Adjuvant-independent induction of immune responses by antibody-mediated targeting of protein and peptide antigens

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parison of abilities of recombinant interleukin-la and -8 and noninflammatory IL.-l8 fragment 163-171 to upregulate C3b receptors (CR]) on human neutrophils and to enhance their phagocytic capacity. ~flammation, 14, 185-194. Palaszynski, E.W. (1987), Synthetic C-terminal peptide of IL-1 functions as a binding domain as well as an antagonist for the IL-1 receptor. Biochem. biophys. Rex Commun., 147, 204-211. Postlethwaite, A.E., Smith Jr., G.N., Lachman, L.B., Endres, R.O., Poppleton, H.M., Hasty, K.A., Seyer. J.M. & Kang, A.H. (1989), Stimulation of glycosaminoglycan synthesis in cultured human derma1 fibroblasts by interleukin 1. Induction of hyaluronic acid synthesis by natural and recombinant interleukin 1s and synthetic interleukin lb peptide 163-171. J. clin. Jnvest., 83, 629-636.

IN IMMUNOLOGY Priestle, J.P., Schlr, H.-P. & Griitter, M.G. (1989), Crystallographic refinement of interleukin lp at 2.0 ,.&resolution. Proc. nal. Acad. Sci. (Wash.), 86,9667-9671. Rao, K.V.S. & Nayak, A.R. (1990), Enhanced immunogenicity of a sequence derived form hepatitis B virus surface antigen in a composite peptide that includes the immunostimulatory region from human interleukin 1. Proc. nat. Acad. Sci. (Wash.), 87, 5519-5522. Reed, S.G., Pihl, D.L. & Grabstein, K.H. (1989), Immune deficiency in chronic Trypanosoma cruzi infection. Recombinant IL-l restores Th function for antibody production. J. Immunol., 142, 2067-2071. Staruch, M.J. & Wood, D.D. (1983), The adjuvanticity of interleukin 1 in vivo. J. Immunol., 130, 2191-2194. Tagliabue, A., Antoni, G. & Boraschi, D. (1989), Defining agonist peptides of human interleukin-lp. f.J~mphokine Res., 8, 31 l-315.

Adjuvant-independent induction of immune responses by antibody-mediated targeting of protein and peptide antigens D.L. Skea and B.H. Barber Department of Immunology, University of Toronto, Toronto. Ontario (Canada) M5S IA8

The induction of effective immune responses to foreign protein or peptide antigens in experimental animals usually requires that the antigen be administered in a potent adjuvant. Unfortunately, most of the adjuvants with demonstrated efficacy in animal models are excIuded from use in human and veterinary applications because of undesirable side effects. In an effort to circumvent the constraint that this places on the design of new subunit vaccines, we developed an adjuvant-independent antigen-delivery system based on the use of monoclonal antibodies specific for celI surface determinants on antigenpresenting cells (APC). By coupling protein or peptide antigens to such monoclonal antibodies, we speculated that this conjugate would serve to “deliver” (i.e. immunotarget) antigen to the surface of APC, and generate an adjuvant-independent serological response to the bound antigen. Our results indicate that immunoconjugates formed between certain mouse monoclonal antibodies and either pro-

tein or synthetic peptide antigens can, when injected subcutaneously in saline, prime the murine immune system for subsequent antigen-specific IgG antibody responses (Carayanniotis and Barber, 1987, 1990; Carayanniotis ef al., 1988). Having demonstrated the ability to generate adjuvant-independent antibody responses by immunotargeting, we are now seeking to better understand the immunological basis of this response and assess its potential as a new approach to subunit vaccine design. Our initial investigations of this system involved the model protein antigen, egg avidin, which was chosen because it could be conveniently and reproducibly coupled to different biotinylated monoclonal antibodies. In terms of the target specificity, we first focussed on the class II gene products of the major histocompatibility complex (MHC), because of their expression on both specialized APC and B lymphocytes. Results from a representative experiment are

CHARACTERISTICS

AND

USE OF NEW-GENERATION

shown in figure 1. (B6x C3H)F, (H-2bx H-2’) mice, when immunized with anti-I-Ak-avidin conjugates and boosted 4 weeks later with free avidin, made significant, secondary anti-avidin IgG antibody responses. In contrast, mice whose primary immunizations were with conjugates composed of avidin and a control antibody of unrelated specificity (i.e. influenza nucleoprotein) or with avidin alone, showed substantially weaker responses. In other experiments, the immunotargeting basis of the response to anti-class-II-MHUavidin immunoconjugates was further demonstrated by the lack of response in mice that did not bear the appropriate class II MHC allele, and by the failure of mixtures of avidin and nonbiotinylated monoclonal antibodies to elicit antiavidin antibody responses (Carayanniotis and

0

Fig. 1. A comparison of the antibody responsesgenerated

to the modelprotein antigen avidin when injected into mice alone, or conjugatedto either control or anti-class-II-MHC antibodies. Mice wereimmunizedS.C.with 100+g of avidin in the form of immunoconjugatein saline,preparedaspreviously described(Carayanniotisel al., 1991)or 100t.rgof avidin alone, in saline.Control mice receivedno primary immunization. Four weekslater, each mousewasboostedi.p. with 10 yg of avidin in saline. One week later, the mice werebledfrom the retro-orbital plexus.The serum IgG antibody responseto avidin wasdeterminedby ELISA. The absolutequantity of IgG anti-avidin antibody wasdeter-

mined using a standard curve which was prepared using a mousemonoclonal 1gGanti-avidin antibody. The data representthe geometricmeanvalue for 5 mice. The error barsrepresentthe upper limit of the rangeof the standard error of the mean.An analysisof variance with Duncan’s multiple range test demonstrated that mice that had received the anti-MHC-class-ll/avidin conjugate at the primary immunization had significantly (p < 0.01) greater serumIgG anti-avidinantibody responses after the boost, compared to all other groups. The mice that receivedthe isotype-matchedcontrol conjugate (anti-influenza virusavidin) gave responsesthat were not significantly different from those of mice that had received free avidin.

ADJUVANTS

569

Barber, 1987). The level of the IgG response to immunotargeted avidin varied from 10 to 150 yg/ml depending upon dose, mouse strain and nature of the conjugate, but was consistently less than the approximate 1 mg/ml responses seen with avidin in Freund’s complete adjuvant (FCA). This quantitative difference notwithstanding, it was of particular interest to note that the isotype distribution of the anti-avidin antibody response elicited by immunotargeting was very similar to that induced by immunization with FCA. That is, in both the FCA-primed and immunotargeted groups of mice, approximately 65-75 070of the response was IgGl, 20-30 % was IgG2a, 2-5 % was IgG2b and less than 5 % was IgM. Little IgG3 and no IgA antibody was detected in either group of mice (manuscript in preparation). By contrast, the antibody response in mice immunized with avidin adsorbed to alum was almost entirely IgGl (> 99 ‘J7o),with barely detectable levels of IgGZa. The isotype distribution of the response to a vaccine is important, since it is considered that, for certain pathogens, particular isotypes are more protective than others. For example, in mice, IgG2a is considered to be an advantageous isotype (Allison and Byers, 1986; Allison, 1989), and the above results demonstrate that immunotargeting effectively elicits the production of antibodies of this subclass. Another important consideration in the development of effective subunit vaccines is the induction of memory. We have found that immunization with immunoconjugates can impart long-term (i.e. > 6 months) immunological memory in mice. Thus, substantial IgG antibody responses to intraperitoneal injections of 10 pg of avidin in saline were elicited in mice that had been primed 6 months previously with anti-class II MHC-avidin immunoconjugates (manuscript in preparation). Although most of our work to date has employed targeting antibodies with specificity for class II MHC, we have also evaluated the efficacy of directing immunoconjugates to other, non-MHC target structures. For example, we recently found (Carayanniotis et al., 1991) that immunoconjugates directed to target structures present on dendritic cells, such as the 33Dl antigen (Nussenzweig et al., 1982) or CD45 (Springer et al., 1978), were particularly effective, while those directed to certain differentiation antigens present on macrophages, namely MAC-l (Springer et a/., 1978) and MAC-2 (Ho and Springer, 1982), were not. Although we have observed low and variable results with immunoconjugates containing antibodies directed to non-immunoglobulin B-cell markers, other groups have demonstrated that IgD is a particularly effective target (Lees et al., 1990; Snider et al., 1990). Identifying which cell surface marker represents the most effective entry point for the promotion of a particular serological response

