Alkyl-esters of polyacrylic acid as vaccine adjuvants

Alkyl-esters of polyacrylic acid as vaccine adjuvants

Vaccine, Vol. 16, No. 16, pp. 15751561, 1998 0 1998 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0264-410X/98 $19+0.00 PII: SO2...

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Vaccine, Vol. 16, No. 16, pp. 15751561, 1998 0 1998 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0264-410X/98 $19+0.00

PII: SO264-410X(98)00047-4

Alkyl-esters of polyacrylic acid as vaccine adjuvants L.A.Th. Hilgers*$, I. NicolaQ, G. Lejeune, E. Dewi& M. Strebelle and B. Boon Previously, we demonstrated that polyacrylic acid (PM) augmented significantly the immune response to inactivated Newcastle disease virus (iNDV) in chickens, but that efJicacy was insufjicient to replace the water-in-mineral oil (WIO) adjuvant applied for boosting primed animals. Attempting to improve its adjuvanticity, PAA with weightaverage molecular weight (M,J of 450 kDa was grafted with a&$-chains by esterthing the carboxylic groups with octanol and butanol. The butyt-PAA and octyl-PAA esters obtained varied in degree of esterification between 10% and 92%. Adjuvant activity of water-soluble esters for humoral responses to iNDV was examined in chickens primed previously with iNDV without adjuvant. The alkyl-PM esters exhibited significantly higher responses than unmodified PAA and titres increased with increasing dose of adjuvant. At doses of 2 mg per animal, octyl- and butyl-PAA esters with a substitution rate of 16% (octyllB-P/C4 and butyll6-PM, respectively) gave similar titres as W/O. In aged animals primed with live NDV at early age, butylI6-PAA and W/O elicited comparable antibody responses. ButylI6-PAA was also more effective than PM in stimulating primary immune responses in mice which was accompanied by stronger local reaction determined by monitoring swelling at the site of injection. Reactogenicity of butyll6-PAA was less than of W/O. We concluded that a&$-PA esters are strong adjuvants for primaly and secondary responses and that they are promising alternatives to the mineral oil-based adjuvants presently used in various veterinary vaccines. 0 1998 Elsevier Science Ltd. All rights reserved Keywords: adjuvants;

polymers; polyacrylate esters; Newcastle disease virus

Water-in-oil (W/O) emulsions of mineral oil belong to the most effective adjuvants presently known. Owing to their reactogenicity, however, application is limited to laboratory and domestic food animals (e.g. poultry, pigs and cattle). In research, W/O emulsions such as Freund’s incomplete adjuvant are often the adjuvant of choice thanks to its activity with a wide range of antigens. In chickens, W/O is used for booster immunisation of animals primed with live viruses’. Immunisation with live ,sttenuated Newcastle disease (NDV), infectious bronchitis (IBV) and infectious bursal disease (IBDV) virus at early age induces antibody titres which last for 2-6 months. When these titres have become too low to ensure sufficient protection, immunisation with inactivated antigens incorporated in W/O is carried out. The application of Solvay Research and Technology, Applied Immunology, Rue de Ransbeek 310, Brussels, Belgium. *Present address: DLO-Institute for Animal Science and Health, Edelhertweg 15, P.O. Box 65, NL-8200 AB Lelystad, The Netherlands. Tel: 00 31 320 238091; Fax: 00 31 320 238050. tPresent address: SmithKline Beecham, Rixensart, Belgium. $Catholic University of Leuven KUL, Heverlee, Belgium. DAuthor to whom all correspondence should be addressed. (Received 19 November 1997; revised version received 12 February 1998; accepted 13 February 1998)

W/O-adjuvanted vaccines can be accompanied by local reactions and introduces risk of auto-injection and of oral intake of mineral oil residues by human consumers via food derived from treated animals. Additional limitations of W/O-adjuvanted vaccines include the necessity of considerable stability of antigens outliving the emulsification procedure, limited antigen volume ( i.e. ~50% of vaccine volume), and high viscosity which hampering manipulation and syringability. Replacement of this adjuvant by formulations devoid of these disadvantages, but with similar adjuvanticity, is therefore desired. Many attempts have been undertaken to develop effective, nonreactogenic adjuvants, but only a limited number has been focused on the replacement of W/O for poultry. Alum* A1P04334, nonmineral-oil emulsions5-’ vitamin E’, acetylated mannan’, quaternary amines“OXrl,double-stranded RNAi2, liposomes’3, and interferon inducersI have been evaluated in naive rather than primed chickens. To exclude possible discrepancy in enhancing primary and secondary immune responses, a model for screening adjuvants for booster immunisation was developed15. Chickens were primed with inactivated NDV (iNDV) without adjuvant to mimic effects of priming with live NDV which resulted in significant, sub-optimal responses. A few

