Feasibility of single-shot H5N1 influenza vaccine in ferrets, macaques and rabbits.

The feasibility of a single-shot, low-dose vaccination against pandemic influenza was investigated. The immunogenicity and safety of whole inactivated, cell culture-derived H5N1 virus plus CoVaccine HT™ as adjuvant was tested in various animal species. In ferrets, doses of 4.0 and 7.5 µg H5N1 (NIBRG-14; A/Vietnam/1194/04; clade 1) without adjuvant gave low geometric mean haemagglutination inhibition (HI) titres (GMTs) of 21-65 three weeks after intramuscular (IM) injection. The addition of 0.25-4 mg CoVaccine HT™ resulted in GMTs of 255-1470 corresponding with 4-25-fold increases. A second immunization caused GMTs of 8914-23,525 two weeks later, which confirmed strong priming. One out of 8 ferrets injected with antigen alone and 5 out of 32 ferrets injected with adjuvanted H5N1 demonstrated minimal transient, local reactions and two animals immunized with adjuvanted H5N1 exhibited increased body temperature one day after injection. In macaques, 5 µg H5N1 with CoVaccine HT™ or aluminium hydroxide as adjuvant elicited GMTs of 172 and 11, respectively three weeks later. A second immunization resulted in GMTs of 1751 and 123, respectively four weeks later. Analysis of cross-reactivity of antibodies after the first immunization with NIBRG-14 adjuvanted plus CoVaccine HT™ revealed GMTs of 69 against NIBRG-23 (A/turkey/Turkey/1/05; clade 2.2) and 42 against IBCDC-RG-2 (A/Indonesia/5/05-like; clade 2.1.3) while titres with aluminium hydroxide were <10. After the second immunization with CoVaccine HT™, GMT against NIBRG-23 was 599 and against IBCDC-RG-2 254, while those with aluminium hydroxide were 23 and 13, respectively. No local or systemic adverse events were detected in macaques. Safety of 5 µg H5N1 plus 0, 2 or 4 mg CoVaccine HT™ was investigated in a repeated dose study in rabbits. Groups of 6 or 9 male and female animals were immunized IM three times at three week intervals. None of the animals exerted treatment-related adverse reactions during the study or at necropsy 3 or 4 days after treatment. We concluded that a low dose of whole inactivated influenza virus plus CoVaccine HT™ is a promising, single-shot vaccine against pandemic influenza.

Oral immunisation of naive and primed animals with transgenic potato tubers expressing LT-B.

The efficacy of edible vaccines produced in potato tubers was examined in mice. Transgenic plants were developed by Agrobacterium tumefaciens-mediated transformation. The antigen selected was the non-toxic B subunit of the Escherichia coli enterotoxin (recLT-B). A synthetic gene coding for recLT-B was made and optimised for expression in potato tubers and accumulation in the endoplasmic reticulum. Introduction of this gene under control of the tuber-specific patatin promoter in potato plants resulted in the production of functional, i.e. Gm1-binding, recLT-B pentamers in tubers. Selected tubers containing about 13 microg of recLT-B per gram fresh weight were used for immunisation. Subcutaneous immunisation with an extract of recLT-B tubers yielded high antibody titres in serum that were similar to those obtained with bacterial recLT-B. The efficacy of oral administration of recLT-B tubers was determined by measuring mucosal and systemic immune responses in naive and primed mice. Animals were primed by subcutaneous injection of an extract of recLT-B tuber plus adjuvant. Naive and primed mice were fed 5 g of tubers ( approximately 65 microg of recLT-B) or were intubated intragastrically with 0.4 ml of tuber extract ( approximately 2 microg of recLT-B). In naive mice, feeding recLT-B tubers or intubation of tuber extract did not induce detectable anti-LT antibody titres. In primed animals, however, oral immunisation resulted in significant anti-LT IgA antibody responses in serum and faeces. Intragastric intubation of tuber extract revealed higher responses than feeding of tubers. These results indicate clearly that functional recLT-B can be produced in potato tubers, that this recombinant protein is immunogenic and that oral administration thereof elicits both systemic and local IgA responses in parentally primed, but not naive, animals.

Adjuvant effects of sulfolipo-cyclodextrin in a squalane-in-water and water-in-mineral oil emulsions for BHV-1 vaccines in cattle.