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will be important for optimizing the use of immunotargeting. Our information suggests that the class II MHC determinants represent the most consistently effective target with respect to the quantity and quality of the IgG response. The immunotargeting approach is not restricted to the generation of serological responses in mice, but has been shown to be effective in other species for which there is an appropriate targeting antibody. For example, using the monoclonal antibody 44H 10, which was raised against a framework determinant on human class 11 MHC HLA-DR molecules, but also cross-reacts with class 11 MHC gene products from a number of other species (Dubiski et al., 1988), we have demonstrated the efficacy of immunotargeting in rabbits, pigs, ferrets, cattle and macaques. Representative data for the responses in rabbits are presented in figure 2. It is interesting to note in the context of the rabbit responses to avidin targeted on the 44H10 antibody, that the IgG anti-avidin response observed was quantitatively about half of that seen with the same amount of antigen emulsified in FCA. Although avidin has proved a useful model protein in our studies of immunotargeting, the technique is generally applicable to any protein antigen which can be effectively coupled to the targeting antibody. Thus, protein antigens may be chemically coupled to the targeting antibody using cross-linking reagents (Kawamura and Berzofsky, 1986; Pierce and Casten, 1988) or may be biotinylated and coupled to biotinylated targeting antibodies via an avidin bridge (Carayanniotis ef al., 1991). We have applied the latter approach to the bromelain haemagglutinin fragment of influenza virus (BHA) and elicited anti-BHA antibody responses in mice, rabbits and ferrets (manuscript in preparation). Successful immunotargeting vehicles have also been created by cross-linking a targeting monoclonal antibody to another monoclonal antibody specific for the protein antigen, thus creating a bispecific conjugate able to deliver antigen to specific sites in vivo (Snider et al., 1990). The immunotargeting approach is also effective at augmenting the immunogenicity of peptide antigens. For example, a synthetic peptide corresponding to a segment of herpes simplex virus glycoprotein D was photochemically coupled to avidin, and then conjugated to biotinylated anti-class-II-MHC antibodies, thereby creating an immunoconjugate able to elicit adjuvant-independent anti-peptide antibody responses in mice (Carayanniotis et al., 1988). Others have also shown that immunotargeting can be used to elicit good secondary antibody responses in the mouse to defined synthetic peptides coupled directly to a murine anti-class-II antibody when the peptide is known to contain both B- and T-cell epitopes (Caplan et al., personal communication). In each case

IN IMMUNOLOGY the magnitude of the anti-peptide response was comparable to that seen with the peptides in FCA (see table I). Because the immunoconjugates are injected in buffered saline, there is no pathology associated with the immunization procedure. Visual and histological examination of the injection sites confirmed the lack of local reactions, a result in striking contrast to the prominent granuloma formation which was observed at the site of antigen injection with FCA. Likewise, histopathological examination of various tissues from rabbits that had been injected with immunoconjugates failed to reveal any negative systemic consequences of the immunization procedure. In summary, we have shown that immunotargeting antigens to appropriate cell surface determinants (such as class II MHC) can effectively prime the im-

2.0

I

1 .5

1 .o

0.5

0.0 1 lb

100

rob0

reciprocal

lOdO

dilulion

looboo

100

000

01 wrurn

Fig. 2. A comparisonof antibody responses for individual rabbits injected rabbits receiving

with avidin emulsified with FCA avidin conjugated to anti-class-II-MHC antibodies in saline.

with

Four rabbits (0) were immunized with immunoconjugates in saline. The immunoconjugates, composed of 44HlO (anti-class-Il-MHC) and avidin, were prepared by a technique previously described (Carayanniotis el al., 1991). Two rabbits (0) were immunized with the same dose (100 pg) of avidin emulsified in Freund’s complete adjuvant. One control rabbit (A) was immunized with the same dose of free avidin in saline. Four weeks later, each “immunotargeting” rabbit received 80 pg of avidin in the form of immunoconjugate in saline, and the other three rabbits, 80 pg of free avidin in saline. All immunizations were administered S.C. One week after the boost, the rabbits were bled from the marginal ear vein. The serum 1gG antibody response to avidin was measured by ELlSA. Since no standardrabbit 1gGanti-avidin antibody was available for a standard curve, the data are shown as titration curves of optical density in ELISA vs. reciprocal dilution of serum.

CHARACTERISTICS

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USE OF NE W-GENERA

Table I. Characteristics

TION ADJUVANTS

of immunotargeting.

Antigen

Protein

Peptide

Name

Avidin mAb to APC (e.g. class II MHC) scd lo-100 pg antigen

HSV gD(z-2,)&

Carrier Route Dose

l-2:1

2-5: 1

(molar ratio of peptide:mAb) NA NA M, P

Side effects Reference :

Carayanniotis

Carayanniotis

T cells Species

None

and Barber (1987)

mune system for secondary IgG responses to the delivered antigen. The enhancement of immunogenicity mediated by immunotargeting can be achieved with no reactogenicity

at the site of injection,

thereby cir-

cumventing many of the problems associated with the use of different adjuvants. This approach has been successfully applied to both protein and synthetic peptide antigens,

and was found to be effective

(26-l-276)

mAb to APC (e.g. class II MHC) scd 5-50 ug peptide

(molar ratio of Ag:mAb) Gl, G2a, G2b, M NA M, Ra, P, ferret, pig None

Isotypes

571

in

a number of different animal species. On this basis, we believe that the immunotargeting approach merits further exploration as an alternative to the use of adjuvants when seeking to generate effective serological responses to protein and synthetic peptide antigens. Acknowledgements D.L.S. is the recipient of a Medical Research Council of Canada Fellowship. This work was supported by Connaught Laboratories Limited and the Province of Ontario Technology Fund.

References Allison, A.C. (1989),Antigens and adjuvants for a new generationof vaccines,in “Immunological adjuvants and vaccines” (G. Gregoriadis, A.C. Allison & G. Poste) (pp. l-12). Plenum Press,New York. Allison, A.C. & Byars, N.E. (1986),An adjuvant formulation that selectivelyelicitsthe formation of antibodies of protective isotypes and of cell-mediated immunity. J. Immunol. Methods, 95, 157-168.

et al., 1988

Carayanniotis, G. & Barber, B.H. (1987), Adjuvant-free IgG responses inducedwith antigencoupled to antibodiesagainstclassII MHC. Nuture (Lond.), 327, 59-61. Carayanniotis, G., Vizi, E., Parker, J.M.R. & Barber, B.H. (1988), Delivery of synthetic peptidesby anticlassII MHC monoclonalantibodiesinducesspecific adjuvant-free IgG responsesin vivo. Mol. Immunol., 25, 907-911. Carayanniotis, G. & Barber, B.H. (1990), Characterization of the adjuvant-free serologicalresponseto protein antigenscoupledto antibodiesspecificfor classII MHC determinants. Vaccine, 8, 137-143. Carayanniotis, G., Skea, D.L., Luscher,M.A. & Barber, B.H. (1991) Adjuvant-independentimmunizationby immunotargetingantigensto MHC andnon-MHC determinantsin vivo. Mol. Immunoi., 28, 261-267. Dubiski, S., Cinader, B., Chou, C.T., Charpentier, L. & Letarte, M. (1988), Cross-reactionof a monoclonal antibody to humanMHC classII moleculeswith rabbit B cells.Mol. Immunol., 25, 713-718. Ho, M. & Springer, T.A. (1982), MAC-2, A novel 32,000Mr mousemacrophagesubpopulation-specific antigen defined by monoclonal antibodies. J. Immunol., 128, 1221-1228. Kawamura,H. & Berzofsky, J.A. (1986),Enhancementof antigenicpotency in vitro and immunogenicityin vivo by coupling the antigen to anti-immunoglobulin. J. Immunol., 136, 58-65. Lees,A., Morris, SC., Thyphronitis, G., Holmes,J.M., Inman, J.K. & Finkelman,G.D. (1990),Rapid stimulation of large specificantibody responses with conjugates of antigen and anti-IgG antibody. J. Immunol., 145, 3594-3600. Nussenzweig,M.C., Steinman, R.M., Witmer, M.D. &