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Alkyd-esters of polyacryiic

acid as vaccine adjuvants: L.A. Th. Hilgers et al.

weeks later, animals were boosted with inactivated antigen plus adjuvant. Analysis of the antibody titres in serum after booster revealed significant effect of adjuvant added”. Among the various adjuvants tested, polyanionic polymers exhibited interesting activity although titres were still lower than with W/O”. An effort was made to increase the activity of these polyanions by chemical modification. Effects of the novel group of synthetic adjuvants on secondary humoral response in chickens and on primary responses and reactogenicity in mice are presented here.

Chickens were bled 3 weeks after the second vaccination (i.e. 10 weeks of age) and in some experiments at additional time intervals. Blood samples of individual animals were incubated at room temperature, blood clots were removed after 2 h, remaining cells were spun down (10 min; 1200g) and serum of individual samples were recovered and stored at -20°C until use.

MATERIALS AND METHODS

Anti-NDV antibody titres measured kit

Animals

Antibody titres against NDV in individual serum samples were determined also by the commercial antiNDV ELISA kit (Flockcheck Newcastle disease antibody test kit; IDEXX Labs Inc., Maine, USA) according to the manufacturer’s instructions.

Female NMRI mice of 8-10 weeks and about 20g (Charles River, Sulzfield, Germany) were maintained at conventional conditions. SPF Leghorns were raised and maintained under conventional conditions at the Catholic University of Leuven (KUL, Heverlee, Belgium) and used at the age of 4 weeks. The chickens were vaccinated against Marek’s disease virus before use, but not against NDV or IBV. Synthesis

of derivatives

of polyacrylate

Polyacrylic acid with a weight-average molecular weight M, of 450000 (PAA; CarbopolTM 907, BFGoodrich, Cleveland, Ohio, USA) was esterified with different aliphatic groups using the respective alcohols according to the method described by Cohen et al.‘“. Briefly, 1 g of PAA was dissolved in 50 ml octanol or butanol and the solution was heated up to 135°C. A sample (50 ~1) of 18 M H,SO, was added and the mixture was maintained at 135°C. Reaction was terminated by adding one volume of cold distilled water and rapid cooling of the reaction mixture. The pH was adjusted to 6 and solvents were evaporated at 80°C at low pressure (10m6bar). The products obtained were dissolved in distilled water, dialysed extensively against distilled water and lyophilised. The compounds synthesised were designated as octyl-PAA and butyl-PAA esters. Analysis

Anti-NDV antibody titres in chicken serum were measured by an ELISA described elsewhere15.

Anti-NDV antibody titres determined neutralisation (VN)

by a commercial

by virus

Anti-NDV antibody titres in serum were measured by VN described elsewhere”. Adjuvanticity

and reactogenicity

in mice

Groups of six mice were injected with 25 ~1 vaccine into the left hind footpad and thickness of the footpad was measured 1 day before and at several intervals after injection by a semi-electronic device especially developed for this purpose by the State University of Utrecht, The Netherlands. The accuracy of the apparatus proved to be about 0.02 mm. Swelling was calculated by subtracting the thickness before and after treatment and was expressed as 0.01 mm. Three weeks after injection, serum was collected and antibody titres against inactivated influenza virus strain MRC-11 (MRC-11) and ovalbumin (OVA) were measured by the ELISA as described for iNDV’ using 5 pg HA of MRC-11 or 25 pg OVA per ml carbonate buffer for coating and prediluting mouse serum samples without pre-treatment with kaolin.

of alkyl-PAA esters

The grades of esterification were determined by ‘H -NMR analysis with a spectrometer (Briicker; model AMXSOO) at 500 MHz and D20 or dimethylsulphoxide as solvent. Vaccines, vaccination

and collection

of blood

Alkyl-PAA esters were dissolved in phosphate buffer (0.11 M Na*HPO, and 0.02 M NaH,P0+2H,O; pH = 7.5) and one volume of adjuvant solution was mixed with one volume of an antigen solution comprising inactivated NDV strain Kimber. At 4 weeks of age, all chickens were immunised intramuscularly (IM) with 0.5 ml vaccine with iNDV without adjuvant. At 7 weeks of age, animals received 0.5 ml vaccine comprising iNDV plus phosphate buffered saline (negative control) or iNDV incorporated into a waterin-mineral oil emulsion (W/O, positive control) or iNDV plus PAA or iNDV plus one of the alkyl-PAA esters.

