrophenyi phosphate; Sigma) and incubation at room temperature for 30 min. The absorbance at 405 nm was read in an automatic ELISA plate reader (Bio-Tek instruments). The average A405n m value from negative control wells, to which three standard deviations were added, was considered as the cut-off value for the calculation of the titre. A curve was constructed in which A405n m values from the series of virus dilutions were plotted against the logarithm of the reciprocal of virus dilutions. The titre was defined as the reciprocal of virus dilution at the meeting point of the cut-off value and the standard curve.

3.3. Preparation of virus-adjuvant mixtures

All suspensions of adjuvants and dilutions of inactivated viruses were prepared in sterile pyrogen-free physiological saline (Travenol, Israel). D D A (Eastman Kodak, U.S.A.) was prepared by suspension of the powder in saline (1.0 mg ml-1). A fine homogenous 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 virus (diluted in saline) and shaken for 60 rain at room temperature. Each injection contained 100/xg of D D A per 0.2 ml per mouse. CFA (Difco, U.S.A.) was emulsi- fied with an equal volume of the inactivated virus suspension by vigorous mixing on a vortex mixer. Aluminium hydroxide (Alhydrogel, Superfos, Denmark), at a 2% concentration, was cen- trifuged at 500 x g for 10 min and resuspended in pyrogen-free saline. The suspension (pH 6.3) was mixed at a 1:1 proportion with the inactivated virus and shaken for 60 rain at room temperature. The final amount of aluminium hydroxide in-

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jected per 0.2 ml per mouse was 2.0 mg. The capacity of aluminium hydroxide and D D A to adsorb the viral antigens was checked.

3.4. Determination of the the adsorbing capacity of aluminium hydroxide and DDA

The relative amount of SFV antigen adsorbed to aluminium hydroxide and D D A was tested in duplicate by the indirect ELISA. The adjuvants were mixed with a 1:100 dilution (2 × 108 pfu m l - 1) of the inactivated virus stock. Each mixture (1.0 ml) was forced through a syringe fitted filter holder equipped with a 0.45 /xm filter (holder: SX0001300; filter: H A W P O 1300, Millipore Cor- poration, U.S.A.). A control preparat ion of the diluted virus in saline (V + SAL) was also filtered in a similar manner. The filters were soaked before use for 60 min in 0.05% (w/v) Tween 20 in PBS + Az to minimize non-specific adsorption. Filtrates were tested for residual unbound antigen in the ELISA. The determination of unbound antigen was also a t tempted by centrifugation of 1.0-ml portions of the preparat ions in a Beckman microfuge for 1 min. This procedure was successful in pelleting the aluminium hydroxide only. D D A could not be pelleted by centrifugation because of its lipoidal nature. Thus, only the supernate of the aluminium hydroxide preparation was examined for free antigen in the ELISA. Net absorbance values were obtained by substracting the m e a n A405n m values of a PBS (no virus) control from the m e a n A405n m values of the samples tested. The percentage of adsorbed SFV antigen in the samples tested was calculated from the net A405n m value obtained with the 1:100 dilution of the untreated virus stock (total).

3.5. Induction and evaluation of humoral immunity

3.5.1. Mice and immunization of mice. Female mice (5-7-week-old; ' I C R ' random

bred, Charles River, U.K.) were used. Inactivated virus preparat ions were mixed with the adjuvants at proportions as ment ioned above. In most experiments five groups of 6 -8 mice were inoculated subcutaneously (sc) with 0.2 ml of the fol- lowing preparations: vaccine + saline (V + SAL),

vaccine + CFA (V + CFA), vaccine + aluminium hydroxide (V + AI), vaccine + D D A (V + DDA) and a control group inoculated with saline only. The first four groups were boosted with the virus alone (without adjuvants). The fifth (control) group received saline alone.

Whenever live virus was used for challenge or immunization it was inoculated intraperitoneally (ip) in 0.5-ml portions. Final concentration of viruses inoculated (live or inactivated) and injection schedules will be mentioned for each experiment in REsucrs.

3.5.2. Evaluation of total anti-SFV antibodies and of antibody-isotypes by ELISA

Ninety-six-well, flat-bottom, polystyrene plates (Dynatech, U.S.A.) were coated with the semipu- rifled, inactivated, SFV preparat ion at a calculated concentration of 107 pfu m1-1 (based on titrations before inactivation). After blocking with 0.25% (w/v) gelatin and washing with PBS + Az + T plates were stored at - 70 ° C until used. In the tests, 50 ~l of individual or pooled sera at a 1 : 100 dilution in PBS + Az + T, were added to each of the duplicate wells for incubation at 37 °C for 1 h. Plates were washed three times in PBS + Az + T. For total antibody determination, a rabbit-anti-mouse alkaline phosphatase conjugate (1:500; Sigma) was added to the wells and incubated for 60 min at 37°C. For isotypes determination a 'Mouse-Typer Subisotyping Kit' (Bio- Rad, U.S.A.) was used according to the manufac- turer 's instruction. The rabbit-anti-mouse isotypes, from the kit, were incubated for 120 min at 37 °C, or for 18 h at room temperature. After washing, a goat-anti-rabbit alkaline phosphatase conjugate was added (1:1000; Sigma) and incubated for 60 rain at 37 ° C.