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Gutchinov, B. (1982), A monoclonal antibody specific for mouse dendritic cells. Proc. nut. Acud. Sei. (Wash.), 79, 161-165. Pierce, SK. & Casten, L.A. (1988), Soluble globular protein antigens covalently coupled to antibodies specific for B cell surface structures are effective antigens both in vilro and in vivo. Progr. Leukocyte Biol., 7, 259-268.

Snider, D.P., Kaubisch, A. & Segal, D.M. (1990), Enhanced antigen immunogenicity induced by bispecific antibodies. J. exp. Med., 171, 1957-1963. Springer, T., Galfre, G., Secher, D.S. & Milstein, C. (1978), Monoclonal xenogeneic antibodies to murine cell surface antigens: identification of novel leukocyte differentiation antigens. Europ. J. Immunol., 8, 539-55 1.

Characteristics and practical use of new-generation adjuvants as an acceptable alternative to Freund’s complete adjuvant DISCUSSION

W. de Leeuw and P. de Greeve:

Initially, the development of adjuvants has been empirical. A number of the conventional adjuvants combine immunopotentiation with undesired side effects such as (local) inflammation, granulomatous reactions, arthritis and pyrogenicity. These deleterious side effects, the rise of a new generation of synthetic vaccines suitable for man and animal, and the increased interest in the welfare of animals used for scientific research, were stimuli to more intensive research into new adjuvants. Due to the explosion of information concerning immune regulation, the development of new adjuvants has been more rational in the past two decades. Modern adjuvants must comply preferably with a lot of terms which inciude : the ability to elicit protective humoral and cellmediated immune responses, also against antigens that are poorly immunogenic, without introducing undesired side effects; being applicable by different routes and with different types of antigen ; being able to enhance the immune response in low responders and immunocompromised individuals ; not leaving toxic residues when used in food animals ; being able to be combined with other adjuvants; having a great influence on the quality of the immune response (isotype, location, cell type) ; being stable ; and being cheap and easy to produce. The new wave of synthetic vaccines demands flexible “adjuvant- and delivery-systems”. Liposomes, iscoms and DL-PLG microspheres especially offer multiple possibilities as regards local application, targeting, antigen presentation, induction of immune responses in the presence of maternal antibodies, and combination with other synergistic adjuvants. However, further research is needed concerning the relation between structure and effectivity and especially the induction of mucosal immunity. Though not reviewed in this Forum, microbiological carriers should also be considered as antigen-delivery systems with the same multiple possibilities as those mentioned above. Further research into this field is desirable.

When used for the production of polyclonal antisera or monoclonal antibodies, the efficacy of adjuvants is often evaluated on basis of titres. FCA is mostly the adjuvant of choice. However, antibodies with specific qualities concerning affinity, isotype, neutralizing activity are often needed. Adjuvants can play a major role in determining these qualities. Therefore a critical selection of one adjuvant or a combination of adjuvants can optimize the production of antibodies. For some qualities SAF, Titermax, lipopeptides, and DL-PLG microspheres have beep shown to be equally or more effective than FCA. Until now, only minimal adverse side effects have been described for the use of these adjuvant-systems. However, additional competitive studies have to be done with different types of antigen. Based on the information that is available now, it is already possible to replace FCA for at least a part of the routine immunizations in laboratory animals ; especially when great amounts of polyclonal antibodies are needed or when highly preserved mammalian antigens are used. Immunization of chickens results in large amounts of high-affinity antibodies in the egg yolk. Egg yolk antibodies have already been used in different types of immunoassays.

C. Hendriksen, M. Koedam, C. Moolenbeek and E. Claassen: and pathological study of rabbits given adjuvant: an evaluation of several regimens Immunological

From the summaries presented in this Forum in Immunology the picture emerges that little is known about adverse side effects of the various types of adjuvants. If on ethical grounds (see summary Claassen et al.) an adjuvant should be selected as an alternative to complete Freund’s adjuvant (CFA), this selection should be based on immunological data as well as on data of clinical findings and gross and histopathological lesions.

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In a recent study we evaluated 5 adjuvants including CFA. Groups of 4 rabbits were immunized by subcutaneous and intramuscular route with CFA, specol, Ribi, “TiterMax” and iscom, respectively, Three different types of antigen were used ; a corpuscular antigen (Mycoplasma pneumoniae), a synthetic peptide (SPEK15A) and a glycolipid (galactocerebroside). Animals were immunized according to the specifications given by the adjuvant suppliers at day 0 and day 28, and animals were bled at day 42. Blood samples for antibody titration and plasma kreatinine kinase levels were taken at 7-day intervals. Body weight and body temperature were recorded twice a week. At clinical examination, soft tissue swelling was found in animals subcutaneously exposed to CFA, Specol, RIB1 and TiterMax. Locomotor activity in animals immunized intramuscularly was not affected. Body weights and temperature were within the normal limits within the post-immunization period for all types of adjuvant. At gross macroscopic examination, inflammatory processes were found in most of the animals immunized with CFA, Ribi and TiterMax. The diameter of the abcesses varied from 0.5 to 8 cm. Many of the lesions were accompanied by small haemorrhages or areas of petechial bleeding. Conflicting results were produced in the specol animals. Large pancake-like sterile inflammatory processes with a diameter of 3 to 8 cm were found in the animals immunized subcutaneously with Mycoplasma pneumoniae. However, most of the animals immunized with SPEKlSA and galactocerebroside were negative at gross examination. In only a few animals was an enlargement of the draining lymph nodes observed at gross examination, which conflicts with the finding of Bokhout et al. (1986) with regard to specol (quoted in the summary of Boersma et al.). Based on preliminary data of gross examination adjuvants are ranked: CFA = Ribi > TiterMax > specol > iscom=control. No information is yet available on antibody titres and levels of plasma kreatinine kinase.

