Adjuvant activity of non-ionic block copolymers. IV. Effect of molecular weight and formulation on titre and isotype of antibody

Robert Hunter,* Margaret Olsen and Stephen Buynitzky

The adjuvant activity o1 block copolymers varies with the lengths o / lhe chains o / polyoxypropylene (POP) and poh,o.vyethylene (POE). This project evaluated the adjuvant activi O, o1' new copoh'mers with long poh'oxypropyh, ne chains in mice immunized with TNP-hen egg albumin in 2% squalone-in-u,ater emulsions. Two oJ'the new copolymers, L 141 and L180.5, not onh' stimulated higher antibody titres to TNP than older preparations, hut also #tduced a higher percentage qlthe IgG2 isot37~e,v. The smaller ohter copolymers, LIO1 and L121, imhlced higher absolute levels 0[' IgG3 antibody and relative increases in IgG1. Incorporation o f immunomodulator,v (cell wall skeletons, monophosphowI lipid A or threonvl muramyl dipeptMe) increa,ved mean IgG titres hut also increased variability o / responsiveness among indivi&tals.

Keywords: Adjuvant; isotypc: block copolymer: IgG antibody

I N T R O D U C T I O N

The development of improved vaccine adjuvants for use in humans is a priority area of research. Modern molecular biology has provided us with a means of producing immunogens with unprecedented case and precision. However, purified immunogens seldom pro- duce strong immtlne responses without effective ad- juvants. Research on adjuvants has lagged seriously behind that on immunogens. Alum, which has been used for decades, is the only adjuvant widely used in humans ~ . Saponin and its purified component Quil A, Freund's Incomplete Adjuvant and other adjuvants used in research and veterinary applications have toxicities which limit their potential for use in human vaccines" s. Newer chemically defined preparations such as muramyl dipeptide and monophosphoryl lipid A are being studied, but much remains to be learned" <~

The non-ionic block copolymer surfactants have a simple structure composed of two blocks or chains of hydrophilic polyoxyethylene {POE) flanking a single block of hydrophobic polyoxypropylene ( POP )t 0. They are considered to be among the least toxic of known surfactants and are widely used in foods, drugs and cosmetics ~. Some of the large hydrophobic copolymers are effective adjuvants while closely related preparations are not 1°12'13. Correlation of adjuvant activity of these copolymers with differences in the chain lengths of POE

Department of Pathology, 762 WMB, Emory University, Atlanta, Georgia 30322, USA. *To whom correspondence should be addressed. (Received 15 March 1990; revised 22 August 1990; accepted 28 August 1990)

0264 410X 91 040250 07 1991 Butterworth-Heinemann Ltd

250 Vaccine, Vol. 9, April 1991

and POP provides the opportunity to develop structure-function relationships. One of the copolymcrs, L I21, is a particularly effective adjuvant for inducing IgG antibody formation and is currently undergoing clinical development as a component of the Syntex adjuvant formulation (SAF-MF) 1~. Copolymer LI01 is similar to L121 except that both its POE and POP chains are approximately 30% shorter. Its ability to serve as an adjuvant for increasing antibody formation is more variable than that of LI21, but it frequently stimulated delayed type hypersensitivity responses 1"'1s. A closely related copolymer, L122, is identical to LI21 in all respects except that the POE hydrophile chains are longer. It is not an effective adjuvant 12. Several other preparations with shorter POP or longer POE chains also had little or no activity as adjuvants. Based oil these observations, we proposed that new copolymers with larger POP chains and a small proportion of POE would be effective adjuvants and might have novel properties. Copolymers were synthesized for us with these considerations in mind. This paper describes studies on their adjuvant activity.

Our early studies on block copolymer adjuvants showed a marked dependence of the antibody response on the relationship of the protein antigen to the oil phase of the emulsion. Protein which was physically incorporated within mineral oil drops induced much higher antibody titres than similar amounts of protein dissolved in the aqueous phase of the emulsion l°. This form of oil-in-water emulsion was adapted from the work of Ribi et al. who found that the adjuvant activity of BCG cell wall emulsions was similarly dependent upon incorpor-

at±on of the cell walls into oil drops 9. In 1987, Byars and Allison reported strong adjuvant effects of copolymer L121 in a squalane-in-water emulsion with the antigen in the aqueous phase 14. The present studies were designed to evaluate the effects of copolymer structure and emulsion formulation in mice immunized with squalane-in-water emulsions of trinitrophenyl conjugated hen egg albumin (TNP-HEA). The isotype and titre of antibody were measured with a newly developed calibrated isotype specific ELISA procedure.

