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.     

Identification of an immunostimulating protein from Mycobacterium leprae.

Despite the recent identification of a number of Mycobacterium leprae proteins, the major immunogenic determinants of this organism remain obscure. We isolated from M. leprae a potent immunostimulatory preparation, designated the MLP fraction, which contains a major protein of 35 kilodaltons (kDa). This protein was precipitated by monoclonal antibody ML03-A1, which recognizes a 35-kDa protein of M. leprae, and by sera obtained from patients with lepromatous leprosy. Neither sera from healthy controls nor sera from patients with pulmonary tuberculosis recognized the 35-kDa protein, and only one of four serum samples from patients with borderline tuberculoid leprosy reacted with this protein. The MLP fraction stimulated T-cell proliferation in patients with leprosy whose T cells proliferate in response to whole M. leprae cells. Apparently, the T-cell epitope associated with MLP is also expressed on M. tuberculosis and M. bovis BCG, since patients with pulmonary tuberculosis and BCG-vaccinated individuals demonstrated significant responses to the MLP fraction. The 35-kDa M. leprae protein, purified to homogeneity in the laboratory of P. J. Brennan, stimulated T-cell proliferative responses in all MLP-responsive subjects. These findings suggest that the 35-kDa protein present in MLP is an immunostimulatory component of M. leprae. In addition to serving as a useful probe for study of the T-cell anergy associated with lepromatous disease, this protein may ultimately be useful as a component of a vaccine designed to provide protection against infection with M. leprae.     

Monocyte and iscom enhancement of cell-mediated response to cytomegalovirus

Cell-mediated and humoral responses to cytomegalovirus were studied in a monkey model. Repeated low doses of virus antigen gave poor reactivities in both respects. High antigen doses gave a good humoral IgG response. When autologous monocytes were incubated with the CMV antigen as the immunizing injection, the specific cellular response to CMV antigen increased. The monocytes themselves did not contribute to the in vitro specific proliferation response. When iscoms were the carrier particles for CMV antigens, cellular response was even more strongly enhanced. In immunization schedules where specific cellular responses are important, we suggest that autologous monocytes or iscoms may be employed as antigen carriers.     

Achieving directed immunostimulating complexes incorporation

In recent years, several studies have been reported with the common aim of generating general expression systems for straightforward production and subsequent coupling of expressed antigens to an adjuvant system. Here, we describe a series of such efforts with a common theme of using gene fusion technology for association of recombinant antigens to immunostimulating complexes (iscoms). In the early stages of vaccine development, uniform antigen preparations are crucial to allow the comparison of immune responses to different antigens, or even subdomains thereof, and we believe that the described systems constitute an important development in this context.     

General expression vectors for production of hydrophobically tagged immunogens for direct iscom incorporation

A new general strategy for the production of recombinant protein immunogens has been investigated. The rationale involves the production of a recombinant immunogen as fused to a composite tag comprising one domain suitable for affinity purification and a hydrophobic tag designed for direct incorporation through hydrophobic interaction of the affinity-purified immunogen into an adjuvant system, in this case immunostimulating complexes (iscoms). Three different hydrophobic tags were evaluated: (i) a tag denoted IW containing stretches of hydrophobic isoleucine (I) and tryptophan (W) residues; (ii) a tag denoted MI consisting of the transmembrane region of hemagglutinin from influenza A virus; and (iii) a tag denoted PD designed to be pH-dependent in such a way that an amphiphatic alpha-helix would be formed at low pH. As an affinity tag, an IgG-binding domain Z derived from Staphylococcus aureus protein A (SpA) was used, and a malaria peptide M5, derived from the central repeat region of the Plasmodium falciparum blood-stage antigen Pf155/RESA, served as a model immunogen in this study. Three different fusion proteins, IW-Z-M5, MI-Z-M5 and PD-Z-M5, were produced in Escherichia coli, and after affinity purification these were evaluated in iscom-incorporation experiments. Two of the fusion proteins, IW-Z-M5 and MI-Z-M5 were found in the iscom fraction following preparative ultracentrifugation, indicating iscom incorporation. This was further supported by electron microscopy analysis showing that iscoms were formed. Furthermore, these iscom preparations were demonstrated to induce efficient M5-specific antibody responses upon immunization of mice, confirming successful incorporation into iscoms. The implications of these results for the design and production of subunit vaccines are discussed.     

The Soviet anti-plague system: an introduction

This article describes the composition of the Soviet Anti-plague (AP) system and presents the methodology used by the authors in their study of the AP system.     