G.L. Gustafson : The influences of adjuvants on quality of immune responses to vaccines were discussed in many of the contributions to this Forum. Attention was focused on qualitative aspects of the immune response in recognition of the fact that the quality rather than the extent of the response determines the development of protective immunity against infectious agents. Indeed, as reviewed in an earlier Forum

ADJUVANTS

(35th Forum in Immunology), it is now clear that potentiation of immune responses in inappropriate directions can exacerbate some infectious diseases. As emphasized in this earlier Forum, the nature of help provided by helper T cells is critical in guiding the selection of appropriate effector cells - vaccine adjuvants which promote selection for TH l-type help over THZ-type help are most efficient in enhancing protective immunity. Studies described by Gajewski and Fitch, and by Williams et a/., in the 35th Forum provide a rationale for comparing the modes of action of different adjuvants in enhancing protective immunity. Their studies imply that adjuvants could promote selection for THl help either (1) by enhancing the role that macrophages play in processing and presenting antigens to helper T cells, and/or (2) by promoting IFNy synthesis. Some selection for THl help is provided simply by ensuring that antigen is associated with a particulate adjuvant delivery system that can be readily phagocytosed by macrophages. Many authors in the present Forum implicated macrophage targeting as a mode of action of their adjuvant vehicles. This may be the dominant effect of those vehicles with the least pro-inflammatory activities (i.e. microspheres, liposomes, iscoms and perhaps nonionic block copolymers). Additional enhancement of macrophage participation may be obtained with vehicles exhibiting greater pro-inflammatory effects vehicles that promote macrophage activation (oilwater emulsions, saponins and cationic detergents). Alum appears somewhat unique among particulate vehicles in eliciting a TH2-guided response (IgG 1 and IgE antibodies). Perhaps, antigens administered with alum are preferentially processed and presented by B cells after they dissociate from the carrier. Among the adjuvant factors relating to constituents of bacterial cell walls that were discussed (monophosphoryl lipid A, lipopeptides and muramyl dipeptide), MLA may be unique as an adjuvant that can both induce IFNy in an antigen-independent manner (i.e. early IFNy derived from NK cells), and also promote pro-inflammatory responses essential for activating macrophage APC activities. However, studies from Unanue’s laboratory (Wherry et al., 1991) have implied that Gram-positive bacteria possess other factor(s) that induce IFNy production by NK cells. In addition, the plant-derived saponins, discussed by Campbell and Peerbaye, might function as early IFNy inducers. In the model presented in our article, it was speculated that TNF would be a cofactor for MLAmediated induction of IFNy by NK cells. Three recent observations have provided support for this hypothesis (Gustafson and Rhodes, manuscript in preparation). First, it has been shown that when very low i.v. doses of MLA are administered to mice, the induction of IFNy by MLA is dependent upon the

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IN IA4iWJNOLOGY

co-administration of exogenous TNF. Second, it has been shown that a high dose of MLA is unable to induce IFNy if TNF induction by MLA is suppressed by pretreatment of mice with dexamethasone; and third, it has been demonstrated that exogenous TNF, administered with MLA, is able to subvert dexamethasone-mediated suppression. In an additional study, we have found that MDP (at i.v. doses as high as 100 pg) did not induce detectable early IFNy in mice. However, like TNF, MDP was able to synergize with low doses of MLA in inducing the response.

W. Nicklas : There are several reasons for research aimed at improving immunization methods. Most importantly, the increased use of subunit antigens in commercial vaccines requires more potent adjuvants which are devoid of undesirable side effects. Knowledge obtained during such studies can be used to reduce distress in laboratory animals caused during immunization for the production of antibodies. For this purpose Freund’s complete adjuvant (FCA) is still commonly used although it can induce severe side effects which have largely prohibited its use in commercial vaccines. This Forum demonstrates that, in addition to the use of FCA, several other methods exist for achieving a good presentation of antigens to the immune system. The classical way is to create an antigen depot at the injection site which is followed by inflammation. It is apparent from experiments with recently developed adjuvants that in vivo toxicity is not essential for adjuvant action. Several papers show that inflammation can be reduced to an acceptable level or that even non-inflammatory immunization procedures exist. Further side effects, too, can be diminished. Two major uses of adjuvants have to be considered. Commercial vaccines can be supplemented with the optimal adjuvant which can be developed or selected in specific studies ; FCA does not play a role in such commercial vaccines because it exhibits strong side effects. Secondly, adjuvants are necessary for experimental immunization of laboratory animals when small amounts of antisera are needed. In such cases, investigators cannot test for the most appropriate antigen-adjuvant combination but require reliable adjuvants for universal use. FCA is frequently used when antigens are available only in limited amounts or are of low immunogenicity. In such cases, the application of specific immunization procedures, coupling methods, or the use of a more appropriate animal species seem more reasonable than standard procedures using a strong

but harmful adjuvant. However, there is no clear answer to the question of alternatives to FCA. It seems that the suitability of some adjuvants or carriers depends on various properties of the antigen and that their use requires expert knowledge. Other adjuvants or adjuvant formulations exist for which only limited information is available as to their suitability for a sufficiently broad spectrum of antigens. Finally, some procedures are not easily accepted as routine methods because vaccine preparation is rather complicated, time-consuming, or requires specific equipment. Presently, we do not have sufficient information regarding the efficiency or toxicological aspects associated with some of the adjuvants reviewed. For that reason, they have up to now only been recommended for specific applications. However, many of the new-generation adjuvants have been demonstrated to be efficient and cause less severe side effects than FCA. Hopefully, this Forum will stimulate many investigators to replace FCA by alternative immunostimulants. One should not be discouraged by sporadically published reports demonstrating that, in specific experiments, FCA induced a better immune reaction than more recently developed formulations. Since then, numerous examples exist of comparative studies where alternative adjuvants were shown to be superior to FCA. At present, it seems that particular adjuvant formulations which are derived from FCA and which contain less harmful constituents can be recommended for a broad spectrum of applications. A number of studies utilizing a variety of antigens have been published indicating that satisfactory adjuvanticity together with acceptable side effects can be expected. As an additional advantage, some of the existing formulations are easier to prepare and to inject than vaccines containing FCA.

H. Snippe, A.F.M.

Verheul and L.A.Th.

Hilgers:

The contributions to the Forum “Characteristics and practical use of new-generation adjuvants as an acceptable alternative to Freund’s complete adjuvants” give a comprehensive overview of adjuvants currently used or under investigation. The objectives of the various papers, however, apparently differ. Some authors describe the potentials of their compound as an alternative for FCA while others focus on possible commercial application in veterinary and human vaccines. Despite explicit indication of the objectives of this Forum (i.e. comparison of effectiveness of different adjuvants for different antigens, their side effects, and animal-friendliness), some confusion seems to exist. Considering alternatives to FCA, it should be em-

CHARACTERISTICS

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phasized that the profile of adjuvants for research purposes is distinct from those aimed at commercial veterinary or human application. Based on the different requirements, we recognize 4 categories of targets for adjuvants (table I). In research, important requirements are high efficacy, broad spectrum and easy handling. The most important criterion for a human adjuvant is safety while in veterinary health, efficacy is important but certain levels of side effects are tolerated. Potentials of the adjuvants described in this Forum have been indicated in table I. For research purposes, ready-to-use formulations such as aluminium salts, “Syntex” adjuvant formulation-l (SAF-l), nonionic block polymers (NBP), saponins, IL1 peptides, dimethyldioctadecylammonium bromide (DDA), monophospholipid A (MPL) and lipopeptides are preferred. The other adjuvants described in this Forum require more complicated preparation procedures but can be very useful in certain cases. Although we realize that this Forum cannot cover all adjuvants described in literature, it is worthwhile mentioning compounds which are utilized momentarily in commercial products as they have proved to meet the appropriate criteria. Adjuvants most widely used in veterinary vaccines are emulsions of

Table I. Relationship

Adjuvant FCA w/o o/w Inert materials SAF-1 Al-salts Liposomes Iscoms Microparticles MPL IL1 peptides Saponins DDA NBP

ADJUVANTS

575

mineral oil (either oil-in-water or water-in-oil) and adsorbents (aluminium hydroxide and phosphate). In a few veterinary vaccines liposomes, saponins, iscorns, Acemannan (Carrington Labs., Inc., TX, USA) or Carbopol (BFGoodrich Co., Cleveland, OH, USA) are incorporated. Acemannan is an acetylated polymer of mannose and is used in Marek’s disease virus vaccine for poultry (Muirihead, 1992). Carbopol is a polymer of acrylic acid and adjuvanticity has been described in different patents (Haver-Lockhart, 1974; Cutter Labs. Inc., 1973; and Bayvet Corp., 1974) and is used in a few viral vaccines. Combinations of Carbopol with various lipophilic adjuvants such as DDA (Duphar, 1991) and NBP (Azko BV, 1991) have been described. Comparison of the efficacy of adjuvants described in the individual chapters is difficult to make. In our opinion, this is due to ‘the following facts : (1) standard criteria for the efficacy of the adjuvants have not been established ; (2) most studies lack standard adjuvant formulations as appropriate references ; (3) levels of immunostimulation by different adjuvants have not been indicated explicitly in the papers; and (4) in general, conditions for reaching optimal efficacy (type of antigens, route of immunization, animal species, etc.) have not been

between applicability

Efficacy is more important than safety Laboratory Food animals animals (mice, rats, (pigs, hamsters, cattle, guinea pigs) poultry)

and the ratio of efficacy/safety. Safety is more important than efficacy Companion animals (dogs, cats, pigeons, Humans horses)

+ + + + + + + + + + + + + +

+ = Applied routinely or in commercial products; (+) = not applied but considered to be applicable on basis of safety; - = not aPplied and considered to be not applicable due to safety problems; ( - ) = not applied and considered to be not applicable except for very specific purposes.