M A T E R I A L S A N D M E T H O D S

Antigen preparation

The trinitrophenyl (TNP) hapten, was conjugated to grade V recrystallized hen egg albumin (HEA) (Sigma, St Louis, MO). TNP was conjugated to HEA using 5 mM trinitrobenzene sulphonate in borate buffer, pH 8.4 according to a published method 16. The extent of trinitrophenylation was determined spectrophotometri- cally using an extinction coefficient of 15 400 at 350 nm. Eight to nine TNP units were bound per mole of HEA.

Adjuvants and formulations

The new non-ionic block copolymer surfactants, L141, L180.5, L181.5 and L182.5, were manufactured by BASF Corporation, Wyandotte, MI for CytRx Corporation, Norcross, GA. The other copolymers were also manufactured by BASF. Each copolymer preparation consists of a central polymer of POP flanked by two hydrophilic chains of POE. The structures of the copolymers used in these studies are shown on Figure 1 and Table 1. The required amount of TNP-HEA was lyophilized and incorporated into oil-in-water emulsions containing 2% squalane with the selected copolymer. The final dose was 50/~g of TNP-HEA and 1 mg ofcopolymer per mouse.

Animals and immunization procedures

Female ICR (outbred) mice, 7 l0 weeks old, were obtained from Charles River Laboratories, Raleigh, NC. Animals were given a subcutaneous injection in the hind footpad (40/A volume) containing the above mentioned dosages of antigen and adjuvant. Mice were bled at

L81

L92

L101 L121

022

L141

L180.5

L181.5

L182.5

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Figure 1 Structure of the non-ionic block copolymers. Each of the preparations used consists of a central hydrophobic polymer of polyoxypropylene (POP) (C3H60)n (black line) flanked by two small hydrophilic polymers of polyoxyethylene (POE) (C2H,O)m (grey lines). The lengths of the representations are drawn to scale to represent the relative sizes of the hydrophilic and hydrophobic components of the preparations used. The POE segments are drawn thicker to represent their hydration shells

Block copolymer adjuvants: R. Hunter et al.

Table 1 Structure of IgG antibody response induced by non-ionic block copolymers

N a m e

IgG antibody t i tres c

MW POP s %POE ~ Day 28 Day 97

L81 2250 10 1 045 ± 117 n.d. L92 2750 20 882 ± 102 n.d. L101 3250 10 24 875 ± 8 751 267 919 ±82 631 L121 4000 10 11 828 ± 4 407 209 891 ± 120 490 L122 4000 20 264± 165 414 ±233 L141 4600 10 112 431 ±22 728 510 272 ± 125 563 L180.5 5200 5 307 863 ±66 575 360 072 ±7 470 L181.5 5000 15 6715± 1 604 152 367 ± 33 649 L182.5 5200 25 1 500±520 n.d.

aMean molecular weight of POP hydrophobic chain. The process of synthesizing these copolymers results in a statistical variation of chain lengths. The distributions are skewed towards small copolymers due to premature termination of the polymerization reaction. ~Mean percent by weight of each preparation which consists of POE. The numbers are truncated to the nearest 5% as per the convention of the manufacturer. ClgG antibody to TNP was measured by ELISA at day 28 after a primary injection and 7 days after a secondary injection at day 90. n.d., not done

various time points throughout the course of the study via retro-orbital plexus using heparinized Natelson capillary tubes and plasma was stored at -70°C.

