Immunostimulating Complexes Clinical Potential in Vaccine Development

Clin. Immunother. 3 (6): 461-475,1995 RESEARCH PERSPECTIVE 1 172 -7039/95/0006-Q461/S07.50/0

Brar Morein, Karin Lovgren, Bengt Ronnberg, Anders Sjolander and Marfa Villacres-Eriksson Swedish University of Agricultural Sciences, Faculty of Veterinary Medicine, and National Veterinary Institute, Department of Virology, Biomedical Centre, Uppsala, Sweden

Contents Summary ...... 461 1. Immunostimulating Complexes (Iscoms) ...... 463 1.1 Quil/ojo Saponins: the Adjuvant Component . 463 1.2 Structure and Construction of Iscoms . . . . . 464 2. Interactions of Iscoms with Cells of the Immune System 466 2.1 Macrophages as Antigen-Presenting Cells ..... 466 2.2 Naive B Cells and Dendritic Cells as Antigen-Presenting Cells 467 2.3 Activation of Unprimed and Primed Spleen Cells 468 3. Immunological Responses to Iscoms 469 3.1 Kinetics and Organ Distribution . 469 3.2 Serum Antibody Responses 470 3.3 Cytotoxic T Cell Responses 470 3.4 Mucosal Responses ... . 470 3.5 Protective Responses .. . 471 4. Visions About Immunotherapy 472 5. Conclusions ...... 473

Summary An immunostimulating complex (iscom) is a particle containing several copies of an antigen, with a built-in adjuvant. It is constructed to provide a physically optimal presentation of antigen to the immune system. An iscom particle without incorporated antigen is called the iscom matrix, or just matrix, and can be used as a conventional adjuvant that is added to the antigen whose immunogenicity is to be reinforced. The unique components of the iscom matrix are saponins (triterpenoids) from the tree Quillaja saponaria, which exhibit a unique affinity for cholesterol and thereby facilitate the stability of the complex. The triterpenoids can be used as a crude preparation of Quillaja saponins or as purified preparations of Quillaja triterpenoids. The various triterpenoids have different characteristics, of which some are relevant to vaccine development such as the iscom-forming capacity, the immunomodulatory capacity, a low cell lytic property and low toxicity in general. Consequently, various compositions oftriterpenoids, including efficient nontoxic adjuvant formulations or inert carrier formulations, can be made. The currently used iscom vaccine and experimental vaccines induce a broad immune response, including major histocompatibility complex (MHC) class I 462 Morein et al.

and II T cell responses. The MHC class II response encompasses a prominent response of T helper I (THI )-like cells, producing interleukin (IL)-2 and inter feron-y and favouring cell-mediated immunity. A TH2-like response may also be evoked, with cells producing IL-4 and IL-IO and promoting humoral immunity. However, the same influenza virus envelope antigen in a micellar nonadjuvanted form induces a more prominent TH2 type of response, with cells producing more IL-IO. The iscom particle is also an interesting nonreplicating candidate for induction of mucosal immunity. Iscoms containing different kinds of antigens in various experimental vaccines evoke secretory IgA or cytotoxic T cell responses when administered orally and intranasally. Experimental iscom vaccine formulations have been shown to induce protective immunity to a number of micro-organisms, including viruses and retroviruses, parasites and bacteria, in several species, in cluding primates.

To date, the only adjuvants registered for human or adjuvant formulation that will cover the diverse use are the aluminium salts, aluminium hydroxide needs of immunopotentiation that different vac and aluminium phosphate. However, the alumin cines require. ium salts are effective only for certain antigens and There are certain criteria that vaccines should they are not free of adverse effects, since they not fulfil for efficient induction of the immune re infrequently cause local reactions. In contrast, a sponse. Most important is the physical presentation number of additional adjuvants are used in animals, of antigens. In a vaccine the antigens should be including various oil emulsions and saponins. presented as several copies in a particle, i.e. in a Classically, the effect of an adjuvant has been multimeric form.[!) If a multimeric presentation is measured by the increase of the antibody response not sufficient to convert an antigen into an efficient it stimulates and, to a limited extent, by its capacity immunogen in a vaccine, the prospective vaccine to increase a cell-mediated immune response. In a has to be supplemented with a suitable adjuvant or modern view of adjuvants, the concept of immuno adjuvant formulation. If the physical presentation modulation has to be included which, in addition form is combined with an adjuvant-active com to the magnitude of immune response elicited, also ponent, the term adjuvant formulation is used, ac encompasses qualitative aspects. These qualitative cording to the nomenclature suggested by Alli aspects include: sonP) • the induction of specific antibodies of the de As mentioned above, the aluminium salts are the sired isotype and IgG subclass only adjuvants registered for human use. Other ef • specific T helper cell responses, as classified by fective adjuvants and adjuvant formulations have cytokine production and cytotoxic T cell (CTL) been considered unsafe, as judged by the systemic response or local reactions they cause. Since adjuvants are • the distribution of the immune response to var often hydrophobic or amphipathic, e.g. saponins, ious lymphatic sites, e.g. mucosal surfaces. avridine and oil-based formulations,[3) they may Any of these factors may be important to obtain interact with cell membranes, thereby causing local protective immunity, which is the ultimate goal for adverse effects. Furthermore, adjuvants induce a vaccine, since one pathogen is different from an production of various kinds of cytokines, which in other and consequently the profile of protective im high concentrations give rise to toxic reactions.[4) munity will differ. With such a perspective in mind, It can, therefore, appear to be an enigma to find an it is obvious that there will be no single adjuvant effective adjuvant that is not toxic. This is the rea-

son why effective adjuvants are still not considered safe for registration for human use.