Colour development was obtained after addition of the substrate (p-ni trophenyl phosphate) and incubation at room tempera ture for 30 min for total antibodies, and 60 rain for isotype determination. The Aa05n m w a s read in 'Bio-Tek ' ELISA reader. Results were expressed as positive to negative ratios ( P / N ) calculated by dividing the average A405n m reading of the test sample to the average A405n m reading obtained from the control non-vaccinated 'saline' group.

3.5.3. Evaluation of neutralizing antibodies by the TCIDso inhibition test

Two-fold dilutions of tested sera (in DMEM containing 2% FCS and antibiotics) were prepared in 96-well flat-bottom tissue culture mi- crotitration plates (Linbro, U.S.A.). Then 1000- 3000 50% tissue culture infective doses (TCIDs0) of the virus (50 /xl in the same medium) were added to each well. After 1 h incubation at 37 °C in the presence of 5% CO 2, 20000 BHK-21 cells in 100 /xl of DMEM, supplemented with 10% FCS, were added to each well and again incubated at 37°C. A cytopathic effect was moni- tored 24-28 h later. The reciprocal of the last dilution of the serum at which no cytopathogenic effect could be detected was considered as the titre of the serum. Duplicates were used, and positive and negative controls were included in each test [12].

3.6. Induction and evaluation of delayed-type hypersensitivity (DTH)

Five groups of 5-week-old ICR mice were first injected subcutaneously with 0.2 ml of preparations as described in paragraph 3.5.1. (V + SAL, V + C F A , V + A 1 , V + D D A and SAL). The amount of inactivated SFV in each virus containing mixture was 2 × 108 pfu. Each group consisted of 12 mice. 6 days later, each mouse in all five groups, received an additional inoculation of inactivated SFV (5 × 108 pfu/0.05 ml) into the right hind footpad. The left hind footpad was

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inoculated with saline. All mice were killed 24 h after the last (footpad) inoculation for the determination of the percentage of weight increase of the right footpad in comparison with the control left footpad [31].

3. 7. Statistical analysis

ELISA or DTH results were expressed as the arithmetic means + standard deviations. The statistical significance of differences between means was determined by Student's t-test.

4. RESULTS

4.1. The adsorbing capacity of aluminium hydroxide and DDA

The relative amount of SFV antigen adsorbed to aluminium hydroxide and DDA was evaluated for portions of virus-adjuvant mixtures (V + A1 and V + DDA, respectively), the results of which are summarized in Table 1. In the filtration experiment, 100% of the viral antigen in the adjuvant-virus mixtures was removed from the filtrates. From the centrifugation experiment (performed with the V + AI preparation) it was de- duced that all the antigen was removed from the supernate. It can be concluded from both experiments, that the aluminium hydroxide adsorbed the total amount of antigen. For DDA, for which centrifugation was not applicable, it is possible to state that at least 66% of the antigen adsorbed to

Table 1

Evaluation of the SFV antigen-adsorbing capacity of aluminium hydroxide and DDA by indirect ELISA

Sample tested Mean Net

absorbance absorbance

'No virus' PBS-control 0.080 0

' V + SAL' (total) 0.826 0.746 Filtrate from 'V + SAL' 0.569 0.489 Filtrate from 'V + DDA' 0.053 - 0.027

Filtrate from ' V + AI' 0.050 - 0.030 Sup' from ' V + Al ' 0.084 0.004

Percent adsorbed

Not relevant Not relevant

34% 103% 104% 99.4%

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Table 2

Neutralization antibodies (expressed as neutralization titres, NT) and ELISA antibodies (expressed as positive to negative ratios, P / N ) in serum pools from groups of mice that received two inoculations, the first at the age of 7 weeks and the second, 18 days later

Group Inoculations Antibodies at days after second inoculation

First Second 0 8 15 27 57

NT P / N NT P / N NT P / N NT P / N NT P / N

A V + S A L V + S A L < 50 0.9 200 5.6 400 2.6 50 3.5 71 2.6 B V + S A L SAL < 50 0.7 < 50 1.7 106 1.i < 50 1.3 100 1.2

C V + C F A V + S A L <50 1.1 282 7.6 566 4.3 282 6.3 200 7.9 D V + CFA V + CFA < 50 1.5 < 50 5.2 50 4.6 50 8.0 < 50 7.9 E V + CFA SAL < 50 2.0 < 50 7.1 < 50 4.0 < 50 6.8 < 50 5.2