ELISA procedures

Evaluation of the immune response induced by each preparation was made using an enzyme linked immunosorbent assay (ELISA). Antigen was prepared by the reaction of picrysulphonic acid with BSA fraction V. Microtitre plates were treated with 100~Lt per well TNP-BSA (25 TNP units per mole BSA) at 0.5/~g ml-1 PBS, pH 8.4 overnight at 4°C. The antigen solution was replaced with 1% BSA in PBS, pH 7.4 and the plates were incubated for 1 h at 22°C in order to block any sites left available for non-specific binding of antibody. The plates were washed four times with 0.05% poloxamer 188 in PBS, pH 7.4. Next, 100/zl of serial dilutions of test plasma with 0.1% BSA and 0.1% poloxamer 188 in PBS, pH 7.4 were added and incubated for l h at 22°C on an orbital shaker (200r.p.m.). The plates were then washed three times and incubated for 90min at 37°C with affinity- purified horseradish peroxidase-conjugated goat anti- body directed against mouse IgG or specific IgG subclass. A 1:2000 dilution of conjugate was used for all except IgG3, for which a 1:1000 dilution was used. Following this step, the plates were washed three times and colour development was achieved with orthophenylene diamine dihydrochloride (OPD) 0.4 mg ml- 1, and hydrogen peroxide 0.009%, in citrate/phosphate buffer, pH 5.0. The reaction was stopped using 2.5 N sulphuric acid (HzSO4) 15 min after the addition of OPD and hydrogen peroxide and read at 490nm using a Bio-Rad (Richmond, CA) model 3550 microplate reader. Titres were defined as the dilution of antiserum required to produce an absorbance of 1.0 under the conditions of the assay.

Frequently, IgG isotype values are simply expressed as end-point ELISA titres. We endeavoured to improve the sensitivity of the assay by determining the binding capacity of the goat anti-mouse IgG l, IgG2a, IgG2b, and IgG3 peroxidase conjugates in terms of nanograms per millilitre of bound myeloma protein and determining the amount of colour in the sensitive range of the ELISA

Vaccine, Vol. 9, April 1991 251

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curve, lsotype quantitation was done by converting the end-point ELISA titres to nanograms per millilitre of each subclass by multiplication with a conversion factor, using a modification of the method of Kenney et al.'*. Ten micrograms per ml of a polyclonal goat anti-mouse lgG (Fisher Biotech, Orangeburg, NY), diluted in PBS, pH 7.3, was used to coat the wells of a 96-well microtitre plate. Washing, blocking, and incubation times were the same as those in the ELISA assay above. Dilutions of mouse myeloma proteins of each isotype (Sigma Chemical Co. St Louis, MO) were used as standards. A goat anti-mouse isotype-specific horseradish peroxidase conjugate (Fisher Biotech) was used to determine the absorbance of the standards at concentrations of 119 0.03ng. The concentrations of isotype-specific standards, resulting in an absorbance value of 1.0, were determined from standard curves of the absorbance (490 nm) versus the concentration, by regression analysis. The titre of hapten-specific isotype at an absorbance of 1.0, was multiplied by the ELISA titre at an absorbance of 1.0, to give the concentrations in ngml '. The lowest levels of detectable antibody for each isotype were 107ngml ' IgG1, 337ngml ' IgG2a, 146ngml t IgG2b, and 280 ng ml- 1 igG3.

R E S U L T S

Groups of five to ten mice were immunized with TNP-HEA in a 2% squalane-in-water emulsion contain- ing 1 mg of each of the copolymers shown in Table l. The time courses of the antibody responses were similar in each of the groups. The titres peaked at approximately 1 month after injection and persisted for over 3 months. The animals were boosted on day 90 after immunization. They were bled again one week post boost. The copolymers with 10% or less POE all induced strong immune responses. Copolymer L122, as expected, was a poor adjuvant. The adjuvant activity of copolymers with a range of POE chain lengths and the POP chains with molecular weights of 5200 (L180.5, L181.5 and L182.5) followed the pattern established previously for the series of smaller copolymers L 121, L 122 and L 123. Copolymers with more than 10% POE were again found to be ineffective adjuvants.

The mean titres stimulated by the most effective copolymer of each length of POP chain increased with increasing molecular weight of the POP hydrophobe, Figure 2. This experiment was repeated several times with similar results. While there was variability between and among groups, the general pattern of increasing titre with increasing molecular weight of hydrophobe was observed repeatedly.

The isotype of antibody was measured at multiple time points using an ELISA assay with calibrated class specific antisera. As shown in Figure 3, the copolymer preparations which were effective adjuvants for inducing antibody induced distinctly different patterns of isotype. The lower molecular weight preparation, L l01, induced a predominant IgG 1 response with lesser amounts of lgG2a and IgG2b. Increasing the molecular weight of the hydrophobe increased the proportion of IgG2, especially lgG2b. Interestingly, the production of the IgG3 isotype followed the opposite pattern with the highest titres produced by the lower molecular weight preparations, L121 and especially L101. The ratio of IgGl to IgG2b antibody increased in a nearly linear fashion with

100

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Figure 2 Influence of molecular weight of POP on antibody titre. Mice were immunized with 501~g of TNP-HEA in a squalane-in-water emulsion containing 1 mg of copolymer with the molecular weights indicated. These were, from left to right, L101, L121, L141 and L180.5. The mean IgG antibody t i tre±s.e, of groups of 5 6 mice on day 28 are shown

Block copolymer adjuvants. R. Hunter et al.