1. Immunosfimulafing Complexes (Iscoms) The iscom is formulated as a particle that com bines a multimeric presentation of antigen with a built-in adjuvant. Consequently, it can be placed within the category of particulate adjuvant formu lations. The iscom particle, first described by Mor ein et al.,[5] is a cage-like structure of about 40nm (fig. I ).[6.7] It is composed of the saponin adjuvant Quil A, cholesterol, antigen and phospholipids.[8] The iscom with its incorporated antigens is a very stable structure that remains intact after lyophilisa Fig. 1. Negative staining electron micrograph of immunostimu lating complexes (iscoms) containing influenza virus envelope tion, limited freeze-thawing and prolonged storage antigens (magnification x 90000). at +4°C. The storage limitations are generally de termined by the stability of the antigen. In pharma ceutical terms, the iscom can be considered as a carried out separation of Quillaja saponins and the carrier for a combination of antigen and adjuvant, subsequent characterisation of important and inter aiming at targeting the construct to antigen-pre esting molecules for iscom technology. senting cells and the lymphatic system. The first saponin preparation used for construc tion of iscoms was the semipurified product Quil 1.1 Quillaja Saponins: A.1 12] In Quil A there are a number of related tri the Adjuvant Component terpenoids, with small molecular structure differ ences but as shown later with a number of physical The backbone or matrix of the iscom is a unique and biological differences. Kensil et al. [I 5] and complex formed between certain Quillaja sapo nins and cholesterol. The adjuvant properties of Kersten et aJ.l16] have published procedures to iso saponins were first described by Ramon,[9] and to late such triterpenoids based on reversed phase a limited extent saponins were used as adjuvants in high performance liquid chromatography (HPLC). These studies reveal the complexity in composi commercial foot and mouth disease vaccines.1 IO] During the 1970s, Dalsgaard[lI] demonstrated that tion of Quillaja extracts with regard to toxicity and the adjuvant activity of saponins derived from the adjuvant activity. South American tree Quillaja saponaria Molina is Three groups of Quillaja components charac superior to that of other saponins. They isolated a terised by reversed phase HPLC (fig. 2) have been 'homogenous' fraction with a consistent high ad identified to be of particular interest for iscoms. juvant activity, which they denoted Quil A.1 II •12] These groups of components, named QH-A, QH-B The action of saponins on biological membranes and QH-C, have different characteristics in terms containing cholesterol has been known since the of adjuvant activity, iscom-forming capacity and early 1960s.[13.14] However, its practical use, by toxicity.[17] combining 2 important features for immuno QH-A is the most complex group of compo enhancement by formation of an antigen-bearing nents, consisting of a mixture of triterpenoids (fig. structure with built-in adjuvant activity, was not 2, left).[17] QH-A has a low or no adjuvant activity, discovered until 1984.[5] With the increasing inter but it can form iscoms and has a low haemolytic est in iscoms and Quil A, we and others[15.16] have activity. Its cellular toxicity in vitro is low, as mea-

0.032 30

E c II) C\i iO ~ 0.016 c € '"o III «.0

O ~----,-----~----~~ ~----,-----~----~~15 o 20 40 60 o 20 40 60 o 20 40 60 Time (min)

Fig. 2. High perfonnance liquid chromatographic analysis of purified Quillaja saponin components OH-A (left). OH-8 (middle) and OH-C (right). Column: CTciL-C8. 51lm particle size. 4.6mm x 15cm; mobile phase was 15 to 30% acetonitrile in 17 mmol/Lsodium phosphate buffer. pH 6.6. in 40 min. A total of 80119 of purified Quillaja saponin was loaded per injection. sured by inhibition of mitochondrial dehydroge both as measured quantitatively and by immuno nase activity in the 3-( 4,5-dimethylthiazol-2-yl)- logical activity. 2,5-diphenyl tetrazolium bromide (MTT) assay.[17] QH-C shows 2 or 3 major peaks in reversed QH-A is of particular interest as an inert carrier for phase HPLC (fig. 2, right). It is highly adjuvant various molecules, particularly for drugs where the active. It is as haemolytic as QH-B, and inhibits immunoenhancing activity of the Quillaja compo enzyme activity in the MTT assay at the same con centration as QH-B. However, it is substantially nents is not desired. Consequently, QH-A can be less toxic for mice, which survive several-fold used for multimeric presentation of antigens sup higher doses of QH-C (800~g) than of QH-B (50 plemented with adjuvants other than those derived to 200~g).[17] from Quillaja saponaria Molina. QH-B shows a double peak on reversed phase 1.2 Structure and Construction of iscoms HPLC (fig. 2, centre). It is highly adjuvant-active, but is the most toxic fraction of Quillaja saponins, The matrix of an iscom is the particle-forming being highly haemolytic and also cell toxic in vitro moiety into which the antigens are incorporated. It as measured by the MTT assay. It is the dominant is a complex between Quil A or selected Quillaja adjuvant active component of Quillaja saponins, components, cholesterol and a phospholipid, for