The antibody and cell mediated immune responses induced by BHV-1 were analysed in cattle after vaccination and challenge exposure to the virulent strain LA of BHV-1. Animals were vaccinated intramuscularly (IM) with inactivated virus vaccines against BHV-1 containing either a water in mineral oil adjuvant (W/O), a water in mineral oil adjuvant plus Avridine (W/O+Avridine) or sulfolipo-cyclodextrin in squalane in-water emulsion (SL-CD/S/W). No significant differences were registered in the antibody response induced by the three evaluated vaccines. However, the BHV-1 specific cell-mediated immunite response was stronger and appeared earlier when SL-CD/S/W was included in the formulation. The efficacy of the vaccines was also evaluated after intranasal challenge of the calves with a virulent BHV-1 LA strain. Animals vaccinated with SL-CD/S/W had reduced virus excretion and clinical symptoms compared with the mock-vaccinated animals. Comparison of levels of BHV-1 specific IgG2 and IgG1 with virus shedding revealed that, regardless of the adjuvant administered, animals showing BHV-1 specific IgG2/IgG1 ratios higher than 1 were those with a significant lower number of individuals shedding virus. Additionally, animals vaccinated with SL-CD/S/W presented no post-vaccinal reactions. These factors, combined with the higher efficacy and the ease of manipulation of the biodegradable oil, makes the vaccine formulated with this new adjuvant an important contribution for the veterinary vaccines industry.

Alkyl-polyacrylate esters are strong mucosal adjuvants.

Synthetic polymers were examined for their potency to enhance mucosal immune responses to inactivated antigens. Aqueous solutions of polyacrylic acid with a MW of 450 kDa (p[AA]) or an butyl-ester thereof with 16% esterification (Butyl16-p[AA]) plus antigen were administered twice intranasally in mice with a 2 week interval. The frequency of IgA-antibody secreting cells (ASCs) in lung cell suspensions was determined 1 week after the second immunisation. Both polymers significantly enhanced the IgA response against inactivated Newcastle disease virus (iNDV), inactivated influenza virus strain MRC-11 (iMRC-11), haemagglutinin/neuraminidase subunits of influenza virus strain A/Texas (HA/NA) and bovine serum albumin (BSA). Butyl16-p(AA) was significantly more effective than non-derivatised p(AA), cholera toxin B subunit (CTB) or liposomes. The factor of increase in IgA-ASCs varied from <10- to >100-fold and depended on the type of antigen, the dose of antigen and the adjuvant. Extremely high responses of about 10,000 IgA-ASCs per million lung cells were detected after immunisation with 5 microg HA/NA plus 50 microg Butyl16-p(AA). Intranasal immunisation with Butyl16-p(AA) resulted in high IgA responses, not only in the lungs, but also in the spleen and in high IgG responses in these organs. We concluded that alkyl-esters of polyacrylate are an interesting, novel category of mucosal adjuvants.

Sulfolipo-cyclodextrin in squalane-in-water as a novel and safe vaccine adjuvant.

Previously, we described synergistic adjuvanticity of combinations of synthetic sulfolipo(SL)-derivatives of polysaccharide (SL-polysaccharides) and squalane-in-water emulsions (squalane/W). In this paper, effects of type of polysaccharide and nature of oil on adjuvanticity, reactogenicity and stability are described. SL-derivatives of the following polysaccharides were synthesised: synthetic polysucroses with weight-average molecular weight (MW) of 400,000 (Ficoll400), 70,000 (Ficoll70) and 39,000 Da (Ficoll39), polyfructose of 5,000 Da (inulin), linear polyglucose of 1,200 Da (maltodextrin) and cyclic polyglucose of 1,135 Da (beta-cyclodextrin). The number of sulphate groups per monosaccharide of the different SL-polysaccharides varied between 0.15 and 0.23 and the number of lipid groups per monosaccharide between 1.15 and 1.29. Adjuvant formulations were prepared by incorporating these SL-polysaccharides into oil-in-water emulsions of either squalane, hexadecane, soya oil or mineral oil. Adjuvanticity of the formulations obtained for humoral responses to inactivated pseudorabies virus (PRV) and inactivated influenza virus strains A/Swine (A/Swine) and MRC-11 (MRC-11) in pigs and MRC-11 and ovalbumin (OVA) in mice depended on the type of oil (squalane = mineral oil > hexadecane = soya oil) but not on the type of polysaccharide backbone of the SL-derivative. Reactogenicity assessed by local swelling in mice decreased with decreasing MW (SL-Ficoll400 = Ficoll70 = Ficoll39 > SL-inulin = SL-maltodextrin > SL-cyclodextrin) when combined with squalane and decreased with the type of oil in the following order: squalane > mineral oil > hexadecane > soya oil when combined with SL-Ficoll400. Stability of the SL-polysaccharide/squalane/W emulsions at elevated temperature increased with decreasing MW of the SL-polysaccharide (SL-Ficoll400 < SL-Ficoll70 = SL-Ficoll39 < SL-inulin = SL-maltodextrin = SL-cyclodextrin). SL-cyclodextrin/squalane/W remained stable for > 2.5 years at 4 degrees C, > 18 weeks at 37 degrees C and > 10 days at 60 degrees C. We concluded that reactogenicity and stability but not adjuvanticity of SL-polysaccharide/squalane/W formulations depended on the MW of SL-polysaccharide and that SL-cyclodextrin/squalane/W is a promising non-mineral oil adjuvant as it combines strong adjuvanticity (i.e. better than the mineral oil-based adjuvant presently applied) with low reactogenicity and good stability.