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indicated. These points were well recognized by Steward-Tull, who proposed the standardization of adjuvant efficacy (Steward-Tull, 1989, 1991) by USing standard antigens (ovalbumin and influenza virus), reference adjuvants, standard parameters of immunity (e.g. antibody titres measured by ELISA), etc. The picture emerging from the papers presented in this Forum is, that depending on the aim of the study (preparation of monoclonal antibodies, development of T-cell clones, characterization of protective epitopes, etc.) a suitable adjuvant can be selected. Alternatives to FCA are abundantly present but due to the lack of an integral picture or general guidelines, adequate selection of the optimal adjuvant is difficult. Although it is tricky to give an integral guideline for the selection of an appropriate adjuvant for research purposes, an attempt is hereby presented. The selection was made based on their stimulating properties with a wide variety of antigens, commercial availability and ease of employ. For the induction of humoral immune responses, NBP (especially L-121) combined with a non-mineral oil-inwater emulsion (including SAF-I), MPL or Ribi adjuvant system (RAS), saponins (including Quil-A) are recommended. For the induction of cellular immunity, the number of alternatives are limited : DDA, NBP (especially L-101) in a non-mineral oil-in-water emulsion (including SAF-1) and lipopeptides are advised.

Specific comments on liposomes This review overestimates the importance and relevance of T-cell interactions and liposomal antigens. In artificially complicated systems (isolated MHC molecules and selected peptides), these interactions could be demonstrated in vivo and in vitro but their general applicability in vaccine development must be doubted. Unless clearly defined crossreactive T-cell epitopes are found and expressed at the surface of, or incorporated into, liposomes, no general conclusions about these interactions may be drawn. We will not disclose those possibilities, but we warn against overoptimism. T-cell involvement in complex protein liposome conjugates may naturally occur as shown by the development of secondary humoral immune responses. Moreover, intracellular targeting of liposomes might be improved by addition of lipophilic adjuvants resulting in an overruling of the observed MHC restriction with peptide antigens. Furthermore, it is suggested that the incoporation of lipid A in haptenated liposomes changes the immunogenic character from TI to TD. In our opinion, these conclusions cannot be drawn from the experiments performed by Zigterman et al. (1986). We came to the conclusion that it was only

possible to change the immunogenic character from TI-2 to TI-1. It was one of the reasons for stopping research on haptenated (e.g. oligosaccharidederivatized) liposomes as a model antigen for vaccination purposes. References Akzo,

N.V. (1991), Adjuvant mixture. Europ. Path. 5,026,543. Bayvet Corp. (1974), Injectable adjuvant and compositions including such adjuvant. USA Pat. 3,919,441. Cutter Labs. Inc. (1973), Adjuvant compositions and medicinal mixtures comprising them. USA Pat. 1,450,557. Duphar, B.V. (1991), Stabilized adjuvant suspension comprising dimethyldioctadecylammonium bromide. Europ. Path. 5,026,546. Haver-Lockhart Labs. Inc. (1974), Injectable adjuvant, method of preparing same and compositions including such adjuvant. USA Pat. 3,790,665. Muirhead, S. (1992), Solvay introduces new immune stimulant product. Feedstuffs, 64, 2. Stewart-Tull, D.E.S. (1989), Recommendations for the assessment of adjuvants (immunopotentiators), in “Immunological adjuvants and vaccines” (G. Gregoriadis, A.C. Allison & G. Poste). Life Sciences Vol. 179 (pp. 213-226). Plenum Press, New York. Steward-Tull, D.E.S. (1991), The assessment and use of adjuvants, in “Vaccines, recent trends and progress” (G. Gregoriadis, A.C. Allison & G. Poste). Life Sciences Vol. 215 (pp. 85-92). Plenum Press, New York. Zigterman, J.W.J., Jansze, M., Snippe, H. & Willers, J.M.N. (1986), Immunomodulating properties of substances to be used in combination with liposomes. Int. Arch. Allergy, 81, 245-252.

A.T.J.

Bianchi and P.J. van der Heijden:

The papers of this Forum give a fairly complete picture of the state of the art in the area of adjuvants.

The compatibility of the tables between the various papers facilitates a first comparison of the potencies of the different adjuvants. Although only some of the papers of this Forum really discuss the application of the adjuvant that is dealt with in the paper as an alternative for CFA, several adjuvants appear to possess qualities that are not provided by CFA at all. One of the most interesting qualities is the possibility of some adjuvants (e.g. iscoms, saponin, lipopeptide, liposomes) to introduce “dead antigens” into the MHC class I pathway of antigen presentation that is needed for induction of CTL activity, and this is normally induced only by live vaccines. The Forum also demonstrates that the knowledge of the mode of action of adjuvants in terms of induction of cy-tokine release is increasing. From the data that

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are available, it now appears that adjuvants (e.g. saponin, bacterial cell di products) often activate a Thl-type response; this coincides with IFNy and IgG2a responses, just as CFA does. This Forum gives only restricted information about the direct use of a cytokine (e.g. an IL1 fragment) as adjuvant. The Forum lacks information about the use of other cytokines as adjuvant. For instance, there is already quite some experience with rHuIL2 and rBoIL2 used as adjuvants during experimental vaccination of domestic food animals (Blecha, 1990). The potential of the mucosal immune system for vaccination has been relatively underestimated until now (McGhee et al., 1992). In general, this may be due to the restricted possibility of adjuvants to potentiate mucosal immunity. Some examples (e.g. aluminum salts, saponin, iscoms, liposomes, microspheres) are given by this Forum and these are mostly based on limited data. To our knowledge, there are two types of mucosal adjuvant at this moment that really seem to be promising. One of them, the use of biodegradable microspheres, is included in this Forum. The other promising mucosal adjuvant is the B subunit of cholera toxin (CTB), which is not discussed in this Forum. CTB is a prerequisite for the induction of protective immunity after intranasal vaccination of mice with HA of influenza virus (Tamura et al., 1988). Further CTB can function as an adjuvant when given orally (Van der Heijden et al., 1991). A future development for potentiation of mucosal protection can be the application of recombinant ThZtype cytokines (e.g. 114,115, 116)that are able to potentiate mucosal IgA responses. The use of these cytokines may enable the induction of mucosal protection by the parenteral route of vaccination, However, this may inhibit the simultaneous induction of IgG2a (Thl-type response) that is supposed to be an important isotype for systemic protection.

References Blecha, F. (1990), In vivo use of interleukins in domestic food animals. Advanc. Vet. Sci. Comp. Med., 35, 23 l-252.

McGhee, J.R., Mostecky, J., Dertzbaugh, M.T., Eldridge, J.H., Hirasawa, M. & Kiyono, H. (1992), The mucosal immune system : from fundamental concepts to vaccine development. Vaccine, 10, 75-88. Tamura, S., Sarnegai, Y., Kurata, H., Nagamine, T., Aizawa, C. & Kurata, T. (1988), Protection against influenza virus infection by vaccine inoculated intranasally with cholera toxin B subunit. Vaccine, 6, 409-413.