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Direct ELISA on anti-NDV antibody titres in serum

Vaccine 1998 Volume 16 Number 16

Statistical

analysis

Antibody titres of the groups were expressed as means of the 2-log values of the regression coefficient of the plot of optical density versus serum concentration (+SEM) and antilogs of these means (2mea”). Student’s t test was carried out to analyse statistical significance of the results and p-values over 0.025 were considered to be insignificant. Groups which are not statistically different are indicated by the same letter. RESULTS Synthesis

and analysis

of alkyl-PAAs

Several alkyl-PAA esters were synthesised with octyl and butyl groups at different degrees of esterification (Table I). The degree of esterification increased with increasing incubation time and varied from 10% to 92%. The general chemical formula of the alkyl-PAA esters obtained is -[CH,-CHCOOR],with iz = 6250

Alkyl-esters of polyactylic acid as vaccine adjuvants: L.A. Th. Hilgers et al. Table 1

Composition

of alkyl-.PAA esters synthesised Solubility in water (g/l)

Compound

Alcohol

% Ester

10

1

0.1

Octyll P-PAA OctyllG-PAA octy127-PAA Octyl45PAA Octyl91 -PAA Octyl92-PAA Butyll 0-PAA Butyll6-PAA

Octanol Octanol Octanol Octanol Octanol Octanol Butanol Butanol

12 16 27 45 91 92 10 16

+ +

_ _

NT NT + * _

NT NT NT + -

+ +

NT NT

NT NT

+: clear solution. + : slightly opalescent solution. - : opalescent suspension/dispersion NT = not tested.

or insoluble material visible.

(with M, of PAA =450000 Da and M,q of acrylic acid = 72 Da), R = H or -(CH&-CH3 or -(CH&-CH3, and the ratio of H:-(Cl&CH3) or H:-(CH&-CH3) varying from lo:90 to 92% Solubility of the compounds in water was determined and only those soluble at a concentration of 1 g/l were tested as adjuvant.

butyl-PAA ester per animal (Figure IA). Low doses of 0.25 and 0.50 mg of butyl-PAA ester gave similar titres as high doses of unmodified PAA (2-4 mg per chicken).

Anti-NDV Anti-NDV ELISA

antibody titres measured

by an indirect

In four independent experiments, adjuvanticity of various alkyl-PAA derivatives were compared with that of unmodified PAA, with W/O (positive controls) and antigen alone (negative controls). Anti-NDV antibody titres measured by an indirect in-house ELBA in negative and positive control animals were constant over the different experiments and increase in titre by W/O ranged from eight- to 14-fold (data not shown). The data of the different experiments were taken together yielding an average lo-fold increase in response by W/O (Table 2). Unmodified PAA, octyl-PAA and butyl PAA esters enhanced significantly the antibody titre to iNDV (Table 2) and the alkyl-PAA esters reached significantly higher titres than PAA. Doses of 2 and 4 mg butyll6PAA and 10 mg octyll2-PAA or octyll6-PAA gave equal responses as W/O. A clear-cut dose-response relationship was observed within the range of 0.25 to 4 mg PAA and Table 2

Effect of PAA and various alkyl-PAA esters on the antibody

antibody titres measured

by the IDEXX kit

Data obtained in the in-house ELISA were evaluated by determining the anti-NDV antibody titres in groups selected from the experiments described above, in a commercially available anti-NDV ELISA kit. The average increase in titre of positive controls was 45-fold (Table 3). The alkyl-PAA esters tested exhibited significantly higher titres than negative controls and butyll6-PAA, but not the octyl-PAA esters attained the activity of W/O.