Effect of various adjuvants on secondary immune response in chickens.

Stimulatory effects of several types of adjuvants on secondary antibody response to inactivated Newcastle disease virus (iNDV) were examined in chickens. For this purpose, animals were primed with iNDV without adjuvant resulting in a low but significant antibody response, boosted with iNDV plus adjuvant 3 weeks later, and analysed for specific antibody titres in serum 3 weeks after the booster. Water-in-mineral oil emulsion (W/O) caused significant increase in antibody titres measured in an indirect enzyme-linked immunosorbent (ELISA), haemagglutination inhibition (HI), and virus neutralisation (VN) assay. The adjuvants tested included three oil-in-water emulsions (i.e. mineral oil-in-water, sulpholipo(SL)-Ficoll400/squalane-in-water and sulpholipo-cyclodextrin/squalane-in-water), three negatively-charged polymers with high molecular weight (i.e. polyacrylate, polystyrenesulphonate and sulpho(S)-Ficoll400) and two surface-active agents (i.e. dimethyldioctadecylammonium bromide (DDA) and Quil A). These adjuvants enhanced significantly the secondary immune response but none reached the titre obtained with W/O. Combinations of adjuvants with distinct physicochemical properties, i.e. polyacrylate and DDA revealed only slight, beneficial effects. We concluded that the various types of adjuvants tested can stimulate secondary immune responses in primed animals but that W/O is superior.

Analysis of oral uptake of a synthetic adjuvant by an immunoassay with monoclonal antibodies.

A blocking ELISA with specific antibodies against a synthetic sulpholipo-polysucrose (SL-Ficoll400) was developed to quantify this vaccine adjuvant component in faeces with the purpose of estimating the uptake thereof by the gastrointestinal tract after oral administration. Specific monoclonal antibodies (MABs) prepared against SL-Ficoll400 did not cross-react with components in murine faeces. SL-Ficoll400 inhibited the reactivity of the MABs in a dose-dependent fashion in the blocking ELISA and the concentration causing 50% inhibition (IC50) was about 1 microg SL-Ficoll400 per milliliter. Derivatives of Ficoll400 lacking sulphate or lipid groups or both were unable to block the MABs (IC50 > 1000 microg/ml) which confirmed the specificity of the test system. Quantification of SL-Ficoll400 in extracts of faeces of untreated mice spiked with SL-Ficoll400 revealed that more than 90% could be recovered. The blocking ELISA was used to quantify SL-Ficoll400 in the faeces of mice given this compound orally. One, two and three days after oral administration, the cumulative recovery of SL-Ficoll400 in faeces was 30, 40 and 92%, respectively. From these data, we concluded that the MABs obtained were specific for SL-Ficoll400, that SL-Ficoll400 in murine faeces could be quantified by a blocking ELISA with MABs and that SL-Ficoll400 was poorly absorbed by the gastrointestinal tract.

Quantitative analysis of a synthetic adjuvant by an immunoassay.