F V + A I V + S A L <50 2.1 200 15.5 141 6.6 282 6.8 100 8.9 G V + A I V + A I < 50 3.1 50 7.8 200 4.3 100 6.3 100 5.2 H V + AI SAL < 50 1.2 < 50 2.6 < 50 1.8 50 2.2 < 50 1.3

I V + D D A V + S A L < 50 1.9 566 14.8 800 5.7 2263 7.1 566 7.8 J V + DDA V + DDA < 50 2.6 200 11.3 200 6.4 200 9.2 400 7.1 K V + DDA SAL 100 2.2 200 5.0 566 3.4 282 3.4 800 3.3

V, Inactivated SFV; SAL, Saline; CFA, Complete Freund's adjuvant; AI, Aluminium hydroxide; DDA, Dimethyl dioctadecyl ammonium bromide.

z Ilu

3O

28 [ ] IgG1

26 [ ] IgG2a 24

22 I~ IgG2b

20 [ ] IgG3 18 16 [ ] IgM

14 • Ig 12 ~

10

8

6

2

0 V+SAL V+CFA V+AI V+DDA

GROUPS OF VACCINATED MICE

Fig. 1. Determination of total antibodies and antibody-isotypes in groups of vaccinated mice. Mice were inoculated twice: first at the age of 7 weeks and second 18 days later. Total antibodies (Ig), IgM, IgG I, IgGza, IgG2b and IgG 3 isotypes were determined by ELISA at 27 days after the second inoculation in sera of individual mice from groups A, C, F, I and L. On the first inoculation date group A received inactivated virus in saline (V + SAL), group C V + CFA, Group F V + AI and group I V + DDA. On the second inoculation date all groups received inactivated virus only (See also Table 2). Group L received saline only on both dates. Sera from

this group served as the controls for the calculation of 'positive to negative' ( P / N ) ratios.

it, since 34% of the virus control (V + SAL) was retained on the filter.

4.2. Effect of adjuvants and vaccination regimens on the antibody response to SFV

4.2.1. Evaluation by ELISA and microneutralization assay

The aim of this experiment was to study the effect of three adjuvants (CFA, A1, and DDA) and of different vaccination regimens on the primary and secondary humoral immune response in mice. Twelve groups (A to L) of six mice each, were inoculated subcutaneously twice: first at the age of 7 weeks and second 18 days later. (See also Table 2.) The final amount of SFV in the virus containing preparations was 8 × 107 pfu per inoculum. Groups A and B received inactivated virus only. Group A received the inactivated virus (in saline) both the first and second (booster) injections, while group B received only one virus injection at the beginning of the experiment and saline (instead of virus) on the second inoculation date. Three other groups of mice were used to study each of the three adjuvants: groups C, D and E for CFA, groups F, G and H for aluminium hydroxide (AI) and groups I, J and K for DDA. The first of each of the three groups received first one injection of the virus-adjuvant mixture and a booster injection of the inactivated virus alone. The second group received the virus-adjuvant mixture in both the first and booster injections. The third group was first inoculated with the virus-adjuvant mixture and then with saline only. Group L, that served as an unvaccinated control, received two injections of saline (SAL) on the two inoculation dates. Blood samples were taken 18 days after the first inoculation, right before the booster injection, and 8, 15, 27 and 57 days later. Blood from the last four bleedings was pooled and tested for antibodies by ELISA and microneutralization (Table 2). Indi- vidual mice from groups A, C, F, and I, bled 27 days after the second injection, were also tested for antibody-isotypes (Fig. 1).

Results in Table 2 indicate that there was a poor correlation between the ELISA antibodies and the neutralizing antibodies. There were no differences in antibody responses between groups

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C (1st, V + CFA; 2nd, V + SAL), F (1st, V + A1; 2nd, V + SAL) and I (1st, V + DDA; 2nd, V + SAL) when tested by ELISA while there were marked differences between those groups when tested in the microneutralization assay. The antibody response in group I was high in both tests, demonstrating the efficacy of DDA as an enhancer of both ELISA and neutralizing antibodies. Neutralizing antibodies in groups A (1st, V + SAL; 2nd, V + SAL), C and F were present, but in much lower amounts. Among these three groups CFA seemed to give better results than the others. DDA was also capable of inducing a significant primary response for both types of antibodies, as seen from the results in group K (lst, V + DDA; 2nd, SAL). CFA induced a high level of ELISA antibodies after one injection but no detectable neutralizing antibodies (group E). Aluminium hydroxide induced a very low primary response by ELISA and no neutralizing antibodies (group H). In spite of the differences, some general agreement between the two tests can be observed in relation to the adjuvants' performance and the effect of the inoculation regimens: (1) For maximum antibody production there was a need for a booster injection (antibodies in groups B, E, H and K were usually lower than in others); (2) there was no advantage in using an adjuvant for the second (booster) injecton. In fact, there seemed to be, in some cases, a lower antibody response in those groups which received two injections with adjuvants (group G and groups D and J) as compared to the groups that were boosted with inactivated virus without adjuvants; (3) the antibody response in group I was high by both tests, demonstrating the efficacy of DDA as an enhancer of both ELISA and neutralizing antibodies; (4) the rate of the antibody response in group K was slow but significant by both tests, indicating that DDA induced a virus-specific, primary humoral response for neutralizing antibodies and ELISA antibodies.