example phosphatidylcholine. Quil A or the QuiZ mains of the antigen. Removal of the solubilising laja components and cholesterol are essential for agent (e.g. detergent) should be started at condi matrix formation. They bind strongly to each other, tions where the antigen exposes hydrophobic do forming a characteristic complex.[14,18] The phos mains ('hydrophobic conditions') so that the anti phatidylcholine seems to facilitate the incorpora gen is incorporated into the iscom. The buffer can tion of amphipathic proteins and also facilitates then be changed to more physiological conditions. particle assembly. The formation of the matrix or 1.2.3 HydrophiliC Antigens iscom is based on solubilisation of its components Hydrophilic antigens without hydrophobic se with a (preferably) non-ionic detergent; the par quence cannot be incorporated into iscoms by the ticles are formed as the detergent is removed. methods described in sections 1.2.1 and 1.2.2, 1.2. 1 Membrane Antigens which rely on hydrophobic interactions. Instead The first iscoms were prepared with membrane these antigens have to be chemically linked to pre derived proteins from enveloped viruses. Such pro formed iscoms[23] or to plain matrix. Synthetic teins have a hydrophobic region anchoring the oligopeptides that require a protein carrier can be protein in situ in the lipid membrane of the virus conjugated to preformed iscoms,[24,25] whereas particle. In order to form an iscom from an envel larger antigens, including T cell epitopes, can be oped virus, the membrane protein is extracted from conjugated to matrix)26,27] The matrix used for the lipid membrane with a detergent. Simulta conjugation can be specially designed for efficient neously, the membrane lipids are solubilised. covalent conjugation by replacing the phospha However, it is often beneficial to add extra choles tidylcholine with an equal amount of phospha terol and phosphatidylcholine along with the sapo tidylethanolamine,l8] which contains an amino group nin. A common composition of the mixture is 1mg available for covalent linking. of each antigen, cholesterol and phospholipid plus An alternative to covalent coupling of the anti Smg of Quiliaja saponin. gen to preformed matrix is to make the antigen During the removal of the detergent, e.g. by di hydrophobic by linkage of a hydrophobic 'tail' , for alysis, ultrafiltration or centrifugation, the iscoms example a fatty acid, to the protein before incorpo are formed) 19] If the membrane proteins are in ration into the iscom.[28-30] Oligopeptides can be completely solubilised, they form micelles instead synthesised with such a hydrophobic tail, enabling of being incorporated into iscoms. It is also impor their incorporation into iscoms,l3l] Some care must tant that the lipids are completely solubilised. be taken not to make short poorly charged peptides Non-ionic detergents are preferred because their too hydrophobic, since such constructs require, in interaction with the antigen is limited to the hydro addition to detergent, large amounts of lipid for phobic part; consequently, they cause less denatur solubilisation and consequently also consume ation of the antigen than do ionic detergents. large amounts of Quil A for incorporation into iscoms, which may lead to unnecessarily toxic 1.2.2 Nonmembrane Antigens preparations. The toxicity can be avoided or dras Essentially nonhydrophobic antigens, e.g. sev tically reduced by the use of defined Quiliaja eral recombinant DNA products containing hydro components. phobic amino acid sequences, can be forced by mild partial denaturation to expose hydrophobic 1.2.4 Principles for Construction domains hidden in the tertiary structure. This can There are several parameters to consider when be achieved by exposure of the protein to low constructing iscoms, since the components build pH[20,21] or to chaotropic agents such as urea.[22] ing the iscom particles are also the immunologi The principle is to mix antigen, detergent and cally active ingredients, i.e. antigen and adjuvant. iscom constituents (lipid and Quillaja saponins) If the antigen requires more lipid for successful under conditions that expose the hydrophobic do- incorporation into iscom, the amount of Quil A