Antibodies against a sulpholipo-derivative of synthetic polysucrose (SL-Ficoll) were prepared with the purpose of developing a detection system for this adjuvant component. SL-Ficolls with polysucrose backbones of 400 kDa (SL-Ficoll400) or 22 kDa (SL-Ficoll22) were not immunogenic. However, SL-Ficoll22 conjugated to bovine serum albumin (SL-Ficoll22-BSA) induced high levels of specific antibodies against SL-Ficoll in mice as determined by an indirect ELISA with either SL-Ficoll400 or SL-Ficoll22 as coating antigen. The specificity of the antibodies was analysed further in a blocking ELISA with SL-Ficoll400 as coat. Dose-dependent inhibition of the ELISA titre was obtained with SL-Ficoll22 and SL-Ficoll400 and the concentration causing 50% inhibition (IC50) was < 1 microg/ml. Non-derivatised polysucrose (Ficoll400) and compounds lacking either lipid or sulphate were not recognised (IC50 > 1000 microg/ml). The reaction of SL-Ficoll400-derivatives with antibodies increased with increasing sulphate and lipid content and maximal inhibition was produced by compounds with a composition similar to the SL-Ficoll22 used for immunisation. Quantitative analysis of SL-Ficoll400 in adjuvant formulations comprising SL-Ficoll400 incorporated in a squalane-in-water emulsion revealed high recovery and sufficient precision. From these data, we conclude that specific antibodies are generated against the synthetic SL-Ficoll400 and that this component of a novel adjuvant formulation can be quantified by an immunoassay.

Alkyl-esters of polyacrylic acid as vaccine adjuvants.

Previously, we demonstrated that polyacrylic acid (PAA) augmented significantly the immune response to inactivated Newcastle disease virus (iNDV) in chickens, but that efficacy was insufficient to replace the water-in-mineral oil (W/O) adjuvant applied for boosting primed animals. Attempting to improve its adjuvanticity, PAA with weight-average molecular weight (Mw) of 450 kDa was grafted with alkyl-chains by esterifying the carboxylic groups with octanol and butanol. The butyl-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-PAA 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% (octyl16-PAA and butyl16-PAA, respectively) gave similar titres as W/O. In aged animals primed with live NDV at early age, butyl16-PAA and W/O elicited comparable antibody responses. Butyl16-PAA was also more effective than PAA 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 butyl16-PAA was less than of W/O. We concluded that alkyl-PAA esters are strong adjuvants for primary and secondary responses and that they are promising alternatives to the mineral oil-based adjuvants presently used in various veterinary vaccines.

A novel non-mineral oil-based adjuvant. I. Efficacy of a synthetic sulfolipopolysaccharide in a squalane-in-water emulsion in laboratory animals.

Sulfolipopolysaccharides (SLPs) were synthesized by reaction of the synthetic polysucrose polymer Ficoll-400 with chlorosulfonic acid and lauroyl chloride in anhydrous medium. Hydrophobic derivatives were obtained by addition of a small number of sulfate and a large number of lipid groups. Gel-permeation high-performance liquid chromatography (g.p.-h.p.l.c.) exhibited a wide range in molecular weight of both Ficoll-400 and SLP polymers. The calculated weight-average molecular weight (Mw) of Ficoll-400 and SLP using polystyrene polymers as references was 187,000 and 380,000 respectively, exhibiting a twofold increase in molecular weight upon derivatization. Adjuvanticity of hydrophobic SLPs with 0.2 sulfate and 1.5 lipid groups per sucrose monomer, a squalane-in-water emulsion (S/W), SLP incorporated into S/W (SLP/S/W), and a mineral oil-based emulsion (O/W) was investigated in combination with different antigens in mice and guinea-pigs. Antibody responses in serum against ovalbumin (OVA), dinitrophenylated bovine serum albumin (DNP-BSA), inactivated influenza virus strain MRC-11 (MRC-11), a mixture of three influenza virus strains (iFlu3) and inactivated pseudorabies virus (iPRV) were measured by either haemagglutination (HA), haemagglutination inhibition (HI) or serum neutralization (SN). Vaccines were prepared by simply mixing one volume of antigen with one volume of adjuvant solution. Antibody titres after one or two injections with these antigens were enhanced significantly by SLP/S/W, SLP, S/W and O/W and in most studies, SLP/S/W was demonstrated to be more effective than either the two constituent components or the O/W adjuvant.(ABSTRACT TRUNCATED AT 250 WORDS)

A novel non-mineral oil-based adjuvant. II. Efficacy of a synthetic sulfolipopolysaccharide in a squalane-in-water emulsion in pigs.

