the invention is further explained in the experimental part of this description which is nonlimiting . until now , four surfactant specific proteins have been described . two hydrophilic proteins , surfactant protein a ( sp - a ) and surfactant protein d ( sp - d ), and two hydrophobic proteins , surfactant protein b ( sp - b ) and surfactant protein c ( sp - c ). the most abundant protein in surfactant is sp - a ( more than 50 %). sp - a consists of 28 to 35 kda peptides assembled into a large multimeric protein composed of 18 similar subunits . also sp - d is a large multivalent molecule comprising 12 monomers of 39 to 43 kda peptides . both proteins have a collagen - like sequence and a cooh — terminal ca 2 ˜ - dependent carbohydrate binding domain . they belong to the c - type mammalian lectins . the surfactant proteins b and c are extremely hydrophobic proteins which in general are dissolved in organic solvents . sp - b is a dimer which consists of two 8 kda peptides whereas sp - c is a monomer with a molecular weight of 8 kda , containing one ( canine ) or two ( human and pig ) palmitoyl groups . the suppression of the immune - system in the lungs and gut is mediated by ( alveolar ) macrophages . in mice after depletion of their alveolar macrophages by intratracheal instillation of clodronate containing multilamelar liposomes , followed by intratracheal instillation of antigen , a local immune response against the antigen can be detected . hence it seems feasible to develop vaccination protocols for vaccination via for example the airways comprising depletion of alveolar macrophages by clodronate containing liposomes , and intratracheal insertion of antigen . antigen , tnp - klh , was entrapped in liposomes consisting of lipids and various concentrations of surfactant proteins and intratracheally instilled in mice which were either depleted of their alveolar macrophages or in control mice . subsequently , the primary immune response against the antigen in these mice was determined after isolation of their spleen cells using an eli - spot assay . in addition , the secondary immune response was measured in mice which were i . p . injected with antigen after the instillation of antigen containing liposomes , in their sera using elisa &# 39 ; s specific for igg , igm and iga . young adult ( 8 - 12 weeks , female ) balb / c mice were obtained from harlan cpb ( the netherlands ). sp - a was isolated from bronchoalveolar lavage fluid of patients with alveolar proteinosis as described ( 1 ). sp - a was dissolved in 5 mm hepes ph 7 . 4 ( 2 . 2 mg / ml ) and stored in small aliquots at − 70 ° c . sp - b and sp - c were isolated from porcine lung lavage . porcine lungs were obtained from the slaughterhouse and lavaged 3 - 5 times with a solution of 154 mm nacl . pulmonary surfactant was prepared from the bronchoalveolar lavage by the method of hawgood et al ( 2 ). lung surfactant was extracted with 1 - butanol ( 1 ). butanol was dried by rotary evaporation , and the residue was dissolved in chloroform / methanol / 0 . 1 m hcl ). ( 1 : 1 : 0 . 05 , v / v / v ). insoluble material was removed by centrifugation . sp - b and sp - c were separated from lipids and purified to homogeneity by sephadex lh - 60 ( pharmacia , uppsala , sweden ) chromatography as described ( 3 ). the proteins were stored in a mixture of chloroform / methanol ( 1 : 1 ; v / v ) at − 20 ° c . the concentration of the proteins was determined by quantitative amino acid analysis . the lh - 60 column fractions which contained the surfactant lipids were dried by rotary evaporation . the lipids were dissolved in chloroform / methanol ( 1 : 1 ; v / v ). the phospholipid concentration was estimated by determining the phosphorus concentration as described by bartlett ( 4 ). dipalmitoylphophatidylcholine ( dppc ), phosphatidylcholine ( eggpc ), phosphatidylglycerol ( eggpg ) and cholesterol were purchased from avanti polar lipids ( alabaster , ala ., usa ). 86 mg eggpc and 8 mg cholesterol were dissolved in 10 ml chloroform . the chloroform was dried by rotary evaporation . the lipid film was suspended in either 4 ml pbs ( control liposomes ) or 10 ml dichloromethylene - diphosphonate ( dmdp ) in pbs ( 0 . 189 g / ml ). the suspension was kept for 2 hours at room temperature . subsequently , it was sonicated for 3 min in a water bath sonicator and kept at room temperature for 2 hours . the dmdp - containing liposomes were suspended in 100 ml pbs and centrifuged at 15 , 000 g to remove free dmdp . the pellet was suspended in 4 ml pbs . lipids were dissolved in the presence or absence of sp - b and / or sp - c in chloroform . the chloroform was dried by rotary evaporation . the lipid film was suspended in 0 . 