The immunostimulating complex (ISCOM) is an efficient mucosal delivery system for respiratory syncytial virus (RSV) envelope antigens inducing high local and systemic antibody responses

ISCOM is an efficient mucosal delivery system for RSV envelope proteins as measured by antibody responses in respiratory tract secretions and in sera of mice following two intranasal (i.n.) administrations. Intranasally administered RSV ISCOMs induced high levels of IgA antibodies both in the upper respiratory tract and in the lungs. In the lungs, a prominent and long-lasting IgA response was recorded, which still persisted 22 weeks after the second i.n. immunization when the experiment ended. Subcutaneous (s.c.) immunization only induced low IgA titres in the upper respiratory tract and no measurable response to RSV was found in the lungs. Differences were also noticed in serum between the i.n. and s.c. modes of immunization. ISCOMs given intranasally induced earlier, higher and longer lasting IgM and IgG1 serum anti-RSV antibody responses than those induced by the s.c. mode of administration. A low serum IgE response was only detectable at 2 weeks after i.n. immunization with ISCOMs and after s.c. immunization with an inactivated virus, but no IgE response was detectable after s.c. injection of ISCOMs. The serum IgA response was more pronounced following s.c. injection of inactivated virus than after i.n. application of ISCOMs, and a clear-cut booster effect was obtained with a second immunization. Virtually no serum IgA response was detected after the s.c. administration of ISCOMs. In conclusion, the high immune responses induced by RSV ISCOMs in the respiratory tract and serum after i.n. administration indicate prominent mucosal delivery and adjuvant properties of the ISCOMs, warranting further studies.     

Induction of antibody responses in the common mucosal immune system by respiratory syncytical virus immunostimulating complexes

Immunostimulating complexes (ISCOMs) containing envelope proteins of respiratory syncytial virus (RSV) were explored as a mucosal delivery system for the capacity of inducing a common mucosal antibody response. Two intranasal (i.n.) administrations of BALB/c mice with ISCOMs induced potent serum IgG, and strong IgA responses to RSV locally in the lungs and the upper respiratory, and remotely in the genital and the intestinal tracts. Virtually no measurable IgA response was found in these mucosal organs after two subcutaneous (s.c.) immunizations. Virus neutralizing (VN) antibodies were detected in serum and in all of the mucosal organ extracts after both s.c. and i.n. immunizations indicating that the neutralizing epitopes were preserved after both mucosal and parenteral modes of administration. While the mucosal IgA response appears to be of mucosal origin, the IgG antibodies to RSV detected in the mucosal organs were likely of serum origin. However, the mucosal VN antibodies correlated with the IgG rather than the IgA levels. An enhanced IgA response to gp120 in various mucosal organs was recorded after i.n. immunization with gp120 incorporated in RSV ISCOMs, indicating a role of RSV envelope proteins in enhancing and targeting mucosal responses to passenger antigens. Received: 23 October 1998     

Improved systems for hydrophobic tagging of recombinant immunogens for efficient iscom incorporation

We have previously reported a strategy for production in Escherichia coli of recombinant immunogens fused to a hydrophobic tag to improve their capacity to associate with an adjuvant formulation [Andersson et al., J. Immunol. Methods 222 (1999) 171]. Here, we describe a further development of the previous strategy and present significant improvements. In the novel system, the target immunogen is produced with an N-terminal affinity tag suitable for affinity purification, and a C-terminal hydrophobic tag, which should enable association through hydrophobic interactions of the immunogen with an adjuvant system, here being immunostimulating complexes (iscoms). Two different hydrophobic tags were evaluated: (i) a tag denoted M, derived from the membrane-spanning region of Staphylococcus aureus protein A (SpA), and (ii) a tag denoted MI consisting of the transmembrane region of hemagglutinin from influenza A virus. Furthermore, two alternative affinity tags were evaluated; the serum albumin-binding protein ABP, derived from streptococcal protein G, and the divalent IgG-binding ZZ-domains derived from SpA. A malaria peptide M5, derived from the central repeat region of the Plasmodium falciparum blood-stage antigen Pf155/RESA, served as model immunogen in this study. Four different fusion proteins, ABP-M5-M, ABP-M5-MI, ZZ-M5-M and ZZ-M5-MI, were thus produced, affinity purified and evaluated in iscom-incorporation experiments. All of the fusion proteins were found in the iscom fractions in analytical ultracentrifugation, indicating iscom incorporation. This was further supported by electron microscopy analysis showing that iscoms were formed. In addition, these iscom preparations were demonstrated to induce M5-specific antibody responses upon immunisation of mice, confirming the successful incorporation into iscoms. The novel system for hydrophobic tagging of immunogens, with optional affinity and hydrophobic tags, gave expression levels that were increased ten to fifty-fold, as compared to the earlier reported system. We believe that the presented strategy would be a convenient way to achieve efficient adjuvant association for recombinant immunogens.     