The adjuvanticity of a sulfolipopolysaccharide (SLP) incorporated into a squalane-in-water emulsion (SLP/S/W) was compared with that of a mineral oil-in-water (O/W) adjuvant currently used in commercial porcine vaccines. Groups of pigs were immunized twice with vaccines comprising either inactivated influenza virus (iFlu3 containing strains A/Swine, MRC-11 and X-79), inactivated pseudorabies virus (iPRV), live pseudorabies virus (PRV) or inactivated porcine parvovirus (iPPV) as antigen and SLP/S/W or O/W as adjuvant. Antibody titres in serum 2 or 3 weeks after the second immunization were measured by haemagglutination inhibition (HI) or serum neutralization (SN) assays. Both adjuvants significantly augmented the antibody responses against the antigens tested. Mean factors of increase obtained by SLP/S/W and O/W were: 315 and 91, respectively, for A/Swine; 478 and 137 for MRC-11; 362 and 128 for X-79; 69 and 49 for iPRV; and 23 and 7 for live PRV. Increased humoral immunity against live PRV was affirmed by reduced levels and duration of virus excreted by pigs after challenge with virulent PRV. Immunization of pigs with iPPV plus adjuvant SLP/S/W gave 36-fold higher titres than with O/W. It was concluded that SLP/S/W is more effective than O/W in stimulating humoral immunity against the viral antigens examined and that the two constituents SLP and S/W interact synergistically. Advantages of SLP/S/W over O/W include stronger adjuvanticity, better biocompatibility and lower doses of active substances.

DDA as an immunological adjuvant.

As compared to other adjuvants, DDA is a moderate or strong adjuvant for humoral responses and a strong adjuvant for CMI, especially DTH responses, against different types of antigens and in both laboratory animals and larger animals. DDA can collaborate with other immunomodulating compounds resulting in further enhanced responses. Mechanisms include interactions with both antigen and components of the host immune system and possibly, multiple beneficial effects contribute to the relatively strong adjuvanticity of DDA. Toxicity of DDA is not known but severe detrimental side effects were not seen. This adjuvant can be applied in experimental vaccines and in commercial vaccines for veterinary purposes, especially if cell-mediated immunity is considered to be important. In immunology, DDA can be of use to study T helper cells responsible for DTH responses (T helper cells type 1) and to characterize T helper cell epitopes on antigens (Snijder et al., 1992).

[Improvement of veterinary vaccines through the use of new adjuvants]. / Verbetering van veterinaire vaccins door gebruik van nieuwe adjuvantia.

A large number of infectious diseases in man and animals may be prevented by vaccination today, but (satisfactory) vaccines are not yet currently available for all forms of infection. Various reasons may be given to account for this fact, such as slight effectiveness of the experimental vaccines so far developed, limitations of large-scale production, storage and distribution of vaccines, etcetera. Recent developments in the fields of molecular biology, virology, bacteriology and immunology offer new opportunities for the development and improvement of vaccines. Two types of vaccine are distinguished, viz. dead and live vaccines. Live vaccines usually produce a stronger and longer-lasting immunity than dead vaccines, and the latter require the assistance of compounds which promote immunity, so-called adjuvants. However, the adjuvants used today tend to give rise to untoward side-effects, or are only moderately effective. The use of new and safer substances as substitutions for adjuvants which are currently administered may help to produce improved vaccines. A number of aspects of adjuvants, such as their possibilities and limitations, the variety of compounds having an adjuvant effect and possible mechanisms of action will be discussed in the present paper.

Detection of Marek's disease virus antigen in chickens by a novel immunoassay.

An immunoassay was developed to detect Marek's disease virus (MDV) antigen on the tips of feathers obtained from MDV-infected chickens. MDV in follicular debris on the feather tip was demonstrated by use of a specific monoclonal antibody. The principle of an indirect ELISA was employed and the feather tip was used as the solid phase. Presence of MDV was reflected by a dark brown precipitate on the feather tip which could be observed by naked eye. This test system proved to be more sensitive than the agar-gel precipitation (AGP) test as all feather tips of MDV-infected animals gave a positive reaction in the feather tip-ELISA whereas about a half yielded a detectable precipitate in the AGP. Advantages of this feather tip-ELISA and applications are discussed.