must also be increased to match. Some antigens, tion of processed antigen fragments by MHC class such as the major outer membrane protein (PI) of I molecules, leading to a CD8+ T cell response, or Neisseria gonorrhoea, require large amounts of by MHC class II molecules, leading to a CD4+ T lipid for solubilisation and incorporation into cell response. iscomsl16] compared with the membrane glycopro The intracellular transport of antigen decides tein of influenza virus.15 ] A dose of 2.5flg of PI in whether immune responses of MHC class I or iscom form contains almost 50flg of Quil A; con MHC class II, or both, will be evoked. In our lab sequently, such a disproportion may cause toxicity oratory we have followed the intracellular distribu problems. As described in section 1.2.3, these tox tion of iscom-borne influenza virus antigens in icity problems may be overcome by the use of de macrophages. By electron microscopy, iscoms fined Quillaja components.!I?] containing influenza virus envelope proteins can Free forms of Quillaja saponins and Quillaja be seen in vesicles, most likely endosomes and ly components have immunoenhancing capacities sosomes, whereas the same antigens in the micelle comparable with that of matrix. The limiting factor form cannot be detected. The micelles appear to for their use in vaccines has been the local reactions disintegrate before or soon after uptake by the cell, they cause, possibly a reflection of their lytic and whereas the iscoms are intact in the cell for a com cell toxic activities.!I?] For instance, it is known paratively long period of time.l3 2] Inside the cell, from animal studies that the dosage of Quil A in iscoms seem to adhere to membranes. They will free form is limited by the local reactions it causes. most likely integrate into the lipid membrane and In a recent study, Ronnberg et al. ll?] showed that expose antigens to both sides of vesicular mem 100 to 200flg of QH-C in the free form causes local branes, i.e. to the inside of intracellular vesicles but reactions at the site of subcutaneous injection in the also to the cytosol. In this way antigen is exposed strain of mice used, whereas QH-C incorporated to cytosolic enzymes. into matrix and given subcutaneously did not cause In a morphometric study, 60% of biotin-labelled local reactions at doses up to 1.6mg (see section 5). antigen was found in the cytosol, whereas 40% was It was also shown in cellular systems in vitro that found in vesicles or associated with membranes. by incorporation of QH-B and QH-C into matrix, These results were confirmed in a quantitative en I O-fold higher doses could be given compared with zyme-linked immunoabsorbent assay (ELISA) of the free form, i.e. 100 mg/L, before haemolytic ac tivity or cell-toxic activity measured by the MTT cell fractions obtained after differential centrifuga 133 test could be observed. tion of disintegrated cells. ] These results partly explain the capacity of iscoms to induce both MHC 2. Interactions of Iscoms with class I and II responses,134] since the key to induc Cells of the Immune System tion of the MHC class I type of immune response is to place the antigen in the cytosol, whereas the MHC class II response requires antigen to be trans 2.1 Macrophages as ported via the endosomal pathway to the lyso Antigen-Presenting Cells somes. It is well recognised that antigen-presenting However, van Binnendijk et aU35] provided evi cells stimulate immune competent cells to immune dence for a nonclassical, probably cytosolic, route responses by 2 different pathways, involving major outside the endosomal pathway for induction of histocompatibility complex (MHC) class I or class MHC class II restricted T cell responses by iscoms. II molecules, respectively. Differences in handling They showed that chloroquine-treated antigen-pre of the antigen between the 2 pathways are found in senting cells pulsed in vitro with iscoms containing the uptake of antigen, its intracellular transport, the F-protein of measles virus stimulated MHC proteolytic processing and finally in the presenta- class II restricted T cells, in contrast to antigen

injected in the form of ultraviolet-killed virus. from antigen-pulsed unfractionated splenocytes, Chloroquine treatment of cells increases their intra dendritic cells or B cellsp3] lysosomal pH, thereby abrogating their enzymatic Antigen initiates the T helper cell response and activity which is dependent on acid conditions. consequently also the B cell response via the MHC This results in inhibition of endosomal antigen pro class II pathway. The upregulation of MHC class cessing. This alternative route makes it possible for II expression on antigen-presenting cells is proba cytosolic enzymes to digest the antigen, possibly bly an important factor of the immune enhancing creating epitopes for MHC class II presentation activity of iscoms. This was indicated by the dem different from those created in the lysosomes. onstration that iscom-bome antigens enhance MHC There are several factors (cytokines) secreted class II expression on monocytes.[32] Using primed by phagocytic mononuclear cells that are impor spleen cells, Bergstrom-Mollaoglu et a1P7] further tant for the initiation of an immune response. The showed that iscom-borne antigen efficiently en capacity of influenza virus iscoms to induce cyto hances MHC class II expression on spleen cells in kine production by antigen-presenting cells has an IFNy-dependent way. been studied in mouse cells. In general, it can be concluded that iscom matrix or influenza virus 2.2 Naive B Cells and Dendritic Cells iscoms stimulate peritoneal or spleen macrophages as Antigen-Presenting Cells to produce high levels of both free and cell-bound IL-I, whereas influenza virus micelles only induce The capacity of B cells from nonprimed mice to production of cell-bound IL-1.[36] Granulocyte function as antigen-presenting cells has been dis macrophage colony-stimulating factor (GM-CSF) cussed extensively. Models used for studies in vitro was detected at low levels following stimulation have generally employed virus-transformed B with iscoms, but not with micelles. Monoclonal cells[38] and cloned B lymphomas.[39AO] In vivo, B antibodies neutralising cytokine activity were used cells are important in the priming of lymphatic T to show that iscoms induced macrophages to pro cells.l41 ] Sanders et al.142] showed that naive B cells duce GM-CSF and tumour necrosis factor-a (TN Fa) require about lO-fold more antigen than memory in addition to IL-l and IL-6.l33] The IL-6 was pro B cells to elicit the formation of antigen-specific B duced at high levels regardless of whether the cell/T cell conjugates. Our data indicate that naive macrophage cultures were pulsed with iscoms or B cells intemalise viral protein antigen in iscoms, micelles, suggesting that nonantigen iscom com process it and present it to primed T cells. These ponents do not enhance the secretion of IL-6. None respond with proliferation and secretion of cyto of these cytokines were detected in supernatants kines as IL-2 and, to a lesser extent, IFNy (table I).