Van der Heijden, P.J., Bianchi, A.T.J., Dol. M., Pals, J.W., Stok, W. & Bokhout, B.A. (1991), Manipulation of intestinal immune responses against ovalbu-

min by cholera toxin and its p subunit in mice. Immunology,

72, 89-93.

J.B. Campbell

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and Y.A. Peerbaye:

One of the points that comes across strongly in this Forum is that there is a need for a more extensive range of adjuvants for human vaccines than aluminum salts, which are the most widely used ones. It is no longer sufficient that an adjuvant simply enhance antibody titres : its effects on immunoglobulin isotype and subtype, as well as cell-mediated responses, must also be appropriate for the purpose used. The fact that aluminum adjuvants stimulate mainly IgG1 and IgE responses (Niklas) limits their effectiveness in providing protective immunity against bacteria, viruses and other pathogens. It is apparent, however, that there exist a number of other adjuvants, such as NBP (Verheul and Snippe), bacterial cell wall products (Gustafson and Rhodes), iscorns (Claassen and Osterhaus) and saponins (Campbell and Peerbaye) that can modify the isotype distribution, possibly by induction of IFNy and selection of IgGZa-secreting B-cell clones. One of the limitations of inactivated or subunit (i.e. non-replicating) vaccines is that the antigens are generally presented in association with MHC class II molecules, recognized by CD4+ cells rather than by CD8+. This generality accounts for the fact that antigen-specific CTL responses, following administration of non-replicating antigen, are poor. It is apparent, however, that several adjuvants described in this Forum have the ability to overcome this drawback, by presenting such antigens in ways that somehow permit processing by the cytosol pathway, resulting in priming of an MHC class-I-restricted CTL response. These adjuvants include amphiphilic compounds such as the NBP, saponins and the unique saponin-containing iscoms. Another interesting point to emerge from the papers in this Forum is that certain adjuvants retain activity when given separately from the antigen, either by different routes or at different times. These include NBP (Verheul and Snippe), DDA (Hilgers and Snippe), LPS/MLA (Gustafson and Rhodes), Al(OH), (Niklas) and saponins (Campbell and Peerbaye). Nothwithstanding the wide variety of adjuvants described, there is still a lack of adequately characterized materials for oral administration. Reference has been made to the fact that MDP and liposomes exhibit adjuvant activity orally (Buiting et al.). Recent work has shown that iscoms may be active by the oral route, although at much higher concentrations than required for parenteral administration (Claassen and Osterhaus). Free saponins may also be effective oral adjuvants. In this Forum, we have restricted discussion so far to Quiiiaja saponins. It is worth noting, however, that many medicinal plants contain high concentrations of saponins. Some of these, such as Panax ginseng (with more than

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20 saponins, or ginsenosides), Glycyrrhiza spp. (licorice; glycyrrhizin) and Bupleurum spp. (saikosaponins) have been shown to have immunostimulatory properties. Certain common foodstuffs, such as legumes, also have a high saponin content. This raises the question of whether saponin-rich diets may have an adjuvanting effect on the immune response to infectious agents, either vaccines or natural infections. At the very least, naturally occurring sources of saponins should provide useful starting materials for future investigations of oral adjuvants. One of the major drawbacks to the study, and implementation, of saponins as immunological adjuvants has been the lack of pure material. This holds true for iscoms as well as for free saponin. The toxicity of some saponins is another disadvantage, although this has perhaps been overemphasized. Mice, the most common test animal species, seem to be particularly sensitive to saponin toxicity : in general, larger animals appear to be much less affected. This notwithstanding, the availability of purified immunogenically active Quillaja preparations with very low toxicity (Kensil and coworkers ; quoted by Campbell and Peerbaye) obviates the concern. According to Kensil et al., QS-18, the major component of Quil-A (the most widely used saponin preparation for iscom formation) is also the most toxic. It is apparent that future studies of saponin adjuvants, both free compound and in the form of iscoms, will benefit greatly from utilization of pure preparations.

N. van Rooijen: Lipopolysaccharide, lipid A or monophosphoryllipid A for eliciting antibodies against surface antigens on liposomes or irradiated tumour cells

The adjuvanticity of bacterial cell wall products has been reviewed in the present Forum by Gustafson and Rhodes. Indeed, the activities of the nontoxic analogue of lipid A, monophosphoryl lipid A are promising. Lipopolysaccharides (LPS) from Gram-negative microorganisms have been shown to produce a wide variety of effects on lymphoid and non-lymphoid cells of the immune system (1). Many effects of LPS are mediated by the high affinity of LPS for mammalian cell membranes (2). The immunopotentiating activity of LPS has been the focus of attention for more than 30 years already (3). It has also been demonstrated that LPS, or its active lipid component lipid A, when inserted into liposomes (artificially prepared spheres of concentric phospholipid bilayers) strongly enhances the antibody response to antigens that are simultaneously exposed on the surfaces of these liposomes (4-7). Obviously,

IN IMMUNOLOGY

the combination of phospholipid bilayers with inserted LPS or lipid A creates a synergistic action to elicit antibody responses against surface antigens, even against those which are normally hardly immunogenic e.g. the phospholipid phosphatidylcholine itself. Both the repertoire of antibody specificities and the magnitude of the response are strongly increased by the incorporation of LPS or lipid A into the phospholipid bilayers. In a similar way to antigens exposed on liposomes, surface antigens on cells such as lymphocytes may elicit an enhanced immune response when LPS or lipid A is simultaneously coupled to their surfaces. Since endotoxins (or their lipid component lipid A) can adsorb to various cells (probably by the same mechanism by which they adsorb to liposomes), it is logical that endotoxins, if adsorbed to such cells, may trigger the production of antibodies and even anti-self antibodies (7). The immunogenicity of a tumour cell antigen is closely related to its presentation to cells of the lymphoid system. So, it is not surprising that liposomes have been proposed as carriers for tumour antigens in order to induce or enhance immune activity against the tumour. Several of such liposome-associated tumour antigens have already been studied (8-11). As in the case of adjuvants for application in vaccines, the synergistic action of liposomes and LPS or lipid A inserted into their bilayers may be expected to enhance the immune activity against tumour antigens that are associated with the same phospholipid bilayers. There is no reason why cells with LPS or lipid A incorporated into their phospholipid bilayers should behave differently from liposomes. For that reason, in our laboratory, we are studying the possibilities of using irradiated and LPS or lipid-Aconjugated tumour cells as a vaccine against liver metastases induced by consecutive administration of viable tumour cells. Although bacterial LPS are considered to be potent immunopotentiating agents, their use as immunological adjuvants has been hindered by the fact that they are toxic and pyrogenic for most animal species (12). These effects have been attributed to the diphosphoryl lipid A portion of the LPS molecule. However, removal of a phosphate group from the reducing end of diphosphoryl lipid A yields monophosphoryl lipid A (MPLA). MPLA retains all of the beneficial properties of LPS and diphosphoryl lipid A (e.g. adjuvanticity and tumour necrosis activity), but is neither toxic nor pyrogenic even in large doses. This has renewed much interest in the use of MPLA as an adjuvant as well as a therapeutic agent for the treatment of tumours (13). Moreover, it has been shown that the toxicity of lipid A is strongly reduced by inclusion of lipid A in phospholipid bilayers (6, 14). Richardson et al. (15) studied the effects of lifelong administration of liposomes and lipid A in

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mice. They failed to find any effects of lifelong injection of lipid A, liposomes or liposomes containing lipid A on the longevity of BALB/c mice, and no unique histopathological changes were found after more than 2 years of regular injection. No increased susceptibility to infections and no enhancement of metastatic spread of tumours were found (15). Several factors might contribute, at least in part, to the ability of MPLA to act as an immunological adjuvant (16), e.g. MPLA is mitogenic for bone-marrow-derived precursors of antibodyforming or B cells, it activates macrophages, stimulates interleukin- 1 production and seems to inactivate suppressor T-cell activity (16). The success of MPLA associated with phospholipid bilayers as a safe and potent adjuvant in the development of a malaria vaccine has been recently demonstrated in human volunteers (17). The mechanism of the immunopotentiating effect of phospholipid-bilayer-associated lipid A has not yet been revealed. Alving et al. (14) have argued that one of the reasons that liposomal lipid A is useful as an adjuvant might be that it exerts an activating effect directly on the macrophages that are processing the liposomal antigen. The role of macrophages as accessory cells in the immune response against particulate thymus-dependent antigens has been recently discussed (18).