4.3. Determination of antibody-isotypes

4.3.1. Antibody-isotype pattern in mice vaccinated with inactivated SFV vaccines

The isotype distribution within the antibodies to SFV was determined at 27 days after the

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second injection in individual mice, of groups A, C, F, and I from the experiment described in 4.2.1. Mice from these groups were first inoculated with V + SAL, V + CFA, V + A1 and V + DDA, respectively and 18 days later with the vaccine without adjuvants (V + SAL) (Fig. 1). No antibodies of the IgM and IgG 3 isotypes could be detected in any of the groups. The only detectable antibodies ( P / N = 2.0 or higher) were of the IgG~, IgG2a and IgG2b subclasses. Relative differences in the proportion of antibody-isotypes in each of the groups of mice were observed. More IgG l antibodies than IgG2a and IgGzb antibodies (P < 0.05 and P < 0.01, respectively) were found in the V + SAL group. A similar pat tern was observed in the V + AI group: higher mean P / N values for IgG~ than for IgGza (P < 0.05) and IgGzb ( P < 0.1, not significant). In the V + CFA group the amount of IgG2a and IgGzb antibodies was higher than the amount of IgG~ antibodies ( P = 0.05 and P < 0.01, respectively). The antibody-isotype pat tern in the V + D D A group was similar to the pattern observed for the V + CFA group: values for IgG2~ and IgG2b antibod-

ies were higher than values for IgG~ antibodies (P < 0.05 and 0.01, respectively).

In another experiment four groups of 5-week- old mice were inoculated with inactivated SFV in saline and with mixtures of the virus and adjuvants ( V + S A L , V + C F A , V + A I , and V + DDA); six mice were used in each group. Each inoculum contained 4 × 10 7 p fu of SFV. The fifth group was inoculated with saline only. 14 days later the first four groups were boosted with 1 × 108 pfu of SFV in saline while the fifth was again injected with saline. This last group of mice served as the control group for the calculation of the P / N values. Mice were bled at 12 days after 1st injection (bleed I) and at 10 (bleed II), 17 (bleed III) and 24 days (bleed IV) after the second (booster) injection. Sera from individual mice were tested by ELISA for total antibodies and antibody isotypes. Results are summarized in Table 3.

No significant primary antibody response was obtained in the V + S A L , V + C F A and V + D D A groups at 12 days after inoculation. A low total antibody response ( P / N = 3.0) was observed

Table 3

Antibody-isotypes and total antibodies in groups of mice at 12 days after the first injection with indicated vaccine preparations (l) and 10 (II), 17 (III) and 24 (IV) days after the second booster inoculation

Group Bleed Antibody-isotypes and total antibodies in P / N ratios (mean ± SD)

IgGl IgG2~ IgG2b IgG 3 IgM Total

V+SAL I 0.6±0.4 0.6±0.4 0.9±0.7 1.0±0.6 0.9±0.5 1.3+0.7 II 2.5±1.7 2.0±2.7 2.3±1.8 1.2±0.7 1.5±1.0 4.4+2.7 III 4.4+1.4 2.6+2.1 2.9+2.7 0.8+0.5 1.5+0.7 4.8±1.2 IV 3.9±1.6 2.3±2.4 2.2±1.8 0.7±0.8 1.3±0.7 5.4±2.4

V+CFA I 1.3±0.7 0.6±0.2 1.3+0.7 1.0±0.4 1.2+0.3 1.5+0.5 II 2.3±1.3 3.0+1.9 3.7+1.9 1.4±0.8 1.l±0.6 5.8+2.7 III 2.4±1.2 3.9±2.3 3.7±2.4 0.3±0.3 1.6±0.2 4.8±1.2 IV 3.2±1.6 4.4±3.4 4.8±1.5 1.4±0.2 1.7±0.4 4.5±3.0

V+AI I 1.7±0.8 0.7±0.2 0.9±0.2 1.2±0.4 1.4±0.5 3.0+1.3 II 6.3±3.0 2.7±1.5 2.5±1.3 1.9±0.5 1.1±0.6 8.1±5.3 III 5.3+1.7 2.3±2.1 2.2±1.5 0.9±0.4 1.6±0.7 8.9+5.7 IV 5.1±2.3 2.8±2.5 2.2±1.8 1.7±1.7 1.4±0.4 6.1±6.5

V + D D A I 1.0±0.4 0.8±0.2 0.5±0.2 1.0±0.4 1.2±0.4 1.4±0.5 II 2.6±1.5 10.3±4.0 6.8±5.1 1.2±0.4 3.0±1.4 12.3±3.6 III 3.0±1.7 9.3±4.8 4.5±3.0 0.3±0.3 1.1±0.2 8.9±1.4 IV 3.5±1.1 9.5±3.1 4.3+2.3 1.2±0.3 1.3±0.2 5.9±4.1

only in the V + A1 group. The value for IgG~ in this group ( P / N = 1.7) was higher than in the others but nevertheless as in all other groups, less than P / N 2.0 and therefore considered as negative.