Anti-NDV antibody titres measured neutralisation assay

by virus

Relevance to protection of the humoral response elicited by the alkyl-PAA esters was assessed by a virus neutralisation test (Table 4). Groups were selected from the experiments described above. An average 79-fold higher antibody titre was observed in the positive control group as compared with the negative controls (Table 4). Unmodified PAA evoked significantly, fivefold enhanced response and the alky-PAA response against NDV in chicken measured by an indirect ELISA

2-log anti-NDV antibody titre

Group 1 2 3 4 5 6 7 8 9 10 11

Adjuvant

(mg per dose)

No adjuvarlt W/O (250) PAA (2) PAA (10) Octyll P-PAA Octyll2-PAA Octyll6-PAA Octyll6-PAA Octyl27-PAA Butyll6-PA4 Butvll8-PA4

(2) (10) (2) (10) (2) (2) (4)

Statistical difference

n

Mean

SEM

Antilog

120 120 80

5.8 9.2 7.2

0.9 0.8 1 .o

57 568 142

a b

40 60 40 40 20 20 80 40

7.6 8.6 9.0 8.6 8.7 8.4 9.0 9.1

0.9 0.7 0.7 0.6 0.9 0.7 0.6 0.7

187 388 495 388 405 339 495 555

zi b,d d b,d d b h

C

Groups of twenty animals were injected IM with 0.5 ml iNDV suspension without adjuvant and 3 weeks later with 0.5 ml of iNDV plus the adjuvant indicated. Three weeks after the booster immunisation, antibody titres were measured by an in-house ELISA. Results of four independent experiments with octyl-PAA and butyl-PAA ester were taken together. Groups which are not statistically different Cp> 0.025) are indicated by the same letter.

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Alkyl-esters

of polyacrylic

acid as vaccine adjuvants: L.A.Th. Hilgers et al.

esters induced significantly higher responses than unmodified PAA. ButyllB-PAA at 4 mg per dose reached high titre which was not significantly different from W/O. A clear dose-response relationship was observed for both unmodified PAA and butyll6-PAA within the range of 0.25 and 4 mg per chicken (Figure 1B).

Adjuvanticity of butyl-PAA esters in chickens with live NDV dose (mg per animal)

8-I 0

1

2

-

PAA

-

butyll6-PAA 4

3

5

dose (mg per animal) Figure Graded groups adjuvant later by

Table 3

1 Dose-response relationship of PA4 and butyll6-PAA. doses of either compound with iNDV were administered in of 20 chickens primed previously with iNDV without and antibody titres against NDV were measured 3 weeks an ELISA (A) and VN (B).

Effect of various alkyl-PAA esters on the antibody

primed

In practice, chickens are primed with live NDV at day of age and at 3 weeks of age and booster immunisation of these animals with inactivated NDV is conducted at the moment that titres are below protective levels, i.e. between 8 and 25 weeks of age. Adjuvanticity of PAA and butyll6-PAA ester was compared with that of W/O in a model approaching field conditions. At the age of 23 weeks, antibody titres were considered sufficiently low to be able to detect effects of booster immunisation with inactivated NDV plus adjuvant. At 23 (Experiment I) or 26 weeks of age (Experiment II), groups of animals received iNDV without adjuvant or with W/O, PAA or butyl-PAA ester. Anti-NDV antibody responses were measured at different time intervals after boost by the in-house ELISA (Figure 2). W/O induced six- to tenfold increase in titre relative to antigen without adjuvant. Unmodified PAA elicited two- to threefold increase and butyll6-PAA ester was significantly more active than PAA giving four- to tenfold increases. Comparison of butyl-PAA and W/O revealed that titres were not significantly different 3

response against iNDV in chicken measured by an ELlSA kit

2-log anti-NDV antibody titre

Group

Adjuvant

1 2 3 4 5 6 7 8

No adjuvant W/O (250) Octyll2-PAA Octyll2-PAA Octyll6-PAA Octyll6-PAA Butyll6-PAA Butyll6-PAA

See Table 2. Antibody

Table 4

(mg per dose)

(2) (10) (2) (10) (2) (4)

titres were measured

n

Mean

SEM

Antilog

80 80 60 40 60 20 40 40

8.1 13.6 12.9 12.7 13.1 12.0 13.6 14.0

2.0 1 .l 1 .l 1.4 0.8 1.9 1.1 0.8

270 12417 7822 6427 8985 4096 11954 16384

in a commercially

Effect of various alkyl-PAA esters on the antibody

available ELISA kit (IDEXX).

response against iNDV in chicken measured by VN

2-log anti-NDV antibody

Group

Adjuvant

(mg per dose)

No adjuvant W/O (250) PAA (2) Octyll2-PAA Octyll2-PAA Octyll 6-PAA OctyllG-PAA Butyll 6-PAA Butyll6-PAA See Tab/e 2. Antibody

1578

(2) (10) (2) (10) (2) (4)

n

Mean

80 80 80 80 60 60 20 40 40

16.0 12.0 14.8 14.9 15.8 13.7 15.4 15.9

titre

9.7

titres were measured by an in vitro virus neutralisation

Vaccine 1998 Volume 16 Number 16

Statistical difference

(VN) test.