Table I. Concentration of cell-associated antigen in B cells and dendritic cells pulsed with 100ng of immunostimulating complexes (iscoms) or micelles, and their capacity to elicit T cell proliferation and cytokine response in primed T lymphocytes (from Villacres-Eriksson[331) Antigen-presenting cell Antigen formulation Cell-associated T cell responses antigen (ng)a proliferation interleukin-2 interieron-y (counts/min)b.c (ng/L)b,d (ng/L)b,d B cells Iscoms 13.3 24105 1153 7609 Micelles 0.2 15852 305 3667 Dendritic celis Iscoms 16,8 32104 873 22831 Micelles 0.6 18241 404 2514 a Cells were pulsed with biotinylated antigen for 1 hour at 37"C. b Celis were pulsed with antigen for 1 hour at 37"C, c Maximal values, scored at day 5 of incubation. d Maximal values, at different time points.

The capacity of dendritic cells to present antigen 12 has been documented both in vivo and in vitro.[43-45] The mechanism of antigen uptake is unclear, since early reports stated that dendritic cells are, at best, poorly phagocytic.l46] Nevertheless, recent stud ies indicate that dendritic cells are as active in endo cytosis as other cells, but their degradative capacity may be weaker than that of macrophages)47] In order to study the capacity of dendritic and B cells to take up iscom-bome antigen, pulse experi • ments with dendritic cells and B cells with bio • tinylated influenza antigen in iscoms or as micelles were carried out. Subsequent quantitative detec tion of cell-associated antigen by immunoassay demonstrated that these cells take up both iscom and micelle-borne antigen. The amount of antigen taken up by dendritic and B cells, even the low amount taken up after exposure to micelles (table I), is enough to elicit recall proliferative responses and cytokine secretion. As reported previously,[48] dendritic cells are superior to B cells and mono cytic cells in eliciting the secretion of IFNy in sec Fig. 3. Supernatants from peritoneal macrophages stimulated ondary responses (table I). with immunostimulating complexes (iscoms) contain factors that enhance the proliferation of cultures containing iscom 2.3 Activation of Unprimed pulsed dendritic cells and naive T lymphocytes as measured by [3HJthymidine uptake. Naive T cells were cocultivated with and Primed Spleen Cells iscom-pulsed dendritic cells alone (right-hand bar) or with iscom-pulsed dendritic cells plus 24-hour culture fluid from peri 2.3. 1 Unprimed TCells toneal macrophages previously stimulated with iscoms (Ieft It is an interesting result that antigen-presenting hand bar) plus antibodies against interleukin (IL)-1, IL-6, tumour necrosis factor-a (TN Fa), granulocyte-macrophage colony cells, including macrophages, dendritic cells and B stimulating factor (GM-CSF) or a mixture of all the antibodies. cells, pulsed with iscoms carrying influenza virus Symbol: • = p < 0,05, antigen stimulate spleen cells from unprimed mice to proliferate in vitro, whereas the same antigen in fact that iscoms devoid of antigen, i.e. matrix only, micelles fails to do this. The proliferative response mediated by iscom-pulsed dendritic cells was en did not stimulate a proliferative response in vitro hanced by addition of cell culture fluid from peri points to a primary antigen-dependent stimulation. toneal macrophages stimulated with iscoms, pro 2.3.2 Primed TCells viding evidence for the synergistic effects of these Immunisation with iscoms stimulates the devel 2 antigen-presenting cells. This enhancing effect opment of memory T cells. As expected, the prolif was probably due to cytokines such as IL-l, IL-6, GM-CSF and TNFu, since neutralising monoclo eration of primed T cell cultures stimulated with nal antibodies to these cytokines partially inhibited unfractionated splenocytes, dendritic cells or B the enhancing effect on proliferation (fig. 3). Fur cells pulsed with iscoms or micelles was higher ther work is in progress to determine whether the than that of their naive counterparts. Generally, re evoked proliferation is a true primary response in stimulation with iscoms resulted in higher prolifer vitro or an oligoclonal proliferative response. The ation than restimulation with micelles)33]