At 10, 17 and 24 days after the booster injection a significant secondary antibody response was obtained in all four groups. P / N values for total antibodies in groups V + SAL and V + CFA were not significantly different. Higher antibody values were observed in the V + A1 group and the highest in the V + DDA group. Since differences in antibody responses within each group were small for the last three bleeding dates, results were compiled within each group for calculations of means and standard deviations as presented in Fig. 2b. For comparison, the primary antibody response is shown in Fig. 2a. The general pattern of antibody-isotypes in the vaccinated groups of mice was similar to that obtained in Fig. 1. In the

313

V + SAL group the mean P / N values of IgG 1 antibodies were higher than the values for IgG2a and IgG2b antibodies (difference not statistically significant). Similar patterns of antibody-isotypes were observed in the V + A1 group, yet in this case the P / N values for IgG1 were significantly higher than the values for IgGza (P < 0.01) and |gGzb ( P < 0 . 0 0 1 ) antibodies. In the V + CFA group IgG2a and IgGzb antibody P / N values were higher than the values for IgG~ antibodies but the difference was not significant. In the V + DDA group IgG2a antibody values were significantly higher than IgG~ P / N antibody values ( P < 0.001). IgG2b antibody values were also higher than IgG 1 P / N values but the difference was not statistically significant.

In a similar experiment, performed in 5-week- old mice, somewhat different vaccination and bleeding schedules were used. Eight mice were used in each group. The amount of SFV in each

] ~ IgG1 [ ] IgG2a E~ IgG2b [ ] IgG3 [ ] IgM BII Ig

14 . . . . . .

12 A 8

10

0 , • , ,

V+SAL V+CFA V+AI V+DDA V+SAL V+CFA V+AI V+DDA

GROUPS OF VACCINATED MICE Fig. 2. Determinat ion of total antibodies and antibody-isotypes in groups of vaccinated mice. Four groups of mice were inoculated with inactivated SFV vaccine in saline ( V + SAL) and mixed with adjuvants ( V + SAL, V + CFA, V + Al, and V + DDA). A fifth group was inoculated with saline only. Fourteen days later the first four groups were boosted with inactivated SFV in saline while the fifth was again injected with saline. This last group of mice served as the control group for the calculation of the P / N values. Mice were bled at 12 days after first injection (a) and at 10, 17 and 24 days after the second (booster) injection. Results from these

last three bleeding dates (Table 3) were compiled for presentat ion in this figure (b). All symbols are as in Fig. 1.

314

virus containing inoculum was 1.3 × 108 pfu. Mice were bled prior to the second injection (24 days after the 1st injection) (Fig. 3a), and at 9 (Fig. 3b) and 60 (Fig. 3c) days after the booster injection. The ELISA for antibody-isotypes was done with pooled sera.

The primary antibody response in the V + SAL group 24 days after the first inoculation and the secondary response at 9 days after booster injection was practically negative (Fig. 3a, b). At the same time points all virus-adjuvant mixtures induced significant amounts of antibodies. The patterns of antibody-isotypes were similar to those shown in Figs. 1 and 2. IgG2~ and IgGzb antibodies were present in higher proportions in the V + CFA and V + DDA groups and the proportions of IgG 1 antibodies was relatively higher in the V + SAL and V + A1 groups. This typical pattern remained unchanged for at least 60 days after the booster injection (Fig. 3c).

4.3.2. Determination of antibody-isotypes in com,a- lescent sera of mice

Ten-week-old mice were inoculated with about 300 pfu of live SFV. Five survivors (out of 71)

were bled 1 month later. The sera were tested by ELISA for antibody isotypes and total antibodies. The individual sera were compared to a normal mouse serum pool for the calculation of P / N values; results were expressed as mean P / N values of the five sera tested (Fig. 4a). In this experiment the total antibody response was relatively low but nevertheless a typical isotype pattern could be observed. The IgG 1 antibody response was remarkably low ( P / N ratios less than 1.0) and the major antibody isotype was IgGz~. IgG2b antibodies had P / N values lower than 2.0 which were significantly lower than the IgG2, P / N values.

In another experiment l l-week-old mice were inoculated with about 300 pfu. Nine survivors (out of 36) were bled 26 days later and their sera were tested for antibody isotypes and total antibodies by ELISA. In these mice the total antibody response was relatively high. However, the very low IgG~ antibody response was observed in this experiment (Fig. 4b). Unlike the results of the previous experiment, similar high P / N values were obtained for both IgG2~ and IgG2b antibody isotypes.