L180.5 L141 L121 LIOI

Figure 3 IgG subclass concentrations induced by copolymer adjuvants. Mice were immunized with 50 l~g of TNP-HEA in a squalane-in-water emulsion containing 1 mg of the copolymer shown. They were bled for determination of antibody of particular IgG isotypes by the calibrated ELISA procedure at day 28. The mean concentration of antibody of each isotype of 5-6 mice_+s.e, are shown. (11), IgG1; (1~) IgG2a; ([:3) IgG2b; (E3) IgG3

molecular weight of the hydrophobe, Figure 4. The distribution of isotypes was measured at multiple intervals following the 28 day determination. The isotype patterns produced by each copolymer tended to persist during subsequent assays.

Several investigations were carried out to evaluate the effect of formulating antigen in the oil or aqueous phase of the emulsions. In keeping with our previous studies, formulations containing antigen incorporated in the oil phase produced significantly higher titres than identical formulations with antigen in the aqueous phase, Figure 5. They also produced a different pattern of distribution of lgG isotypes. Antigen in the aqueous phase produced similar concentrations of IgG2b antibody but less IgG2a and IgG1. Immunizations of the same preparations without the copolymer produced only very low con- centrations of antibody. Several other experiments were conducted to compare the antibody titres induced by single injections of copolymer emulsions with antigen in

252 Vaccine, Vol. 9, Apri l 1991

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Figure 5 Effect of incorporation of the antigen in the oil or aqueous phase of the emulsion. Mice were immunized with 50#g of TNP-HEA and 100/~g of tMDP in squalane-in-water emulsions. The emulsions contained 1 mg copolymer L141 or no copolymer as shown. The antigen was incorporated in either the oil or saline phase of the emulsion. Antibody titres were determined 28 days after a single injection and are expressed as mean-t-s.e. (11), IgG1; (1~1)IgG2a; (D) IgG2b; (rq) IgG3

the oil or aqueous phase. In each case, higher titres were induced by antigen in the oil phase.

The effects of multiple injections of an emulsion containing the immunomodulator tMDP and antigen in the aqueous phase were different. Even though the preparation produced a small primary response, mice were primed for an anamnestic response almost equivalent to that of the mice immunized with the

Block copolymer adjuvants: R. Hunter et al.

emulsion containing antigen in the oil phase, Figure 6. tMD P was used in this study to facilitate comparison with published data on the SAF-MF formulation 4'14. In previous studies with the copolymer emulsions without immunomodulators, the magnitude of the anamnestic response varied in a direct way with that of the primary response. Several of the mice in this and other experiments were followed for over 1 year. Antibody titres persisted at levels of approximately 1000 ELISA units for at least this long following single or multiple injections of each of the emulsions containing antigen in the oil phase.

Outbred mice were used in these studies to facilitate identification of possible genetic influences that might influence antibody responses. In studies with tMDP as well as other immunomodulating agents, we consistently observed greater variability among the titres of mice immunized with emulsions containing immunomodula- tors than with those which contained only the copolymer as adjuvant, Figure 7. The occurrence of two apparently

50

40

30

20

10

20 40 60 80

I 00

Time (days)

Figure 6 Effect of emulsion formulation on primary and secondary antibody titres. Mice were immunized with 50,ug of TNP-HEA with 1 mg of L121 and 100 #g of the threonyl derivative of MDP on day zero and boosted with the same emulsion without tMDP on day 90. Antigen was incorporated !n either the oil (@) or saline (O) phase as shown. The mean titres of IgG antibody +s.e. for groups of 5 or 6 mice are shown

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Vacc ine , Vol. 9, Apr i l 1991 253

Block copolymer adjuvants: R. Hunter et al.

distinct groups of high responders and low responders was observed on multiple occasions when any one of the immunomodulators was used with an adhesive copolymer adjuvant. The copolymcr adjuvants, by themselves, consistently produced less wmation m antibody titres.