The supernatants of primed T cell cultures stim and (b) study the kinetics and organ distribution of ulated in vitro with iscoms contain IL-2 and the resulting antibody and T cell responses. IFNy.!49.50] Overall, cultures stimulated with iscoms Influenza virus protein in iscoms was rapidly contain more IL-2 and IFNy than those stimulated distributed from the subcutaneous injection site with micelles. On the other hand, micelles are more throughout the body (A. Sjolander et aI., unpub efficient in priming T helper memory cells and lished work). Thus, the immunopotentiating ca stimulating them to produce IL-lO in vitro.!51] pacity of iscoms is not dependent on the depot Furthermore, micelles induce IL-4-producing T mechanism described for aluminium hydroxide helper cells as efficiently as do iscoms. B cells, and oil emulsion adjuvants. After intraperitoneal dendritic cells and unfractionated splenocytes administration, influenza virus iscoms are rap pulsed with iscoms mediate more secretion of IL-2 idly transported from the site of injection to the in spleen cell cultures from primed mice than do spleen.[53] Compared with micelles, higher amounts peritoneal cells. Dendritic cells were superior to of influenza virus protein in iscoms are detected in other antigen-presenting cells in mediating secre the spleen for longer periods of time following tion of IFNy, provided the cells were pulsed with intraperitoneal injection.!53] iscoms (table I). This observation is in agreement We have studied antibody and T cell responses with previous studies indicating that dendritic cells in lymph nodes and spleens taken at different time are important for the presentation of antigen to points from mice immunised subcutaneously with cells able to produce IFNy.[48] influenza virus iscoms. The T cell response in In recent experiments, the adjuvant activity in draining lymph nodes was characterised by a rapid vivo of the QH-C component (see section 1.1) in and transient expansion of responding cells, as free form was compared with that of oil adjuvants, measured by cell proliferation (A. Sjolander et aI., muramyl tripeptide and lipid A. [52] The high capac unpublished work). The response in the spleen de ity of the QH-C component to induce TH1-type veloped more slowly than in the lymph nodes, but cytokines, i.e. IL-2, IFNyand GM-CSF, in vivo was was of long duration, more than 3 months. Primed striking. The results clearly indicate that QH-C has spleen cells produced high levels ofIL-2 and IFNy similar immunomodulating properties to the crude after restimulation in vitro, whereas the production Quillaja saponins generally used in iscoms, and of IL-4 was approximately lOO-fold lower (fig. 4). suggest that immunologically efficient iscoms can After a booster injection, there was a rapid expan be produced with a relatively nontoxic purified sion of responding cells in the draining lymph Quillaja component. nodes. IL-2 production by both lymph node and spleen cells taken after the booster and restimu 3. Immunological Responses to Iscoms lated in vitro decreased as compared with that of cells taken before the booster, whereas the produc tion of IL-4 and IFNy was enhanced. 3.1 Kinetics and Organ Distribution The primary antibody response to influenza There is surprisingly little information available virus iscoms, recorded as the number of antigen on the influence of antigen, adjuvant and delivery specific antibody-secreting cells using the enzyme systems on the kinetics and distribution of immune linked immunospot (ELlS POT) technique,[54] was responses, in view of the importance these factors primarily localised to the draining lymph nodes may have for a protective immune response. To and reached a peak around day 7 after immuni address this question, studies were recently initi sation (A. Sjolander et aI., unpublished work). The ated in our laboratory to: (a) analyse the distribu serum levels of IgG reacting with influenza virus tion of influenza virus antigen in iscoms and in protein in ELISA increased during the first weeks protein micelles after parenteral immunisation; after immunisation and remained on a plateau level

antibody response is obtained compared with that • Interleukin-2 o Interferon-y induced by the same amount of antigen in a similar o Interleukin-4 size particle, for example as a micelle or in situ in ~ 51()'1 a virus particle. Of particular interest is the capac c: .2 ity of iscoms to induce a broad serum antibody re ~ sponse, encompassing both IgG I and IgG2a in 3 ~u 10 c: mice. This broad antibody response is seen with a o u Q) variety of antigens, for example influenza virus c: :ii1()2 antigen, gp 160 of HIV-I or the B subunit of cholera ~ u toxin. Conversely, micelles containing influenza virus antigen or cholera toxin B subunit stimulate