In situ detection by monoclonal antibody D-35.1 of cells infected with Marek's disease virus that interact with splenic ellipsoid-associated reticulum cells.

Immuno- and enzyme-histochemical staining procedures were used to investigate in vivo the interaction of Marek's disease virus (MDV) with splenic non-lymphoid cells. The newly developed monoclonal antibody D-35.1, which recognizes all three MDV serotypes, was used to study the localization of MDV at various times after intramuscular inoculation of 1-day-old chicks with MDV strain K. The D-35.1-positive cells were detected in the bursa of Fabricius, spleen, thymus, proventriculus, and cecal tonsils, and the number of chickens showing the cells increased between days 4 and 10. From day 21, the skin of the chickens contained D-35.1-positive feather follicles. The D-35.1 monoclonal antibody did not stain any cells in peripheral blood, nerves, kidney, and gonads at any time. In addition, D-35.1-positive cells were not detected in lymphoproliferative lesions in visceral organs and peripheral nerves. Double staining procedures on serial sections using monoclonal antibody CVI-ChNL-68.2, specific for splenic ellipsoid-associated reticulum cells, revealed that the majority of D-35.1-positive cells were situated in the peri-capillary sheath of reticulum cells at day 10. The sheath of cells detected by monoclonal antibody CVI-ChNL-68.2 was disrupted, and they were clustered around D-35.1-positive cells. These results support the hypothesis that ellipsoid-associated reticulum cells are involved in the early pathogenesis of Marek's disease.

Serum amyloid P component induction by immunomodulators.

The capacity of immunoadjuvants to enhance serum amyloid P component (SAP) levels and to modulate the humoral immune response to sheep red blood cells in a number of different mouse strains was investigated. Although the synthetic adjuvants dimethyldioctadecylammonium bromide, dextran sulphate and bacterial-derived lipopolysaccharide did not enhance SAP levels in some of the mouse strains tested, these strains responded normally to the immunomodulating effects of the adjuvants. We conclude that increased SAP levels and modulation of immune responses are induced via at least partially different pathways. For these reasons, it is impossible to screen drugs for potential adjuvant activity by only measuring SAP levels in mice.

Synthetic sulpholipopolysaccharides: novel adjuvants for humoral immune responses.

Referring to the strong immunostimulating activity of combinations of lipophilic agents and dextran sulphate, conjugates with chemical determinants of both types of adjuvants were synthesized and then examined for immunostimulatory capabilities in mice. Saturated fatty acids with varying chain lengths and sulphate groups were coupled covalently at defined ratios to the polysaccharide Ficoll (MW 400,000). Chemical analysis of 60 of the sulpholipopolysaccharides synthesized revealed that the number of sulphate groups per monosaccharide unit varied from 0 to 1.6, and the number of lipid groups from 0 to 0.8. Adjuvanticity of these conjugates for the humoral immune response was determined using sheep red blood cells (SRBC) and dinitrophenyl-haptenated bovine serum albumin (DNP-BSA) as antigens. Five days after intraperitoneal injection of adjuvant and antigen, the numbers of direct anti-SRBC plaque-forming cells (PFC) in the spleen were determined. Anti-DNP antibody titres were measured from 1 to 4 weeks after immunization. PFC responses to 2 X 10(6) SRBC were augmented up to a 100-fold by conjugates of Ficoll and sulphate (sulphopolysaccharides: SPs) or lipid groups (lipopolysaccharides: LPs). Introduction of low or moderate numbers of lipid groups in SPs reduced adjuvanticity. Adjuvant activity of sulpholipopolysaccharides (SLPs) with varying sulphate and high lipid content depended on the sulphate contents and the chain length of the lipids. Sulphate reduced adjuvanticity of the SLPs, and the number of sulphate groups required for complete annihilation increased with the chain length of the lipid. LPs and SLPs, including conjugates that did not enhance anti-SRBC PFC responses, augmented serum antibody responses to DNP-BSA while SPs were hardly effective.

Suppression of the cellular adjuvanticity of lipophilic amines by a polyanion.