D I S C U S S I O N

This paper reports that non-ionic block copolymer adjuvants which are larger than those previously available are more effective adjuvants for stimulating IgG antibody and that they increase the proportion of IgG2a and IgG2b isotypes. Since protection against infection frequently depends upon stimulating an appropriate isotype of antibody and/or type of cell mediated immunity, an understanding of the properties of immunogens which influence the stimulation of different types of immune response would be of significant value in developing improved vaccines 4'~7 2o. It has been reported that determination of antibody isotype is largely dependent upon B cell epitopes 2~. This implies that the only way to modulate isotypes is to prepare immunogens with different B epitopes. More recent evidence has demonstrated that the situation is far more complex. The synthesis ofisotypes is now thought to be influenced by an array of interacting cells and soluble factors. It is thought that the antigen presenting cell somehow induces the proliferation or activation of different subclasses of helper cells, THI and TH2, which tend to increase the production of lgG2 or lgG1 antibody, respectively ~2 2s In addition, gamma interferon tends to be associated with the production of IgG2a while I L-4 is associated with IgE and IgG126. While much remains to be learned, it is clear that the system can be influenced at multiple levels.

The structural similarity of wmous non-ionic block copolymer surfactants is deceptive and has led to confusion in the literature. Variation in the length and mode of linkage of blocks of POP and POE produce great diversity of physical-chemical and biological properties. Some promote adhesion of proteins to the surface of oil drops while others inhibit i t 1 ° ' 1 1 ' 2 7 Closely related copolymers induce markedly different inflammatory responses TM. The block copolymers ewduated in this study belong to a distinct group. They share a common single chain structure, are large, insoluble, and act as adhesive molecules in that they bind protein antigen to hydrophobic surfaces ~3. The octablock copolymer, TI50RI, is also an adjuvant, but acts via a different mechanism. It has only weak adhesive properties and is a potent ionophore for monovalent cations 2s'2'. As an adjuvant, it can act synergistically with adhesive copolymcr LI213°. Other investigators have reported adjuvant activity of block copolymers, such as 25R1, which have neither adhesive nor ionophoric properties 32. They induce intense inflammation which suggests that they may act as immtmomodulating agents ~°. Although much remains to be learned, it is clear that copolymers with the POE POP POE structure are a distinct group and should not be confused with those with other structures.

The availability of a series of copolymers which differ only in the lengths of chains of POP and POE provides a unique opportunity to evaluate structure function relationships ~o,~2,13, The new copolymers investigated in this project were synthesized for this purpose. The

prcparations with larger h_xdrophobcs than those previously available proved to be superior adjuvants: they induced higher titrcs o[ antibody and a hight: proportion of the IgG2 isotype. The preparations which induced higher concentrations of IgG2 antibody were larger, less soluble and would be expected to form more stable structures at oil water interfaces than the smaller preparations which more cflectively stimulated the synthesis of lgGl and IgG3 antibody. One can anticipate that these differences would influence interaction with antigen presenting cells, the retention or distribution of antigen in the tissue or other fi~ctors which might influence the response.

We previously reported that the adhesive copolymcr adjuvants are able to fold in such a way as to produce it hydrophilic surface which binds protein antigens to hydrophobic surfaces such as oil drops. The hydrophobic chains of preparations with fewer than 56 polyoxypropy- lene moieties arc unable to fold sufficiently to place all of the hydrophile on the surface t 3. This appears to explain the lack of efficacy of adjuvants of preparations smaller than LI01, Fi~.lm'(' b,'. As illustrated by the comparison:~ among the two series of copolymers with identical hydrophobes (L121, L122, LI80.5, L181.5 and L182.5) the length of the hydrophilic chain which is exposed at the surface is also important. Excessive hydrophile (POE) chain length is correlated in a reduced ability to bind protein antigens TM. It has been suggested that hydrogen bonding to the POE chains is important in binding of antigen by these copolymers ~~. Since a high proportion of hydrophobic POP is required for binding protein, hydrophobic bonding is probably important also. In addition, POE by itself is umtble to bind native proteins ~ . Copolymers with long chains of POE actually inhibit bonding of proteins to hydrophobic surfaces ~ ~,2-" The adhesion of protein is, therefore, not due solely to either hydrogen bonding or hydrophobic interactions, but involves both.