8 mg trinitrophenyl - keyhole limpet hemocyanin ( tnp - klh )/ ml pbs by vortexing . subsequently , the liposomes were sonicated for 1 min on ice using an mse ultrasonic disintegrator . for the experiments with sp - a , sp - a was either added to the lipid suspension together with the antigen or after the preparation of the antigen - containing liposomes . the mice were fixed in an upright position under anesthetization with 20 μl of a 4 : 3 ( v / v ) mixture of aescoket ( aesculaap , gent , belgium ) and rompun ( bayer , leverkussen , germany ), intramuscularly injected . using a nylon tube connected to a 1 ml syringe , 100 μl liposomes were injected through the glottis into the trachea . at day 0 , mice were intratracheally instilled with 100 p . 1 of either pbs or dmdp - liposomes or pbs liposomes . at day 2 , mice were intratracheally injected with 100 p . 1 of antigen - containing liposomes . to study the primary immune response , animals were autopsied at day 9 and their spleen cells were isolated to determine the number of tnp - antibody secreting cells using a spot - elisa . animals used to study the secondary immune response were injected intratracheally with the same suspensions at the same times as the mice used for the primary response , but were injected with 100 p . 1 tnp - klh ( 0 . 8 mg / ml pbs ) intraperitonealy at day 23 . the animals were sacrificed at day 30 , and their sera were collected in order to determine the tnp - antibody titers using an elisa . this assay was based on the method described by sedgwick and holt ( 5 ). briefly , microtiter plates were coated overnight at 4 ° c . with 5 p . g per well trinitrophenylated ovalbumin ( tnp - ova ). the plates were emptied and incubated with 1 % ( w / v ) bovine serum albumin ( bsa )/ ml pbs for 1 h at room temperature . the plates were rinsed twice with pbs . a single cell suspension of spleen cells ( 10 7 / ml rpmi 1640 supplemented with 1 % hepes ( w / v ), 1 % bsa ( w / v ) and 10 % fetal calf serum ( v / v ) was added to the first well of each row ( 150 p . 1 / well ) and serial diluted 1 : 2 in the same medium . after incubation at 37 ° c ., the wells were rinsed with 0 . 1 % tween 20 ( v / v ) in pbs ( pbt ) till the cells were lysed . subsequently , goat anti mouse igm ( 1 : 500 ; in 1 % bsa - pbt ; sanbio ) conjugated with alkaline phosphatase was added to each well ( 100 μl / well ) and the plates were incubated for 2 h at 37 ° c . after rinsing the plates four times , the alkaline phosphatase substrate , 5 - bromo - 4 - chloro - 3 — indolyl - phosphate ( 1 mg / ml amp buffer ( sigma ) supplemented with 1 % low melting agarose ( w / v ) was added to the wells ( 100 μl / well ) at 37 ° c . the plates were incubated overnight at 4 ° c . and the cells secreting tnp antibodies were counted . in order to detect specific antibodies against tnp in the sera of mice , an elisa was developed based on the method described by delemarre et al ( 6 ). in short , microtiter plates were coated with tnp - ova and incubated with 1 % bsa as described above . the plates were rinsed four times with pbt . for the determination of the igm and igg in the sera , the sera were diluted with pbt supplemented with 1 % bsa ( 1 : 7 ). in order to measure iga , the sera were diluted four times with same buffer . the diluted sera were added to the first well of each row ( 200 μl / well ) and serially diluted 1 : 1 in the pbtbsa bufter . after incubation of the plates for 2h at room temperature , the plates were rinsed four times with pbt . to each well , 100 μl of either biotinylated anti igm , or anti igg or anti iga was added . the plates were incubated for 1 h at room temperature and rinsed four times with pbt . the plates were incubated for 1 h at room temperature avidin conjugated hrp ( 1 : 5000 in pbt - bsa ), washed and the bound antibodies were visualised by incubation for 1 h at room temperature with the substrate o - phenylenediaminedihydrochloride ( 2 mg / ml 0 . 1 m phosphate - citrate buffer ph 5 . 5 supplemented with 0 . 015 % h 2 o 2 100 μl / well ). the reaction was terminated by adding 50 μl of 2 . 5 m h 2 so 4 . the absorbance at 492 nm was measured and the values were expressed as compared to positive control sera for either igm , or igg or iga . the control sera were included on each plate , and were raised in mice by immunisation with tnp - ova . depletion of am by dmdp containing liposomes followed by intratracheal instillation of antigen incorporated in small unilamellar vesicles ( suv ) leads to the production and secretion of specific igm antibodies in the spleen cells of mice ( fig1 ). the mice remained healthy throughout the experiment and no inflammatory responses were observed in the lungs of the treated mice . a maximal immune response is obtained at lower phospholipid concentrations , 5 mg / ml versus 20 mg / ml , when tnp - klh is incorporated in suv &# 39 ; s which contain the surfactant lipids and also the surfactant proteins sp - b and sp - c compared to suv &# 39 ; s which consists of only the surfactant lipids . however , at high lipid concentrations , 20 mg / ml , no difference is observed between suv &# 39 ; s containing the surfactant proteins and suv &# 39 ; s devoid of the hydrophobic proteins . the half maximal immune response for the surfactant suv &# 39 ; s which contained sp - b and sp - c was observed at a phospholipid concentration of 3 mg / ml . this concentration was used in the following experiments . in order to determine which surfactant protein ( s ) entrapped in antigen surfactant liposomes can enhance the immune response , the following experiment was performed . antigen liposomes were prepared which consisted of the surfactant lipids , and the various surfactant proteins . the lipid to protein ratio was approximately the same as the physiological ratio , i . e ., for sp - a : phospholipid / sp - a ( 15 : 1 ( w / w ), for sp - b : phospholipid / sp - b ( 100 : 0 . 