SEM

Antilog

1.2 1.8 1.9 1.9 1.8 1.3 3.0 0.9 1.9

630 65 536 3956 28 526 29 875 55 749 13308 43 238 61147

Statistical difference

Alkyl-esters of polyactylic acid as vaccine adjuvants: L.A.Th. Hilgers et al. and 6 weeks after boost, but differed in one experiment at 9 and 12 weeks. The calculated half-life of the antibodies elicited by W,‘O was 36 and 68 days, by PAA 81 and 60 days and by butyl-PAA 25 and 31 days.

Reactogenicity

Effect of alkyl-PAA esters on primary immune response in mice

Adjuvanticity of butyl-PAA ester for primary responses was investigated in mice. For this purpose, groups of animals were immunised with influenza virus strain MRC-11 plus OVA with or without adjuvant. PAA octyl-PAA ester and butyll6-PAA increased significantly the antibody responses against both antigens and titres with the alkyl-PAA were equal or higher than those with PAA (Table 5).

13

7-

Polyacrylic acids (PAAs) are available under several trade names (Carbopol, Carbomer, Acritamer, etc.) and applied in cosmetics, house-hold products and pharmaceutical preparations as suspending and thickening agents’7”8. In addition, PAAs are applied as adjuvant in veterinary vaccines, e.g. equine encephalomyelitis virus, equine influenza virus, porcine parvovirus, Mycoplasma hyopneumoniae and Actinobaccilus pleuropneumoniae, avian paramyxovirus and feline and canine rabiesvirus. Adjuvanticity of PAAs has been described several years algo19,20 and has been confirmed more recently by others* . In addition, covalent linkage of proteins to PAA increased the immunogenicity of the antigen22-24. Recently, we demonstrated that polyanionic polymers including PAAs are more effective than various wellknown adjuvant formulations in enhancing secondary immune responses in primed animals”. Efficacy of these polyanions in chickens was insufficient to replace the W/O adjuvant presently used in inactivated poultry vaccines. Attempts to increase the activity of PAAs by supplementing adjuvants from a distinct physicochemical category such as DDA and nonionic blockpolymer LlOl, revealed some beneficial effect?. These observations taken together with examples of positive interference between polyanionic polymers and lipophilic compounds25-‘7 led to the development of the novel group of compounds presented here. The additional or synergistic collaboration between polyanionic polymers and the lipophilic adjuvant DDA was thought to be the consequence of physical association of the negatively charged polymer and the positively charged DDA. These complexes with improved adjuvanticity were mimicked by covalent linkage of aliphatic chains to PAA resulting in a series of so-called alkyl-PAA esters

I

I

*

4

6

10

8

12

Weeksafter booster 15

( b

11 1

o

104-----y 2

=------L

1

4

Weeks

after

r

8

8

10

I 12

booster

Figure 2 Coarse of antibody titres against NDV in chicken after booster immunisation with different adjuvants. In two experiments (A and B), groups of chickens primed with live NDV were boosted with inactivated NDV plus different adjuvants and the specific antibody titre in serum was measured by an ELBA.

Table 5

Adjuvanticity

in mice

DISCUSSION

1

a

of alkyl-PAA derivatives

Local toxicity of alkyl-PAA was investigated in mice (Figure 3). PAA gave moderate swelling of the footpad after injection with maximum values after several days and which declined slowly within 2 or 3 weeks. The two alkyl-PAA esters tested elicited significantly stronger and more persistent reactions than PAA (Figure 3A). W/O provoked significant swelling which was slightly less than that seen with the alkyl-PAA esters during the first weeks, but significantly higher after 3-5 weeks (Figure 3B).

of PAA and a butyl-PAA ester in mice 2-log antibody

titre against:

MRC-11

OVA

Group

Adjuvant (mg per dose)

n

Mean

SEM

1 2 3

No adjuvant PAA [0.05] Butyll 6-PAA [0.005]

12 12 12

10.5 13.1 14.9

0.4 0.5 0.3

Statistical difference* a b C

Mean

SEM

Statistical difference*

4.7 8.9 9.5

0.6 0.6 0.7

z b

Groups of 6 mice were injected in the footpad with 25 ~1 vaccine comprising one volume of antigen solution containing 1 mg ovalbumin per ml and one volume of the adjuvant formulation mentioned above. Three weeks after injection, measured by ELISA. Results of two independent experiments were combined. *Groups which are not statistically (P>O.O25) are indicated by the same letter.

10 pg MRC-11 and antibody titre were

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Alkyl-esters of polyacrylic acid as vaccine adjuvants: L.A. Th. Hilgers et al.

PAA [O.OSl

-

0-l

I

I

1

2

I

days

10.051 [O.OSl

-

Butyll6-PAA

[0.05]

r 17

22

10

after

-

PAA LO.0251

-

PAA [O.OSO]

20

after



a

injection

10

0

CmyllZ-PAA OctyllB-PAA

I

6

days

-

30

injection

Figure 3 Reactogenicity of various alkyl-PAA derivatives in mice. Vaccines containing alkyl-PAA or underivatised PAA (A and B) or W/O (B) were injected into the footpad and swelling was monitored during the subsequent days. The dose of PAA and alkyl-PAA-ester was 0.050 or 0.025 mg per mouse and the dose of W/O corresponded with 10 mg mineral oil per mouse.

varying

in chain

length

of the [email protected]

added

and

the degree of esterification. Depending on the composition, solubility of these esters in water varied from readily soluble to completely insoluble (Table I). Although water-insoluble compounds may have interesting activity, research was focused on the watersoluble products by reason of easiness of manipulation. Adjuvanticity of the alkyl-PAA esters was measured in a chicken model developed to examine stimulation of secondary immune responses by adjuvants and to identify alternatives to the W/O adjuvant presently applied. Antibody titres against NDV were measured by three different test systems, namely an in-house ELISA, a commercially available ELISA kit and a virus neutralisation test. Results of both ELISA systems corresponded closely (correlation coefficient = 0.861; p ~0.01; data not shown), but increase in titres by the adjuvants were more pronounced in the commercial ELISA kit. Determination of the virus neutralisation titres of the samples revealed a significant correlation with the in-house ELISA titres (correlation coefficient = 0.761; p < 0.01; data not shown) which supports the significance of ELISA data. The VN test revealed a larger difference between positive (W/O) and negative control animals than either ELISA. The alkyl-PAA esters and PAA increased the antibody titres in a dose-dependent fashion. Activity of alkyl-PAA esters depended on the esterification grade, but hardly on the length of the carbon-chain. Butyland octyl-PAA with lo-16% esterification exhibited

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1998 Volume 16 Number 16

similar stimulatory effect measured by ELISA and VN. The alkyl-PAA esters generated significantly higher antibody titres than PAA and reached similar responses as W/O. In addition to this model with animals primed with inactivated antigen, the polymers were tested in aged chickens primed with live NDV at early age which is the actual situation in the field. In this situation, butyll6-PAA gave similar antibody titres as W/O, but with slightly shorter half-life (Figure 2). Improved adjuvanticity of butyll6-PAA as compared with PAA was observed also in mice after a single immunisation and in pigs after two immunisations (data not shown). Besides efficacy, factors such as reactogenicity, costeffective production and syringability are important criteria for the commercial success of a vaccine. Depending on the target animal species and the alternatives available, certain degree of local reactions is considered acceptable. In mice, reactogenicity of the novel alkyl-PAA esters manifested by swelling of the footpad after injecting vaccine therein was significantly higher than that of unmodified PAA. The local reaction elicited depended on the dose of alkyl-PAA esters and disappeared within 2 or 5 weeks (Figure 3A). As compared with alkyl-PAA ester, W/O induced stronger, more persistent swelling (35). In chickens and pigs, macroscopic analysis of the site of injection 3 weeks after intramuscular administration of vaccine containing 8 mg alkyl-PAA ester per dose did not display any abnormalities (data not shown) suggesting limited predictive value of the mouse model. Alkyl-PAAs esters have several advantages over W/O formulations. The antigen volume of W/O-adjuvanted vaccines is maximally 50% of the total vaccine volume while that of vaccines on base of alkylPAA ester can be as high as 90%. This is important feature as it facilitates incorporation of multiple antigens into a single shot. During W/O preparation, antigens are exhibited to the emulsification process characterised by increased temperature, high shear stress, etc. These physical/mechanical factors may affect the immunogenicity of the antigens. Emulsions including W/O-adjuvanted vaccines are subjected to destabilisation manifested by separation of phases which limits the shelf-life and implicates strict storage conditions. Finally, W/O adjuvanted vaccines are often considerably viscous products which are difficult to manipulate. Vaccines adjuvanted with alkyl-PAA esters lack these restrictions as they are homogeneous, transparent, aqueous products with low viscosity. It is very likely that at least partially different mechanisms are involved in the regulation of immune responses in naive and primed animals. Circulating antibodies elicited by a previous contact may accelerate antigen elimination and thereby reducing secondary immune responses. A similar mechanism has been suggested to underlay low responsiveness in animals with titres of high maternal-derived antibodies. On base of the relatively strong adjuvanticity of alkyl-PAA esters for secondary immune responses, it can be suggested that these novel adjuvants are also of interest for use in animals with high maternal-derived antibody titres. We concluded that adjuvanticity of polyacrylic acids (PAAs) can be enhanced by introducing alkyl-esters