References (1) Morrison, D.C. & Ryan, S.L. (1979), Bacterial endotoxins and host immune responses. Advanc. Immunol., 28, 293. (2) Davies, M. & Stewart-Tull, D.E.S. (1981), The affinity of bacterial polysaccharide-containing fractions for mammalian cell membranes and its relationship to immunopotentiating activity. Biochim. biophys. Acta (Amst.), 643, 17. (3) Johnson, A.G., Gaines, A. & Landy, M. (1956) Studies on the O-antigen of Salmonella typhosa. V. Enhancement of antibody response to protein antigens by the purified lipopolysaccharide. J. exp. Med., 103, 225. (4) Schuster, B.C., Neidig, M., Alving, B.M. & Alving, C.R. (1979) Production of antibodies against phosphocholine, phosphatidyl-choline, spbingomyelin and lipid A by injection of liposomes containing lipid A. J. Immunol., 122, 900. (5) Van Rooijen, N. & Van Nieuwmegen, R. (1980), Endotoxin enhanced adjuvant effect of liposomes, particularly when antigen and endotoxin are incorporated within the same liposome. Immunol. Commun., 9, 747. (6) Alving, C.R. & Richards, R.L. (1990) Liposomes containing lipid A: a potent nontoxic adjuvant for a human malaria sporozoite vaccine. Immunol. Letters, 25, 275-279. (7) Van Rooijen, N. (1989) Are bacterial endotoxins involved in autoimmunity by Ly-I+ (CD5+) B cells? Immunol. Today, 10, 334-336. (8) Gerlier, D., Bakouche, 0. &Dore, J.F. (1983) Lipo-

(9) (10)

(11)

(12) (13) (14)

(15)

(16)

(17)

(18)

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somes as a tool to study the role of membrane presentation in the immunogenicity of a Mu-LV-related tumor antigen. J. Immunol., 131, 485. Le Grue, S. (1984), Carrier and adjuvant properties of liposome-borne tumor-specific antigens. Cancer Immun. Immunother., 17, 135. Raphael, L. &Tom, B.H. (1984), Liposome facilitated xenogeneic approach for studying human colon cancer immunity: carrier and adjuvant effect of liposomes. Clin. exp. Immunol., 55, 1. Steele. G., Ravikumar. T.. Ross. D.. King. V.. Wilson. E. & Do&on, T. (1984) Speciric active%nmunother: apy with butanol-extracted, tumor-associated antigens incorporated into liposomes. Surgery, 96, 352. Ribi, E. (1984) Beneficial modification of the endotoxin molecule. J. biol. Resp. Modif., 3, l-9. Ribi, E., Cantrell, J.L., Takayama, K., Qureshi, N., Peterson, J. & Ribi, H.O. Lipid A and immunotherapy. Rev. infect. Dis., 6, 567-572. Alving, C.R., Verma, J.N., Rao, M., Krzych, U., Amselem, S., Green, S.M. & Wassef, N.M. (1992), Liposomes containing lipid A as a potent nontoxic adjuvant. Res. Immunol., 143, 197-198. Richardson, E.C., Swartz, G.M. Jr., Moe, J.B. & Alving, C.R. (1989) Lifelong administration of liposomes and lipid A in mice : effects on longevity, antibodies to liposomes and terminal histopathological patterns. J. Liposome Res., 1, 93-110. Baker, P. J., Hiernaux, J.R., Fauntleroy, M.B., Prescott, B., Cantrell, J.L. & Rudbach, J.A. (1988), Inactivation of suppressor T-cell activity by nontoxic monophosphoryl lipid A. Infect. Immun., 56, 1076-1083. Fries, L.F., Gordon, D.M., Richards, R.L., Egan, J.E., Hollingdale, M.R., Gross, M., Silverman, C. & Alving, C.R. (1992), Liposomal malaria vaccine in humans; a safe and potent adjuvant strategy. Proc. not. Acad. Sci. (Wash.), 89, 358-362. Van Rooijen, N. (1992), Macrophages as accessory cells in the in vivo humoral immune response : from processing of particulate antigens to regulation by suppression. Semin. Immunol., 4 (in press).

W.G. Bessler and G. Jung: This Forum comprises various aspects and novel developments of adjuvant research. One of the major goals of the ongoing research is the replacement of the classic Freund’s adjuvant which causes severe irritation in animals by novel atoxic and apyrogenic agents. Especially for the application to humans, various new aspects are discussed. As to the use of synthetic analogues of bacterial surface components as adjuvants, MDP analogues, lipopeptides, and lipid A analogues are suggested. These substances constitute, by themselves, potent leucocyte activators. Especially lipopeptides, which are non-toxic and non-mitogeuic in the human system, can be covalently coupled to low molecular weight antigens including T and B-cell epitopes, thus acting as carriers with built-in leucocyte-activating properties. A further

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class of carriers is constituted by inert membrane carriers and non-ionic block polymers which effectively can present antigen to the immune system. Iscoms and saponins combined with antigen effectively activate cell-mediated immunity. To replace Freund’s complete adjuvant by other antigen delivery systems, the mineral oil in this adjuvant is replaced by novel biodegradable substances like Squalene emulsions or specol, which could also be applied to human systems, where up till now mostly aluminium salts are used. Other systems constitute liposomes or microspheres by which the antigen can be delivered over a long time range. The adjuvant effect of many of the above-mentioned compounds can be further enhanced by other adjuvants like DDA. A novel approach also is the application of IL1 fragments as natural immune-enhancing mechanisms. Lastly, instead of passively presenting the antigen to the immune system, the targeting of the antigen to immune cells by antibodies is described. In summary, the tendence to use novel chemically well defined adjuvants is obvious. Also, the antigen becomes more defined by using defined epitopes to which adjuvants like lipopeptides or MDP can be covalentiy coupled. Lastly, the immune cell population activated by the adjuvants (T helper cells, cytotoxic T cells, B cells and/or monocytes) is dependent on the adjuvant used or the mode of presentation; the adjuvant also decides which immunoglobulin isotype will be induced. Since the molecular mechanism of adjuvanticity is mostly unknown, a lot of work is required in this field in the near future. However, using the knowledge available, the unwanted side reactions of Freund’s complete adjuvant in animal experiments can now be avoided, and in the human system the efficacy of the vaccines presently available can be enhanced by specifically stimulating defined cell populations of the immune system.