Our current conception of the mechanism of action of the copolymer adjuwmts is illustrated in Fiqure 9. The copolymcrs which arc effective adjuvants appear to be adhesive molecules which bind the protein antigens to the surface of oil drops. Such surfaces also bind and activate host mediators such as complement ~2. Each of the

Figure 8 Model of the conformation of representative copolymers at ar oil water interface. The copolymers which are adjuvants are able to form hydrophilic surfaces which bind proteins by hydrophobic interactions. The effective adjuvants (underlined) (L101, L121, L141 and L180.5) have small hydrophilic portions which are just sufficient to form hydrophilic surfaces The longer POE chains of L122, L181.5 and L182.5 retard access ot proteins to the underlying hydrophobic adhesive regions and prevent these preparations from being effective adjuvants. The small copolymers (L81 and L92) have POP chains which are too short to fold sufficiently for the surface to be uniformly hydrophilic. This causes surfaces covered by them to have high surface tensions which become distorted in multiple ways. The figures are drawn to scale to represent the relative lengths of the POE (in water) and POP (in oil) chains on each preparation

254 Vaccine, Vol. 9, April 1991

Figure 9 Model of the adhesive activity of copolymer adjuvants. The block copolymers are depicted as in Figure 8 on a hydrophobic surface such as an oil drop. In this configuration, they are able to bind protein antigens (A) and complement (C) in a way which facilitates presentation to cells of the immune system. The copolymer also induces expression of class II (la) antigen on macrophages. Each of these activities appears to be due to the adhesive properties of the copolymers

copolymers which are adhesive adjuvants also induce increased expression of class II (Ia) molecules on macrophages and increase their ability to present antigen to T cells 32. However, the effectiveness of various copolymers in inducing expression of Ia does not correlate with their potency as adjuvants. Consequently, we proposed that this may be a necessary, but not sufficient, condition for adjuvant activity of this group of copolymers. Antigen bound by copolymers to the surface of oil drops is presented in a highly concentrated form to cells of the immune system in conjunction with a variety of activated mediators. We believe that this is the essential activity of this class of block polymer adjuvants. Immunomodulators such as tMDP may add additional effects by stimulating the production of lymphokines or otherwise activating and recruiting cells.

The effect of immunomodulators on within-group variability of antibody titres was unexpected. In the present experiments as in our previous work, the variability of immune responses produced by the copolymer adjuvants with the antigen incorporated in oil drops was relatively small even in outbred mice. Addition ofimmunomodulators such as LPS or MPL increased the mean titres primarily by selecting very high responses in a small proportion of the mice. The immunomodulators tMDP and MPL induced heterogeneity of immune responses which was not observed with the copolymers alone. The mechanisms of this effect are purely speculative but may be related to the differential ability of individual mice to produce a cytokine response to the im- munomodulators. Outbred mice were used in these studies in order to evaluate individual effects. An adjuvant that induces very high titres in some individuals but relatively low ones in others may be of less value than one that induces uniform responses at an intermediate level.

The importance of placing the protein antigen in different phases of the emulsion requires comment. In the present experiment and in our previous work, the magnitude of the anamnestic response induced by block copolymer adjuvants was roughly proportional to the magnitude of the primary response. Results with the SAF-MF preparation were different. This preparation with antigen incorporated in the aqueous phase induced

Block copo lymer adjuvants: R. Hunter et al.

only a very weak primary antibody response but primed the animals for a secondary response equivalent to that of the emulsion with the antigen incorporated in the oil phase. The extent to which this effect was due to the immunomodulating effect of tMDP, to faster removal of antigen from the site of injection with altered kinetics of delivery to the lymph node or other factors is unknown.

In summary, we have demonstrated that various non-ionic block copolymer adjuvants are able to influence the isotype as well as the intensity of immune responses in multiple ways. Investigations reported in the accompanying paper demonstrate that the production of the highly protective IgG2 isotype can be further enhanced by incorporation of immunomodulating agents into the copolymer emulsions.

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

This work was supported in part by USPHS Grant AI-25856 and CytRx Corporation. The authors are grateful to Ms Beth Bennett for much help and advice in carrying out the studies and writing the manuscript, to Ms Marcia Kalish for advice and assistance in the development of antibody assays, and to Ms Linda McGuire for expert secretarial assistance.