5 - 1 ( w / w ) and for sp - c : phospholipid / sp - b ( 100 : 0 . 5 - 1 ) ( 3 , 7 ). the antigen liposomes which contained sp - b yielded a higher immune response than the other liposomes ( fig2 ). the liposomes which contained only sp - c of sp - a gave a similar immune response as the liposomes which consisted of the surfactant lipids . in addition , incorporation of sp - c in sp - b containing antigen liposomes did not influence the sp - b mediated enhancement of the immune response . in this experiment , sp - a was encapsulated in the antigen liposomes together with the antigen , when sp - a was added to the antigen - liposomes , i . e ., was not present in the liposomes , similar results were obtained ( results not shown ). to determine the concentration dependency of the sp - b induced enhancement of the immune response , various concentrations of sp - b were incorporated in antigen containing liposomes . an optimal immune response was observed as a phospholipid to protein ratio of 200 : 1 ( 3 mg phospholipid : 15 μg sp - b ) ( fig3 ). for stimulation of the immune response by antigen sp - b liposomes , the presence of the negatively charged phospholipid , phosphatidylglycerol ( pg ), in the liposomes is required ( fig4 ). since no effect of sp - b inclusion in the antigen - liposomes on the immune response was noted when the antigen - liposomes consisted of a lipid mixture of dppc , eggpc , cholesterol ( 61 : 30 : 9 ; w / w / w ) whereas the sp - b activity was restored when a mixture of the lipids dppc , eggpc , cholesterol and pg ( 55 : 27 : 8 : 10 ; w / w / w ) was used . influence of am depletion on the sp - b mediated stimulation of the immune response in order to determine if depletion of am by intratracheal instillation of dmdp - containing liposomes is needed to elicit an immune response via the airways when antigen sp - b liposomes are used , the following experiment was performed . mice were divided in three groups , group 1 was intratracheally instilled with only the antigen containing liposomes , group 2 was intratracheally injected with pbs liposomes followed two days later by the antigen liposomes , and group 3 received the dmdp liposomes and the antigen liposomes ( fig4 ). the use of pbs - containing liposomes instead of dmdp - containing liposomes or no liposome treatment at all prior to the intratracheal instillation tnp - klh encapsulated in the sp - b - dppc / pc / cholesterol / pg liposomes , yields an immune response in the spleen cells which is approximately the same as when the am were depleted , also when surfactant lipids were used instead of the dppc / pc / cholesterol / pl lipid mixture in combination with the hydrophobic surfactant proteins for antigen liposomes , similar results were obtained ( fig5 ). influence of sp - b containing antigen liposomes on the secondary immune response in these experiments , mice were either intracheally injected with dmdp - liposomes or not on day 0 . at day 2 , mice received intratracheal antigen - liposomes of different composition . at day 23 , mice were boosted with an ip injection of antigen . at day 30 , mice were sacrificed and the sera were collected to determine the titers of specific antibodies against the antigen as well as their classes . a high igm titer was detected in the sera of mice which were not depleted of their am , intratracheally primed with sp - b containing antigen liposomes , and boosted with antigen ( fig6 a ). additional depletion of am caused only a slight enhancement of the igm titers . in contrast , only a poor igg titer is observed when the animals were not depleted of their am with marginal effects of incorporation of sp - b in the antigen liposomes ( fig6 b ). higher igg titers are observed when the mice were depleted of their am prior to the intratracheal instillation of antigen liposomes , which can be further increased by the use of sp - b containing antigen liposomes . antigen entrapped in surfactant liposomes can induce a systemic immune response in mice , as shown after intratracheal instillation . no depletion of the alveolar macrophages by an intratracheal injection of dmdp - containing liposomes is needed to observe this immune response . a preferred method for the induction of the immune response via the airways is a method whereby the antigen is entrapped in liposomes which contain at least sp - b and phosphatidylglycerol . the sp - b effect is concentration dependent reaching a maximum at the physiological phospholipid : sp - b ratio of 100 : 0 , 5 - 1 . the observed