Z O.

30 28 26 24 22 20 18 16 14 12 10

8 6 4 2

J ~ IgG1 [ ] IgG2a

V+SAL V+CFA V+AI V+DDA

[ ] IgG2b [ ] IgG3 [ ] IgM • Ig ]

V+SAL V+CFA V+AI V+DDA V+SAL V+CFA V+AI V+DDA


Fig. 3. Determination of total antibodies and antibody-isotypes: This experiment was performed similarly to that described Fig. 2, except that different vaccination and bleeding schedules were used. In this experiment, the second injection was given 24 days after the first inoculation and mice were bled at 24 days after the first injection (a), and at 9 (b) and 60 (c) days after the second booster

injection. The ELISA was done with pooled sera. All symbols are as in Fig. 1.

315

Z

I1.

a b

4 1

.:,:,:,:,+: 1 !

: , : , : , : , : , : - :

!i!i!iiiii!ili a 1 ilil]!i~ii~ii i ] ..... " ........ ~ ~ i!ii!i!iiiiiii iii!iiiiii!ill

...... i!iiiii!}ii! i!! ! i ii!iii :::::::::::::! :,:,:+:,:,: I:!:~:U ":~

ii!i!i!iiiiii 2 -t ~ IgG1 IgG2a IgG2b IgG3 IgM lg IgG1 IgG2a IgG2b IgG3 IgM

ANTIBODY ISOTYPES

T

ii,i

i:!:?:i:i iiiii'iiii

Fig. 4. Determinat ion of total antibodies and antibody-isotypes in sera of two groups of mice (a and b) which were inoculated with live SFV and recovered: (a) 10-week-old mice, inoculated with about 300 pfu of live SFV. Five survivors were bled 1 month later and sera were tested for antibody-isotypes and total antibodies (Ig) by ELISA. (b) 11-week-old mice were inoculated with about 300 pfu. Nine suvivors were bled 26 days later and their sera were tested for antibody isotypes and total antibodies by ELISA. Sera were tested individually and compared to a normal mouse serum pool for the calculation of P / N values. Results are expressed as

mean P / N values.

z O.

20

18

16

14

12

10

8

6

4

2

0

[ ] IgG1

[ ] IgG2a

[ ] IgG2b

[ ] IgG3

[ ] IgM

• ig

V+SAL V+CFA V+AI V+DDA


Fig, 5. Total antibodies and antibody-isotypes after challenge of vaccinated mice with live SFV. Five groups of mice were first inoculated with the indicated vaccines and 33 days later they were challenged with 300 pfu of live SFV. Blood from surviving mice was drawn 16 days later. Sera from surviving mice in each group were pooled and tested by ELISA for total and antibody-isotypes. For calculation of P / N values a pool of normal mouse serum was also tested in the assay. Symbols are identical to those used in

Fig. 1.

316

Results in Fig. 4a and b depict a typical negative lgG I antibody response in mice which recovered from an SFV infection. This very low IgG~ antibody P / N value is different from the responses that were obtained in previous experiments (see paragraph 4.3.1.), with the inactivated virus with or without adjuvants.

4.3.3. Effect of liue L, irus challenge on the pattern of antibody-isotypes in mice which were preuiously t~accinated with inactiuated SFV

As shown in the previous experiment (see paragraph 4.3.2.) immunization of mice by infection with live virus induced a typical antibody-isotype response. This response differed from the responses obtained after one injection with inactivated SFV, with or without adjuvants, and a second booster inoculation with the inactivated virus (see paragraph 4.3.1.). The purpose of this experiment was to study the nature of the secondary response in mice immunized with the inactivated SFV and boosted by an infection of live virus.

Five groups of 7-week-old mice were first inoculated as described for the second experiment in paragraph 4.3.1. The calculated amount of SFV in each inoculum was 2 × 10 s pfu. 33 days later mice were inoculated with 300 pfu of live SFV. All control mice in the 'saline' group died. Most of the mice in the vaccinated groups resisted challenge (71-100% in the different groups). Sera from surviving mice in each group were obtained 16 days later, pooled and tested by ELISA for total antibodies and antibody-isotypes. For calculation of P / N values a pool of normal mouse serum was used in the assay. As shown in Fig. 5, the three groups of mice which were inoculated with adjuvant virus mixtures (V + CFA, V + AI and V + DDA) produced more antibodies than the group which received inactivated virus alone (V + SAL). In the adjuvanted groups the general pattern of the antibody-isotype response resem- bled the pattern obtained in previous experiments (see paragraph 4.3.1.) in which the booster inoculation consisted of the inactivated virus. This result indicates that once an antibody-isotype pattern was determined it could not be changed by subsequent infection. However, the response in

the V + SAL group was somewhat different. As much as twice the amount of IgG2a antibodies was produced in comparison to IgG t antibodies. This profile is different from the one obtained in the experiments described in paragraph 4.3.1., in which the proportions of IgG t antibodies was relatively higher for the V + SAL groups.

