Karin Lövgren

The ISCOM: an immunostimulating complex.

Vaccine development is based largely upon the use of killed or live attenuated organisms. But most of the components of an organism are irrelevant to the generation of an immune response and some may be positively harmful. Vaccines based on protective antigens have also had a chequered history, largely because of poor antigen immunogenicity and the obligatory use of damaging quantities of adjuvant. Here Bror Morein and colleagues report on an alernative vaccine vehicle -the immunostimulating complex (ISCOM). Composed of the adjuvant Quil A and immunizing peptides, ISCOM particles contain low concentrations of adjuvant and can significantly enhance immunogenicity. If early successes are confirmed, ISCOMs may be significant part of the future vaccine programmes.

Antigenic presentation of small molecules and peptides conjugated to a preformed iscom as carrier.

The aim of the present study was to elaborate a carrier system for haptens and synthetic peptides, making them immunogenic without addition of Freunds adjuvants. As carriers, preformed iscoms and micelles as well as BSA have been compared. The iscoms and micelles were prepared with envelope proteins of an influenza virus. As a model hapten, the small molecules of biotin were coupled to iscoms to determine the optimum epitope density for induction of an enhanced antibody response to the hapten. The most efficient carrier tested was the preformed iscom at an epitope density of ten biotin molecules per viral protein in the iscom. This carrier system exceeded the efficacy of both the preformed micelles and BSA, the latter with or without addition of Freunds adjuvant. A favourable epitope density could not be achieved when each of two different synthetic peptides was conjugated to iscoms. Epitope densities higher than one to three peptide molecules per protein lead to polymerization of either the peptide or the carrier. The coupling agent was glutardialdehyde.

ISCOMs and Immunostimulation with Viral Antigens

With the dissection of microorganisms followed by biochemical and immunological characterization, antigens inducing protective immunity became recognized. Early attempts to use these isolated antigens as vaccines, i.e. subunit vaccines, showed that although immunogenic in situ as part of the microorganism, they were not immunogenic as purified antigens. Subsequent studies showed that the formation of antigen into defined multimeric forms such as protein micelles or into liposomes made them considerably more immunogenic. In a way, micelles and liposomes mimic a submicroscopic particle of a microorganism with several copies of surface antigens. By contrast, monomeric forms of antigens, e.g., envelope proteins of parainfluenza-3 virus or Semliki forest virus, not only had a low immunogenicity but had a specific suppressive effect on the immune response as well; this was shown when the monomers were given simultaneously with the same antigen in a micelle (Morein et al., 1982, 1983; Morein and Simons, 1985; Jennings, 1987).

Influenza virus ISCOMs: antibody response in animals

A monovalent experimental ISCOM vaccine has been prepared with the envelope glycoproteins haemagglutinin and neuraminidase of the equine virus strain A/Solvalla/79 (H3N8). In vaccination trials on BALB/c mice the ISCOM vaccine induced more than ten times higher serum antibody titres measured in ELISA than a corresponding experimental micelle vaccine. Similarly, in guinea-pigs the ISCOMs induced about tenfold higher haemagglutination inhibition (HI) and neuraminidase inhibition (NI) titres than a micelle vaccine or a conventional killed influenza whole virus vaccine. Horses vaccinated with a divalent experimental ISCOM vaccine, containing the equine strains A/Prague/56 (H7N7) and A/Solvalla/79 (H3N8), responded with ELISA antibody titres against haemagglutinin which were higher and lasted considerably longer than those in horses vaccinated with conventional whole virus vaccine. ISCOMs induced complete immunoprotection in mice vaccinated with a dose of 1 microgram envelope glycoproteins of the mouse pathogenic strain A/PR/8/34 (H1N1).

The combined CTA1-DD/ISCOMs vector is an effective intranasal adjuvant for boosting prior Mycobacterium bovis BCG immunity to Mycobacterium tuberculosis.

ABSTRACT Infection with Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), remains one of the leading causes of mortality worldwide. The current “gold standard” vaccine Mycobacterium bovis BCG has a limited efficacy that wanes over time. The development of a vaccine to boost BCG-induced immunity is therefore a highly active area of research. Mucosal administration of vaccines is believed to provide better protection against pathogens, such as M. tuberculosis, that invade the host via mucosal surfaces. In this study we demonstrate that an intranasal vaccine, comprising the antigenic fusion protein Ag85B-ESAT-6 and the mucosal combined adjuvant vector CTA1-DD/ISCOMs, strongly promotes a Th1-specific immune response, dominated by gamma interferon-secreting CD4-positive T cells. Mucosal administration of Ag85B-ESAT-6 mixed with CTA1-DD/ISCOMs strongly boosted prior BCG immunity, leading to a highly increased recruitment of antigen-specific cells to the site of infection. Most importantly, we observed a significantly (P < 0.001) reduced bacterial burden in the lung compared to nonboosted control animals. Thus, the results demonstrate the effectiveness of mucosal vaccination with Ag85B-ESAT-6 mixed with CTA1-DD/ISCOMs as adjuvant for stimulating TB-specific protective immunity in the lung.