0. Nilsson : It appears that the Ribi adjuvant system which uses cell wall components as adjuvants is highly interesting and has probably good potential for further development. There is, however, the risk of cross-reaction and autoimmunity. An advantage is that MLA is easily available. IFNa determinations could perhaps be used to judge the efficiency of various adjuvants. Of the mineral salts discussed, aluminium works well but seems to be less useful with weak antigens and with small amounts of antigen, It sometimes causes granuloma and therefore intramuscular deposition seems less appropriate compared to oral administration. Present discussions on Alzheimer’s

disease which might be related to aluminium could hamper its use. Dimethyl dioctadecyl ammonium bromide (DDA) has been used in both animals and humans. It can be combined with other adjuvants and does not cause granulomas. Since it also mixes easily with antigen, it should be appropriate for human vaccinations. The water-in-oil emulsion specol is approved by the FAO. It is not expensive and easy to obtain, compared to some other adjuvants, and is appropriate for human vaccinations. The application of non-ionic block polymers (NBP) is approved for use in humans. They are well defined and easily available. A drawback is the requirement of various proportions of NBP, o/w, or w/o for different antigens. Saponins as adjuvants require more well defined and purified compositions than those available at present. Once these are established, it is expected that they will provide a good method for oral vaccination. Iscoms are appropriate for application with hydrophobic antigen. They are in clinical use already for vaccination, butare probably less useful as adjuvant when immunizing mice for monoclonal antibodies, since there could be loss of immunogen during iscom production. In addition, electron microscopic check of the shape of the iscoms, if required, could be cumbersome. Llposomes as universal carrier and as adjuvant offer an interesting approach, since an immune response is obtained with minor amounts of antigen and liposomes are biodegradable, non-toxic and inert. It would be valuable to have further information on loss of antigen during production, the length of time a liposome preparation can be stored, whether lipids in clinical use (like “Intralipid”) can be used for liposome production. Lipopeptide derivatives as adjuvant have advantages, since they are inert, non-toxic, nonimmunogenic and non-irritating, and should be useful for human vaccination. Availability of lipopeptides which can be coupled to another peptide (for instance, N-hydroxy-succinimide-lipopeptide) might enhance the use of this type of adjuvant. Biodegradable and biocompatible materials, now applied as microspheres with adjuvant activity have been used for many years for sutures in surgery and are thus well-tested. Their slow degradation could perhaps facilitate vaccination by making boosters less necessary? There is a risk, although small, of granuloma formation. With the application of cytokines as adjuvants, there is the risk of raising antibodies against the adjuvant at vaccination and, in addition, creating crossreactions which could upset the immune system. Also, antibodies against cell components close to the

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site of injection might appear. Even if the cytokines are expensive at present, they will in the future be produced to lower cost. Adjuvant-independent immunotargeting offers an interesting approach with potential for development. There are problems, however, the most appropriate target is not yet known, and HAMA appear which is an increasing clinical problem ; this might perhaps be avoided by using the antigen-binding peptide instead of the whole antibody.

J.H. Eldridge: The papers compiled within this Forum provide a comprehensive overview of current thinking in the area of using adjuvants to improve the magnitude and quality of the immune response to nonreplicating vaccine antigens. The diversity of the approaches which have been successful in potentiating antibody responses illustrates that a large number of compounds and formulations can function as adjuvants for this arm of the immune system. However, it is evident that a much smaller subgroup of adjuvants has the capacity to support CTL generation. In general, the underlying mechanisms by which the reviewed adjuvants exert their activity is not completely understood, but there are features common to many which are worthy of note. 1) The majority of these adjuvants present antigen as a particulate, although the form of the particles may vary from a large inert carrier such as a piece of nitrocellulose paper to the oil droplets within the emulsions used in several approaches. These particles appear to enhance immune responses by directing delivery to antigen-presenting cells and/or depot release, with the contribution from each mechanism varying between formulations. As pointed out by Allison and Byars, it is appropriate that the classic water-in-oil emulsion employed in complete Freund’s adjuvant is being superseded by the less inflammatory and more readily phagocytized oil-in-water emulsions. However, it remains to be established whether these oil-in-water preparations will be sufficiently benign for human application. 2) Unlike many past adjuvant formulations, few of the preparations discussed in this Forum rely on bacteria-derived components for their activity. Although many bacterial components are powerful immunopotentiators, they are often polyclonal activators with the potential to induce autoantibody production and inflammatory side effects which will likely bar them from widespread use in all but veterinary application. 3) Those adjuvants with the ability to induce vaccine-specific CTL responses possess surface-active

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properties for the cells of the host. However, precise data as to the mechanism by which each of these adjuvants results in the presentation of antigen-derived peptides in the context of class I MHC molecules is lacking. It is reasonable to hypothesize that the antigens are either directly inserted into host cell membranes or introduced into the cell cytoplasm. Determining whether either or both of these possibilities is correct, and the way in which these adjuvants accomplish this, are important topics for future research. The toxicologic data available for several of these adjuvant formulations suggests that they are reasonable candidates for use in human immunization in the near future. Hopefully, one or more of these will prove to be capable of receiving regulatory approval and providing an alternative to aluminum salts for enhancing the immunity of the world’s population to a number of infectious diseases.

A. Tagliabue

and D. Borascbi:

The papers included in this Forum represent a fairly comprehensive update of the field of immunological enhancement. The problem of achieving optimal humoral and cellular immune responses and stimulating immunological memory is still present in human vaccinations, in particular for poorly immunogenic antigens. The search for effective adjuvants of limited toxicity is thus of significant importance for the development of novel immunization strategies. Optimal adjuvants should prolong the availability of antigen to allow adequate response, recruit and trigger the immunocompetent cells of the host, selectively stimulate the desired type of response (cellular vs humoral, IgG2a vs IgGl, etc.). In comparison with aluminum salts, nowadays the major adjuvant of human use (see paper of Nicklas), a wide array of particulate carriers and membraneactive compounds are being studied. Inert carriers such as beads, resins and membranes with transferred antigens are particularly useful for obtaining antibodies to rare antigens in experimental animals (Nilsson ef al.). Very stable structures such as iscoms allow proper presentation of viral antigens and can stimulate class-I-restricted T-cell responses, and are already in use in veterinary vaccines (Claassen and Osterhaus). Liposomes can selectively direct the antigen to macrophages and stimulate T-dependent responses but do not allow antigen presentation by B cells (Buiting et ai.). Water-in-oil and oil-in-water emulsions induce non-specific enhancement of immune responses, also in the absence of antigens (Boersma et al.), and are being used together with

44th FOR I/M IN IMMUNOLOGY

polymeric surfactants in the formulation of improved adjuvants (Verheul and Snippe). Among adjuvants which prolong the release of antigen in vivo, very interesting from the practical viewpoint are polymeric biodegradable microspheres, which can allow a controlled repeated release of antigen and achieve the same result as multiple inoculations with single administration (Eldridge et al.). Substances which can target protein antigens to cell membranes, such as synthetic lipopeptides, are effective adjuvants also for small non-immunogenic haptens and may thus allow the development of particularly useful immunization strategies (Bessler and Jung). On the same principle, antigens can be coupled to antibodies specific for selected structures of the cell membrane and can be thus specifically directed to interact with the desired cell population (Skea and Barber). A different approach in the study of adjuvanticity consists of the analysis of the cascade of amplifying immunostimulatory events initiated by classical experimental adjuvants (such as bacterial products), in the attempt to isolate immunoenhancing factors devoid of the highly inflammatory effects of bacterial moieties. Cytokines induced by the adjuvant are of Key-words: Forum.

Immunostimulation,

major importance in the stimulation of the immune response and are thus good candidates for the development of a second generation of adjuvants. IFNy is apparently a major immunostimulatory molecule induced by modified lipid A adjuvants, which can preferentially trigger a Thl response and IgG2a production, and inhibit Th2 stimulation (Gustafson and Rhodes). The efficacy of IFNy as adjuvant for malaria peptide vaccines opens the possibility of introducing cytokines or cytokine fragments as adjuvant moieties of third generation synthetic vaccines. A first attempt in this direction is described for ILIP, where an immunostimulatory domain can be isolated from the entire molecule, which maintains adjuvant activity in the absence of inflammatory effects (Tagliabue et a/.). The hope for the future is the achievement of fully synthetic vaccine molecules containing adjuvant domains and B-cell and T-cell epitopes of antigens from several microorganisms, which could stimulate immunological memory and protective immunity against a group of different infections. The enormous possibilities offered by genetic engineering techniques make this goal possible.

Adjuvant ; New products, Vehicles, Associations,

Comparison,

FCA ;