4.4. The effect of adjuvants on the delayed-type hypersensitiL,ity response

D T H was evaluated in groups of mice which were injected subcutaneously. Three groups were inoculated with mixtures of inactivated SFV (2 × 108 pfu per mouse) and adjuvants (V + FCA,

I,u (n < uJ n - O 30 Z

I - "1- _o

I- 2 0 0 0 ilk

X

10 u .

0 ) - Z W o E W o. 0

SAL V+SAL V+CFA V+AI V+DDA


Fig. 6. The effect of adjuvants on the DTH response. Three groups of mice were inoculated with mixtures of virus and adjuvants (V+FCA, V + A I and V + D D A ) , one group was inoculated with virus alone (V+ SAL) and the fifth group with saline (SAL) only. All mice were inoculated subcutaneously. The virus amount in the virus containing preparations was 2× l0 s pfu per mouse. Six days later the right hind footpads of all mice were injected intradermally with 5 × 108 pfu of the inactivated SFV, and the left hind footpads with saline. The weight of the hind footpads was determined 24 h later. Results are expressed as the mean percentage of weight

increase of the right footpad as compared to the left one.

V + AI and V + DDA), one group was inoculated with virus alone (V + SAL) and the fifth group with saline only (SAL). 6 days later the right hind footpads of all mice were inoculated intradermally with the inactivated SFV preparation (5 x l0 s pfu per mouse) and the left hind footpads with saline. 24 h later all mice were killed and the weights of their hind footpads were determined. Results (Fig. 6) were expressed as the mean value of the percentage weight increase of the right footpad as compared to the left footpad. DDA induced the highest DTH response which was significantly higher than the DTH induced by saline (P < 0.001), by the virus alone (P < 0.001), by A1 ( P < 0.001) and by CFA (P < 0.05). The DTH response induced by CFA was higher than that induced by saline (P < 0.001), by the virus (P < 0.01), and by AI (P < 0.05). The adjuvant that induced the lowest DTH was aluminium hydroxide. The DTH in the V + A1 group was significantly higher only than the DTH in the control group (SAL) ( P < 0.05). Although the mean weight increase in the V + AI group was higher than that of the V + SAL group, this difference was not statistically significant. Histologi- cal examinations revealed typical cellular infiltra- tions in DTH-positive footpads. The intensity of the local reaction correlated with the amount of DTH measured.

5. DISCUSSION

In the present study DDA, a synthetic quaternary ammonium compound, was compared to CFA and Al for its immunostimulatory properties. Inactivated SFV was used as a model vaccine in outbred mice. Results of vaccinations were evaluated by monitoring antibody production, antibody-isotype distribution and DTH reaction. We have not used protection as a parameter for immunity, since in our experimental system mice which were immunized with the inactivated virus without adjuvants were as resistant to challenge as mice which were vaccinated with adjuvant-containing mixtures. This is in contrast with the findings of other authors [24] and can be ex- plained as due to differences in virus strains,

317

strain of mice and other experimental parame- ters. Our results emphasize some interesting fea- tures of DDA in comparison to CFA and A1. One such feature, which was not described before, is the effect of DDA on the switching of im- munoglobulin isotypes.

The primary antibody response with DDA was similar, in general, to the response obtained with CFA and Al when tested by ELISA. At 18 days (Fig. 1) or 24 days (Fig. 3) after inoculation low amounts of antibodies were produced. At 12 days post-inoculation a low amount of antibodies was induced only by Al; no antibodies could be detected in any of the mice of the other groups (Fig. 2). Only DDA induced a substantial amount of neutralizing antibodies after a single inoculation. Booster inoculations with the inactivated SFV (without adjuvants) induced typical secondary antibody responses in all experimental groups. DDA induced the highest amount of secondary neutralizing antibodies (Table 2). Other authors did not obtain antibodies to SFV (with or without DDA) after both intracutaneous or footpad booster inoculations, but obtained antibodies only after in- traperitoneal injections. DTH however, was induced by any one of the inoculation routes [23,24]. Differences in immunization results may be ex- plained as being due to differences in inoculation regimens [2,3,11,20]. In our study, DDA induced an enhanced DTH response to SFV at 6 days after subcutaneous inoculation at a time when antibodies could not be demonstrated by ELISA. This capacity of DDA to enhance cell-mediated immune responses was found also by other authors [17-20,23-25,27]. In two studies, in which a comparison was done, DDA was found to be superior to CFA in inducing DTH [18,19]. Here we have demonstrated that DDA was superior to CFA and aluminium hydroxide in the induction of DTH to SFV.

In addition, we have found in a study with chickens immunized by inactivated Newcastle dis- ease virus vaccines that DDA was superior to CFA and mineral oil in inducing DTH and in vitro lymphocyte stimulation, and of equal potency to a commercial oil vaccine in inducing resistance to challenge (Katz, D., Inbar, I., Sam- ina, I., Peleg, B. and Heler, D. (1990) presented

318

at the NATO Advanced Studies Institute, "Vac- cines: Recent Trends and Progress", Cape Sounion Beach, Greece).