11 84 an antibody response dominated by IgG I. Time (days) after immunisation 3.3 Cytotoxic T Cell Responses Fig. 4. Secretion of interleukin-2. interleukin-4 and interferon-y by spleen cells from mice immunised with immunostimulating The capacity of iscoms as a nonreplicating anti complexes (iscoms) containing influenza virus antigens. Mice gen formulation to induce MHC class I restricted were immunised subcutaneously with 31-1g of influenza virus iscoms. After 11 or 84 days. spleen cells (2.5 x 105/well) were CTL is striking, This was first shown with gp160 restimulated in vitro with influenza virus protein 1 mg/L in the of HIV-I and the envelope proteins of influenza form of micelles. Values represent concentrations of cytokines virus.[55] Later, CTL have been induced to a num in spleen cell culture supernatants as measured by capture enzyme-linked immunoabsorbent assay. The cytokine concen ber of other antigens in iscoms, e.g. influenza virus trations in supernatants from controls (rnedium only) were below nucleoprotein (D. Burt et aI., unpublished work), the detection limit. measles virus F-protein,[35] heat shock protein of mycobacteria[56] and ovalbuminpo.57.58] After intra thereafter. A booster injection resulted in a drastic nasal mucosal administration, influenza virus[59] increase of antibody-secreting cells producing and respiratory syncytial virus[60] antigens have antigen-specific IgG in both the draining lymph been shown to induce CTL. Additionally, CTL nodes and the spleen (fig. 5), and in a strong serum were induced by oral administration of ovalbumin antibody response. iscoms.[61] Taken together, these results indicate that func Likely reasons for the efficiency of induction of tionally different Band T cell activities, including CTL by iscoms are their capacity to: (a) deliver both THl- and TH2-like characteristics, are in antigen to the cytosol of antigen-presenting cells, volved in the immune response to influenza virus leading to antigen presentation on class I MHC iscoms. Moreover, both the activation of regulatory molecules and a CD8+ T cell response; and (b) in T cell functions as well as the induction of antibody duce the production of the relevant cytokines, e.g. responses vary with regard to kinetics and organ IL-I, GM-CSF, IL-2 and IFNy, by T cells. distribution. 3,4 Mucosal Responses

3.2 Serum Antibody Responses It is well established that it is difficult to evoke mucosal immunity with nonreplicating antigens, A large number of articles have been published since mucosal immune responses are preferentially showing the capacity of iscoms to induce Band T evoked at the site where the antigen is applied. cell responses.l 19,34] The serum antibody response However, oral administration of antigen has been is a classical one, with an initial short-lived IgM shown to induce a specific secretory IgA response response followed by a long-lasting IgG response. in milk via the gut-associated lymphoid tissue Generally, a lO-fold or more enhancement of the (GALT). Similarly, bronchus-associated lymphoid

We have conjugated peptides representing the • Lymph nodes o Spleen V-3 loop of HIV gp 160 to influenza virus envelope proteins in iscoms. In this experimental model, one intranasal immunisation of mice with 51lg of this conjugate induced a clearcut serum antibody re sponse against the peptide (our unpublished work). We have incorporated the recombinantly produced B subunit of cholera toxin into iscoms. The immuno logical evaluation of these constructs is in prog ress, in collaboration with Dr J. Holmgren (Depart ment of Microbiology, University of Gothenburg, Sweden). The first results show that clearcut re sponses are obtained after one oral or intranasal Pre-booster After booster immunisation with a dose of 101lg. Therefore, the envelope proteins of influenza Fig. 5. Booster effect of immunostimulating complexes (iscoms) on the production of antigen·specific IgG. Mice were immunised virus and probably also the B subunit of cholera subcutaneously with 3~g of iscoms containing influenza virus toxin are able to induce systemic immune re antigens; 6 weeks later some of them received a booster im sponses via the mucosal surfaces. Recently, it was munisation with the same iscom preparation. Serial dilutions of lymph node and spleen cells taken at the time of and after the also shown that iscoms containing antigens from booster injection were tested in triplicate by enzyme·linked im protoscoles of Echinococcus granulosus induce a munospot assayl541 for production of IgG reactive with influenza serum antibody response in mice after intranasal virus protein. Values represent means ± S.E. of the number of antigen-specific IgG-producing cells/106 lymph node or spleen immunisation (H. Carol et aI., unpublished work). cells. No IgG-producing cells were detected in wells incubated In conclusion, the iscom is an interesting carrier with lymph node and spleen cells from nonimmunised mice. system for local immunisation. Techniques have been partially established for various experimental approaches, but the immunological responses to tissue (BALT) has been reported to distribute the such constructs need to be further evaluated. immune response to mucosal surfaces other than at the site of administration of the antigen. [62] Conse 3.5 Protective Responses quently, parenteral administration may not be an efficient route of immunisation for induction of Protective immunity induced by iscoms was local mucosal immunity. first demonstrated against influenza virus in mice[64] However, there are now data claiming that and horses.[65] The first iscom vaccine available iscoms carrying the Hand N antigens of influenza commercially was a veterinary influenza virus vac virus induce a strong secretory IgA response in the cine for horses. lung after intramuscular administration similar to Table II lists infections to which protective im that obtained after intranasal immunisation with munity has been induced by antigens borne on live virus.[63] Protection is induced in mice[64] and iscoms. Some of these are infections against which horses[65] after subcutaneous injection or intra vaccines do not presently exist or against which nasal administration of influenza virus iscoms, and protection has not previously been obtained. It is strong serum antibody responses are induced. Var of particular interest that: (a) an experimental ious routes of administration of iscoms induce se iscom-bome subunit rabies vaccine efficiently pre cretory responses in the genital tract of mice.f66] vented disease and death in post-exposure experi Mowat et al.[57,61,67] showed that oral administra ments in mice, whereas a commercially available tion of ovalbumin iscoms induces both antibody vaccine did not;[68] (b) an experimental iscom vac responses and MHC class I restricted CTL. cine induced full protection against Trypanosoma