Alkyl-esters

of polyacrylic

and that these alkyl-PAA esters are promising alternatives to the W/O-adjuvant presently used in poultry vaccines.

12

13

ACKNOWLEDGEM:ENTS The authors thank A. Devos for technical assistance.

REFERENCES Glisson, JR. and Kleven S.H. Poultry vaccines. In: Vaccines for Veterinary Applications (Ed. Peters, AR.). Butterworth Heinemann, Oxford, 1993, pp. 170-173 2 Boney, W.A. and Stone, H.D. Immunologic response to two-day-old passively immune and susceptible chicks to inactivated Newcastle disease virus. I. Alum-precipitated and sodium hydroxide-conjugated vaccine. Avian Dis. 1970, 14, 445-454. 3 Yamanaka, M., Okabe, T., Nakai, M. and Goto, N. Local pathological reactions and immune response of chickens to ISA-70 and other adjuvants containing Newcastle disease virus antigen. Avian Dis. 1993, 37, 459-466. 4 Solyom, F., Masek, I., Geresi, M., Rethy, L., Bacskai, L. and Magyar, T. Inactivated virus antigens’ immunogenicity in adsorbed and freeze-dried multivalent toxoid-virus-bacterium vaccines. Ann. Immunol. Hung. 1986, 25, 29-36. 5 Stone, H.D. and Xie, Z.X. Efficacy of experimental Newcastle disease water-in-oil emulsion vaccines formulated from squalane and squalene. Avian Dis. 1990, 34, 979-983. 6 Stone, H.D. Efficacy of experimental animal and vegetable oil-emulsion vaccines for Newcastle disease and avian influenza. Avian Dis. 1993, 37, 399-403. 7 Brugh, M., Stone, H.D. and Lupton, H.W. Comparison of inactivated Newcastle disease viral vaccines containing different emulsion adjuvants. Am. J. Vet Res. 1983, 44, 72-75. 8 Franchini, A., Canti, M., Manfred, G., Bertuzzi, S., Asdrubali, G. and Franciosi, C. Vitamin E as adjuvant in emulsified vaccine for chicks. Poultry Sci. 1991, 70, 709-715. 9 Chinnah, A.D., Baig, M.A. and Tizard, I.R. Antigen dependent adjuvant activity of a polydispersed beta-(1,4)-linked acetylated mannan (acemannan). Vaccine 1992, 10, 551-557. M.M., Umehara, O., de Lucca-Neto, D., de 10 Rweyemamu, Baltazar, M., Vicente, F.E. and Medeiros-Neto, R.R. Efficacy of avridin as an adjuvant for Newcastle disease virus antigen in chickens. Am. J. Vet. Res. 1986, 47, 1243-1248. 11 Katz, D., Inbar, I., Samina, I., Peleg, B.A. and Heller, E.D. 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. FEMS Immunol. Med. Microb. 1993, 7, 303-314.

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acid as vaccine adjuvants: L.A. Th. Hilgers et al.

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