The antibody-isotype pattern in mice which recovered from an experimental SFV infection was compared to the antibody-isotype pattern in mice vaccinated with the inactivated SFV vaccines. We have found, in agreement with others, that aluminium hydroxide induces higher proportion of IgG 1 antibodies (Figs. 1, 2 and 3) and that infection with viruses does not induce IgGj antibodies at all (Fig. 4) [7,8,10]. In our hands IgGzb, as well as IgG2a antibodies, were induced after infection and not IgG2, t exclusively as claimed by others [3,6,9]. Differences between results in Fig. 4a and b could stem from differences in the total antibody response: in Fig. 4a IgG2b is practically absent while in Fig. 4b this antibody-isotype is relatively abundant. The reason for the difference in the total antibody response of the two groups of mice is not known. It could be due to differences in resistance which is age-dependent. It is possible that the 1 week age difference at 10 weeks was critical, but we have no direct evidence for this.

DDA and CFA had a similar influence on the antibody-isotype pattern, namely, it induced low amounts of IgG~ and higher amounts of IgG2a and IgG2b (Figs. 1-3). It is also evident that mice inoculated with inactivated virus without adjuvant produced an isotype antibody response which was similar to the 'aluminum hydroxide' group~ although the total antibody response in the latter group was usually higher (Figs. 1, 3). Once an isotype pattern was established under the influence of an adjuvant, this pattern did not change with time (Figs. 2, 3) and was not influenced by the nature of the booster antigen, be it inactivated (Figs. 1-3) or live virus (Fig. 5). However, some deviation from the typical pattern of antibody-isotypes was obtained in the group of mice which was vaccinated with inactivated virus without adjuvants and was later challenged with live virus. In this group the proportions of IgG2~ , antibodies obtained were higher than usual. This result is similar to that obtained with influenza purified surface glycoproteins (HANA-flu) which induced mainly IgG1 antibodies but after infec-

tion the production switched to high proportions of IgG2a antibodies [9]. Since mice that recovered from infection did not produce IgG 1 antibodies it can be assumed that those antibodies are less important for recovery. In fact, IgGl antibodies may interfere with recovery processes by compet- ing with complement-binding antibodies (lgGz~ , and IgG2b) [8].

The adjuvant activity of DDA is often ex- plained on the basis of its physicochemical nature. DDA presumably binds to negatively charged proteins through its positive charge [17] and to lipid membranes of cells and enveloped viruses due to its lipoidal and surfactant properties through hydrophobic bonds [32]. In this re- gard its activity is similar to the immunostimulatory activity of lipids covalently linked to proteins or peptides which provide a hydrophobic handle to the antigen [17,32,33]. Attached lipids are be- lieved to increase the retention of the antigen in the lymph nodes and direct the antigens towards the T cell rich area in the paracortical region [17]. We have shown that our viral antigen preparation actually complexed with the DDA (Table 1). However, binding of DDA to antigens is not always essential for its adjuvant activity. Interac- tion with macrophages brings about release of cytokines (including interferon), depresses antigen degradation and prolongs antigen persistence [20,21,25,34]. Moreover, since isotype switching and DTH, which are affected by DDA so effi- ciently, are both dependent on the TH1 subset of T helper cells through the production of IL-2 and interferon y [35] it can be assumed that DDA has a direct or an indirect influence on those lympho- cytes.

In conclusion, the efficacy of DDA as an adjuvant, in comparison to the two well-known adjuvants, CFA and AI, was confirmed in our studies. DDA, like CFA, had a beneficial stimulatory effect in inducing antibodies of the complement- binding isotypes. It also induced neutralizing antibodies to a greater extent than CFA and AI. In addition, DDA was superior to CFA in enhanc- ing DTH, a cell-mediated immune response of great importance for acquired immunity. In spite of its remarkable potency, as an adjuvant, we, as other authors reported, have not observed any

u n d e s i r a b l e g e n e r a l o r loca l s i d e - e f f e c t s a f t e r in-

j e c t i n g m i c e w i t h D D A .

A C K N O W L E D G E M E N T S

W e a r e g r a t e f u l to Prof . E. K u t i n a n d M r . M.

M e s h u l a m fo r p r e p a r i n g a n d e v a l u a t i n g t h e h i s t o -

log ica l p r e p a r a t i o n s f r o m t h e D T H e x p e r i m e n t ,

to P ro f . A . K o h n , D r . R. B a r z i l a i a n d D r . P.

F u c h s fo r t h e i r c r i t i ca l c o m m e n t s , a n d to M r s . L.

A s h a n i fo r p h o t o g r a p h y se rv ices .

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adjuvant effects of dimethyl dioctadecyl ammonium bromide, complete freund's adjuvant and...

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