R E F E R E N C E S

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2 Louis, J.A. and Lambert, P.H. Lipopolysaccharides: from immunostimulation to autoimmunity. Springer Seminars in Immunopathology 1979, 2, 215-229

3 Bomford, R.H.R. Differential adjuvant activity of AI(OH) 3 and saponin. In: Immunopharmacology of Infectious Diseases: Vaccine Adjuvants and Modulators of Non-Specific Resistance (Ed. lvlajde, J.). Alan R. Liss, 1987, pp. 165-170

4 Kenney, J.S., Hughes, B.W., Masada, M.P. and Allison, A.C. Influences of adjuvants on the quantity, affinity, isotype and epitope specificity of murine antibodies. J. Immunol. Methods 1989, 121, 157-166

5 Verheui, A.F.M.M., Versteeg, A.A., De Reuver, M.J., Jansze, M. and Snippe, H. Modulation of the immune response to pneumococcal type 14 capsular polysaccharide-protein conjugates by the adjuvant Quil A depends on the properties of the conjugates. Infect. Immun. 1989, 57, 1078-1083

6 Takayama, K. Qureshi, N., Ribi, E. and Cantrell, J.L. Separation and characterization of toxic and nontoxic forms of lipid A. Rev. Infect. Dis. 1984, 6, 439~443

7 Chedid, L., Aldibert, F., Lefrancier, P., Choay J. and Lederer, E. Modulation of the immune response by a specific adjuvant and analogs. Prec. Natl Acad. Sci. USA 1976, 73, 2472

8 Takayama, K., Qureshi, N., Ribi, E. and Cantrell, J.L. Separation and characterization of toxic and nontoxic forms of lipid A. Rev. Infect. Dis. 1984, 6, 439-443

9 Ribi, E., Cantrell, J.L., Takayama, K., Qureshi, N., Peterson, J. and Ribi, H.O. Lipid A and immunotherapy. Rev. Infect. Dis. 1984, 6, 567~72

10 Hunter, R.L. Strickland, F. and Kezdy, F. Studies on the adjuvant activity of nonionic block polymer surfactants. I. The role of hydrophile-lipophile balance. J. Irnmunol 1981, 127, 1244-1250

11 Ceresa, R.J. Block and Graft Copolymerization, Vol. 2, John Wiley, New York, 1976

12 Hunter, R.L. and Bennett, B. The adjuvant activity of nonionic block polymer surfactants. II. Antibody formation and inflammation related to the structure of triblock and octablock copolymers. J. Immunol. 1984, 133, 136~1375

13 Hunter, R.L. and Bennett, B. The adjuvant activity of nonionic block polymer surfactants. III. Characterization of selected biologically active surfaces. Scand. J. Immunol. 1986, 23, 287-300

14 Byars, N.E. and Allison, A.C. Adjuvant formulation for use in vaccines to elicit both cell-mediated and humeral immunity. Vaccine 1987, 5, 123-128

Vacc ine , Vol. 9, Ap r i l 1991 255

Block c o p o l y m e r adjuvants. R. Hun te r et al.

15 Snippe, H., DeReuver, M.J., Strickland, F., Willers, J.M.N. and Hunter, R.L. Adjuvant effect of nonionic block polymer surfactants in humoral and cellular immunity. Int. Arch. Allergy Appl. Immunol. 1981, 85, 390 398

16 Garvey, J.S., Cremer, N.E., Sussdorf, D.H. DNP- and TNP-conjugated proteins. In: Methods in Immunology 3rd edition (Eds Garvey J.S., Cremer, N.E. and Sussdorf, D.H.) W.A. Benjamin Inc., Reading, Mass. 1977, pp. 153 158

17 Kaminski, M.S., Kitamura, K., Maloney, DG. Campbell, M.J. and Levy, R. Importance of antibody isotype in monoclonal anti-idiotype therapy of a murine B cell lymphoma. A study of hybridoma class switch variants. J. Immunol. 1986, 136, 11231130

18 Brodskyn, C.I., Da Silva, A.M.M., Takehara, H.A. and Mota, I. Characterization of antibody isotype responsible for immune clearance in mice infected with Trypanosoma cruzi. Immunol. Lett. 1988, 18, 255 256

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256 Vaccine, Vol. 9, Apr i l 1991