pg dependency of sp - b is not surprising , since the positive charges of sp - b were reported to be essential for its activity ( 8 ) such as the phospholipid insertion in the surface film , which covers the alveoli , via the negatively charged pg ( 9 , 10 ). in a previous study from our laboratory , thepen et al ( 11 ) demonstrated that only a local , iga , immune response can be elicited in the lung after prior depletion of the alveolar macrophages by dmdp - containing liposomes . depletion of alveolar macrophages was necessary to induce an immune response via the airways , because alveolar macrophages were shown to inhibit immune responses probably via the suppression of t - cells and dendritic cells . depletion of alveolar macrophages which leads to the destruction of cells which may compete with the type ii cells for the uptake of antigen containing liposomes as well as a reduction of the suppression of the immune response is no longer needed to induce a primary immune response when animals are intratracheally injected with sp - b - containing antigen liposomes . the advantages for the use of sp - b containing liposomes are : a . naturally occuring in the form of surfactant , b . the homology of sp - b derived from different species ( canine , human , porcine rabbit ) is high ( more than 80 %), c . sp - b ( porcine , bovine ) containing liposomes , in the form of surfactant , have already been used to treat premature children . 1 ) sp - b containing liposomes can be used , as in this study , for immunisation via the airways . a systemic immune response , igm , can be obtained . no depletion necessary . 2 ) in combination with am depleting liposomes , sf - b - antigen liposomes can be used for immunisation . 3 ) since sp - b and surfactant analogue material is present in the intestines , immunisation via the intestines using sp - b containing antigen liposomes is effective . 4 ) in the lungs or in the intestines sp - b containing liposomes can be used to transport the following substances to the alveolar type ii cells ( or similar cells in the intestines ) a ) dna , sp - b coupled to modified sp - b has been shown to enhance transfection . probably sp - b , dna containing liposomes may work to get dna in vivo into the type ii cells . b ) transport of corticosteroids to alveolartype ii cells . these substances are used for immature children in order to induce the surfactant production by type ii cells . c ) transport of antioxidants to type ii cells . has been shown for sp - a containing liposomes . 1 . haagsman h p , hawgood 5 , sargeant t , buckley d , white r t , drickamer k , benson b j . 1987 . the major lung surfactant protein , sp28 - 36 , is a calcium - dependent , carbohydrate binding protein . j . biol . chem . 262 : 13877 - 13880 . 2 . hawgood et al . 1985 . effects of a surfactant - associated protein and calcium ions on the structure and surface activity of lung surfactant lipids . biochemistry 24 : 184 - 190 . 3 . oosterlaken - dijksterhuis m a , van eijk m , van buel b l m van golde lmg , haagsman h p . 1991 . surfactant protein composition of lamellar bodies isolated from rat lung . biochem . j . 274 : 115 - 119 . 4 . bartlett , g r . 1959 . phosphorous assay in column chromatography . j . biol . chern . 234 : 466 - 468 . 5 . sedgwick j d , holt pga . 1983 . solid - phase immunoenzymatic technique for the enumberation of specific antibody - secreting cells . j immunol meth . 57 : 301 - 309 . 6 . delemarre f g a , claassen e , van rooijen n . 1989 . the primary in situ immune response in popliteal lymph nodes and spleen of mice after subcutaneous immunisation with thymusdependent or thymus - independent ( type 1 and 2 ) antigens . anat . rec . 223 : 152 - 157 . 7 . phizackerley p j r , town m h , newman g e . 1979 . hydrophobic proteins of lamellated osmiophilic bodies isolated from pig lung . biochem j 183 : 731 - 736 . 8 . cochrane c g , revak s d . 1991 . pulmonary surfactant protein ( sf - b ): structure - function relationships . science . 253 : 566 - 568 . 9 . yu s h , possmayer f . 1992 . effect of pulmonary surfactant protein b ( sp - b ) and calcium on phospholipid adsorption and squeeze - out of phosphatidylglycerol from binary phospholipid monolayers containing dipalmitoylphosphatidylcholine . biochim . biophys . acta . 1126 : 26 - 34 . 10 . yu sh , fossmayer f . 1992 . studies on surfactantassociated protein b mediated adsorption of surfactant phospholipids . am . rev . respir . dis . 145 : a874 . 11 . thepen t , van rooijen n . kraal g . 1989 . alveolar macrophage elimination in vivo is associated with an increase in pulmonary immune response in mice . j . exp . med . 176 : 499 - 509 .
the invention relates to the field of immunology , more specifically to adjuvants used to increase the immunogenicity of antigens , for example , for incorporation into vaccines . the invention provides use of a pulmonary surfactant or a component thereof as an adjuvant substance that is able to stimulate an immune response , but , in general , are homologous to substances native to the body .