Modulation of delayed-type hypersensitivity reaction (DTH) in mice by synthetic adjuvants and the mode of their action were investigated. Intracutaneous injection of azobenzenearsonate coupled to phosphatidylethanolamine (A-PE) without adjuvant did not induce DTH. Administration of A-PE with the quaternary amines dimethyldioctadecylammonium bromide (DDA) or N,N-dioctadecyl-N',N'-bis(2-hydroxyethyl)propane diamine (CP-20,961) induced a strong response. Other surfactants, dextran sulfate (DXS) and dextran were not effective. In combination with 200 nmol DDA the optimal dose of antigen was 5 nmol A-PE, while at higher antigen doses DTH was diminished. Responses on combination of two adjuvants and A-PE revealed that DXS counteracted the stimulatory effects of both DDA and CP-20,961. In vitro, DDA formed insoluble complexes with 14C-A-PE and at optimal antigen concentration more than 90% of the antigen was bound to the adjuvant. The percentage of 14C-A-PE bound to 200 nmol DDA decreased with increasing doses of 14C-A-PE. Addition of DXS to the mixture of 14C-A-PE and DDA reduced the percentage of 14C-A-PE bound to DDA. Dose-response curves demonstrated a close relationship between the inhibitory effects of DXS on the DTH and the A-PE/DDA complex formation. Nonsulfated dextran affected neither the DTH nor the formation of complexes in vitro. In conclusion, cellular adjuvanticity of DDA for the lipophilic antigen A-PE is probably the result of formation of insoluble complexes with the antigen. Free A-PE suppresses the cellular response to A-PE/DDA complexes. The adjuvant DXS inhibits DTH by reducing the amount of immunogenic A-PE/DDA complexes and thus increasing the amount of free, immunosuppressive A-PE.

Route-dependent immunomodulation: local stimulation by a surfactant and systemic stimulation by a polyanion.

Immunomodulatory activity of the two synthetic adjuvants dimethyldioctadecylammonium bromide (DDA) and dextran sulfate (DXS) in relation to route and time of injection was investigated in mice. Humoral responses to sheep red blood cells (SRBC) were measured as the number of direct anti-SRBC plaque-forming cells (PFC) in the spleen 5 days after immunization. Both adjuvants stimulated the anti-SRBC response if adjuvant and antigen were injected simultaneously via the same route (either intraperitoneally or intravenously). Administration of adjuvant and antigen via different routes (intraperitoneally or intravenously, respectively or vice versa) resulted in enhanced humoral responses after DXS, but not after DDA. Intraperitoneal immunization of mice which were injected intraperitoneally with either adjuvant 4 days earlier resulted in diminished humoral responses. Immune responses in pretreated mice were not suppressed when the antigen was injected intravenously instead of intraperitoneally. In conclusion, DDA and DXS differ in immunostimulating properties as DDA enhanced only a response to antigen injected via the same route whereas DXS induced a systemic state of increased immunoresponsiveness. The immunosuppressive state induced by intraperitoneal injection of either adjuvant prior to immunization is restricted to the peritoneal compartment. Mechanisms underlying differences between both adjuvants and aspects of systemic immunopotentiation are discussed.

Synergistic effects of synthetic adjuvants on the humoral immune response.

The effect of combinations of adjuvants on the humoral immune response to sheep red blood cells (SRBC) as antigen was investigated. Adjuvants belonging to two categories differing in physicochemical properties were used: surfactants (N,N-dioctadecyl-N',N'-bis-(2-hydroyethyl)propanediamine (CP-20,961), dimethyldioctadecylammonium bromide (DDA), neutrally charged liposomes, polyol (L 101 and L 121) and polyanions [dextran sulfate (DXS), liquoid and suramine]. All adjuvants but suramine augmented humoral responses to 2 X 10(7) SRBC measured by the number of direct anti-SRBC plaque-forming cells (PFC) in the spleen. The response to 2 X 10(6) SRBC was enhanced considerably by L 121 and DXS but hardly or not at all by the other adjuvants. Combinations of two adjuvants were made at distinct ratios (1:3, 2:2, and 3:1) and injected intraperitoneally with 2 X 10(6) SRBC. Low responses (5 X 10(3) PFC per spleen) were induced by combinations of liquoid or suramine with DDA or DXS, and by combinations of CP-20,961, liposomes, L 101 or L 121 with DDA. Combinations of the surfactants DDA, CP-20,961, liposomes, L 101 or L 121 with DXS evoked responses which were significantly higher than the sum of responses supported by the single adjuvants. Ratios of 1:3 or 2:2 (surfactant: DXS) resulted in the most effective combinations. The data obtained suggest that only adjuvants derived from two different physicochemical groups are able to act synergistically.