Table II. Protective immunity induced by immunostimulating For several infections, there is increasing evi complexes (iscoms) containing various microbial antigens dence that T cell responses of the T HI type mediate Antigen and micro·organism Experimental Disease protective immunity, whereas the T animal prevented H2 type of im Haemagglutinin and Mice Pneumonia mune response is linked to disease progres neuraminidase, sionP I.721 This is true for HIV infection and leish influenza virus maniasis in humans, or T. cruzi infection or murine Haemagglutinin, Mice Encephalitis measles virus leprosy in the mouse. Knowledge of this kind sug Fusion protein, measles virus Mice Encephalitis gests that vaccines eventually should be formu Haemagglutinin and Seal Lethal lated to induce specific types of immune response. fusion protein, infection phoid distemper virus For example, in order to cure a persistent infection, Haemagglutinin and Dog Pneumonia it may be necessary to switch the evoked immune fusion protein, response from the T H2 type to the T HI type, or canine distemper virus superimpose a T HI response on a T H2 response. It G protein, rabies virus Mice Lethal infection, is also claimed that progression towards AIDS after postexposure infection with HIV-I is preceded by a change in immunisation immune response from T HI to T H2, or to THO, fol gp120, simian Monkey Lethal immunodeficiency virus infection lowed by a decrease in the CD4+/CD8+ ratio.[73- 751 gp-125, HIV-2 Monkey Viraemia An efficient induction of IL-12 may cause rever Envelope protein, Sheep Abortion sion to a T HI response. bovine diarrhoea virus An example of a broadening of the immune re gp70, feline leukaemia virus Cat Viraemia gp360, Epstein-Barr virus Tamarin monkey Lethal tumour sponse has been demonstrated with cholera toxin Surface antigens. Mice Lethal subunit. Alone, the B subunit of cholera toxin in Toxoplasma gondii infection duces an almost pure IgG 1 response and no IgG2a Immunoaffinity purified Mice Lethal response. However, when it is integrated into protein. Trypanosoma cruzi infection iscoms or administered with iscom matrix as an adjuvant, a strong IgG2a response is added to the cruzi in a mouse model;[691 and (c) long-lasting IgG 1 response (our unpublished results). The IgG 1 protection has been induced in macaques against subclass is generally evoked in connection with a simian immunodeficiency virus (P. de Vries et aI., T H2 response, and the IgG2a subclass is associated unpublished work) and HIV-2POl with a THI response. These experiments show that an adjuvant component can superimpose a differ ent immune response than that intrinsically evoked 4_ Visions About Immunotherapy by the antigen alone. In the case of T. cruzi infection, a protein similar A number of diseases are caused by infectious micro-organisms that persist in the host, for exam to a-fetoprotein exerts an immunosuppressive ef ple herpes viruses, retroviruses and various para fect, particularly on T-cell proliferation, IL-2 and sites such as African and South American trypano interferon-y production, thus evading the protec somes. There are in general no vaccines against tive mechanisms of the host (D. Hansen et aI., un these pathogens, which persist in the presence of published work). This provokes the question of an immune response evoked by themselves and whether an immune response should first be elic that is apparently favourable for their survival in ited against this imrnunomodulatory protein, be the host. To devise a vaccine against such micro fore immunisation with other antigens that may be organisms, it is necessary to understand what type protective. The outcome could be that a neutralis of immune response should be evoked. ing immune response to the immunomodulatory

protein is evoked, but surprising results in the op 4. Beutler B, Milsark IW. Cerami A. Passive immunisation against cachectinltumour necrosis factor protects mice from lethal posite direction might also occur. effects of endotoxin. Science 1985; 229: 869-71 Thus, the prospects for immunotherapy against 5. Morein B, Sundquist B, Hoglund S, et at. Iscom: a novel struc pathogens causing persistent infections lie partly ture for antigenic presentation of membrane proteins from enveloped viruses. Nature 1984; 308: 457-60 in being able to modulate the immune response and 6. Ozel M, Hoglund S. Gelderblom H. et at. Quaternary structure partly in defining immunomodulating components of the immunostimulating complex (iscom). J Ultrastruct Mol in the pathogen. Struct Res 1989; 102: 240-8 7. Akerblom L, Stromstedt K. Hoglund S. et at. Formation and characterization of FELV ISCOMs. Vaccine 1989; 7: 142-6 5. Conclusions 8. Lovgren K, Morein B. 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