Immunostimulating Complexes: Clinical Potential in Vaccine Development

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 of triterpenoids, 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 and II T cell responses. The MHC class II response encompasses a prominent response of T helper 1 (TH1)-like cells, producing interleukin (IL)-2 and interferon-γ and favouring cell-mediated immunity. A TH2-like response may also be evoked, with cells producing IL-4 and IL-10 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-10. 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, including primates.

Increased immunogenicity of a non-amphipathic protein (BSA) after inclusion into iscoms

Bovine serum albumin (BSA) was used as a non-amphipathic model protein to be included into iscoms. Pretreatment at an acidic pH (2.5) was used to reveal hydrophobic regions, after which BSA could be integrated. In immunization experiments in mice the BSA iscoms induced long lasting and considerably higher serum antibody responses than non-treated monomeric BSA or BSA aggregated by acidic treatment.

Interactions between Immune‐Stimulating Complexes (ISCOMs) and Peritoneal Mononuclear Leucocytes

Studies were undertaken in mice using immune‐stimulating complexes (ISCOMs) or micelles prepared from envelope glycoproteins of human influenza virus (PR8) and matrix (i.e., ISCOM skeleton without incorporated antigen). Electron microscopic studies showed that ISCOMs, in contrast to micelles, have a remarkable affinity for cell membranes and seem to rapidly promote their own internalization by cells to which they adhere. PR8 ISCOMs, but not matrix nor micelles, significantly increased the expression of membrane Ia by peritoneal mononuclear leucocytes 24 hr after intraperitoneal immunization.

Influenza virus ISCOMs: biochemical characterization.

Immunostimulating complexes (ISCOMs) have been prepared from influenza A virus envelope glycoproteins, i.e. haemagglutinin (HA) and neuraminidase (NA). An ISCOM consists of a matrix, which is the micellar form of the glycoside, Quil A, in hydrophobic interaction with both the envelope glycoproteins (HA/NA). The Quil A bound to the ISCOM amounted to 50 micrograms mg-1 (5%) of ISCOM protein. ISCOMs were morphologically identified as symmetrical cage-like structures of approximately equal to 40 nm in diameter with hexagonal or pentagonal subunits of approximately equal to 12 nm. The sedimentation coefficient was approximately equal to 19 S as compared to 30 S for the glycoprotein micelles. The biological activities of the HA and NA are preserved in both ISCOMs and micelles.

High antibody responses in rabbits immunized with influenza virus ISCOMs containing a repeated sequence of the Plasmodium falciparum antigen Pf155/RESA.

Immunostimulating complexes (ISCOMs) are spherical structures where immunogens are presented as multimers in a matrix of the adjuvant Quil A. ISCOMs have been shown to enhance the immunogenicity of several antigens important to both human and veterinary vaccine development. We have coupled a fusion protein, designated ZZ-M2, comprising eight copies of the C-terminal repeat subunit EENV of the Plasmodium falciparum blood-stage antigen Pf155/RESA and two IgG-binding domains of staphylococcal protein A (SpA), to preformed influenza virus envelope protein ISCOMs. Rabbits immunized with the conjugated ISCOMs produced high titres of antibodies even after the first injection. These antibodies reacted with the EENV repeat sequence in ELISA and with Pf155/RESA in immunofluorescence on infected erythrocytes. The antibody response, which was sustained for more than 20 weeks, was efficiently boosted and superior or equal to that obtained after immunization with ZZ-M2 in Freunds complete adjuvant. In contrast, the antibody response induced in rabbits immunized with ZZ-M2 in Syntex Adjuvant Formulation-MF (SAF-MF) was weak and of short duration. The antibodies produced after immunization with ZZ-M2 coupled to influenza virus ISCOMs mainly recognized epitopes formed by two or more EENV subunits and were highly specific for Pf155/RESA. Furthermore, the antibodies efficiently inhibited merozoite reinvasion of erythrocytes in vitro, indicating that they recognized epitopes exposed on the native antigen. In addition, the ZZ-M2-conjugated ISCOMs also induced high titres of antibodies reacting with SpA or the influenza virus envelope protein.(ABSTRACT TRUNCATED AT 250 WORDS)