In vivo and in vitro lipidation of recombinant immunogens for direct iscom incorporation

We have previously reported strategies for Escherichia coli production of recombinant immunogens fused to hydrophobic tags to improve their capacity to be incorporated into an adjuvant formulation (J. Immunol. Methods 222 (1999) 171; 238 (2000) 181). Here, we have explored the possibility to use in vivo or in vitro lipidation of recombinant immunogens as means to achieve iscom incorporation through hydrophobic interaction. For the in vivo lipidation strategy, a general expression vector was constructed encoding a composite tag consisting of a sequence (lpp) of the major lipoprotein of E. coli, fused to a dual affinity fusion tag to allow efficient recovery by affinity chromatography. Upon expression in E. coli, fatty acids would be linked to the produced gene products. To achieve in vitro lipidation, the target immunogen would be expressed in frame with an N-terminal His6-ABP affinity tag, in which the hexahistidyl tag was utilized to obtain lipidation via a Cu2+-chelating lipid. A 238 amino acid segment DeltaSAG1, from the central region of the major surface antigen SAG1 of Toxoplasma gondii, served as model immunogen in this study. The two generated fusion proteins, lpp-His6-ABP-DeltaSAG1 and His6-ABP-DeltaSAG1, both expressed at high levels (approximately 5 and 100 mg/l, respectively), could be recovered to high purity by ABP-mediated affinity chromatography, and were evaluated in iscom-incorporation experiments. The His6-ABP-DeltaSAG1 fusion protein was associated to iscom matrix with pre-incorporated chelating lipid. Both fusion proteins were found in the iscom fractions after analytical ultracentrifugation in a sucrose gradient, indicating successful iscom incorporation/association. Iscom formation was further supported by electron microscopy analysis. In addition, these iscom preparations were demonstrated to induce high-titer antigen-specific antibody responses upon immunization of mice. For this particular target immunogen, DeltaSAG1, the induced antibodies demonstrated poor reactivity to the native antigen, although slightly better for the preparation employing the in vitro lipidation strategy, indicating that DeltaSAG1 was suboptimally folded or presented. Nevertheless, we believe that the presented strategies offer convenient alternative ways to achieve efficient adjuvant incorporation for recombinant immunogens.     

Characterization of purified gp 51 from bovine leukemia virus integrated into iscom

Summary It is proposed that the envelope glycoprotein, gp 51, is the protective antigen of bovine leukemia virus (BLV). An experimental iscom vaccine has been prepared from immunoaffinity purified gp 51. To overcome the problem of integrating a nonamphipathic protein, gp 51 was partially denatured at pH 2.4 before integration into the iscom. The recovery of gp 51 into the iscom was calculated to be 85%. The gp 51 incorporated into iscom retained its physicochemical properties and the neutralizing epitopes F, G and H were found to be intact. The iscom preparation was shown to induce a specific immune response to gp 51 after inoculation into mice and calves, as tested by ELISA and Western blotting. Sera from the immunized calves specifically inhibited the VSV-(BLV) pseudotypes. Thus the gp 51-iscom preparations appear to be highly immunogenic and to induce a gp 51 specific response.     

Opposite effects of a glucocorticoid and an immunostimulating agent on prostaglandin production by two different cell types

Studies were performed to investigate the effects of several agents known to modulate wound healing on prostaglandin production by mouse embryo fibroblasts and adult thymic phagocytic cells in culture. Dexamethasone (10(-6)M) induced in both cell types a significant inhibition of the production of PGE2 and 6 keto PGF1 alpha, and a moderate inhibition of PGF2 alpha. Using an antiglucocorticoid compound, RU 38486, we were able to demonstrate that the inhibition of PG secretion represents a classical receptor-mediated effect of the steroid. In contrast, LPS and RU 41740 (5 micrograms/ml) induced a significant stimulation of PGE2 and 6-keto-PGF1 alpha secretion in the two types of cells. These results suggest that agents which modulate in different ways the process of tissue repair have opposite effects on PG production by cells involved in inflammatory and/or immunological reactions.     

Experimental analysis of the immunostimulating properties of vitamin A

Bulletin of Experimental Biology and Medicine -     

The primary visual system of flatfish: an evolutionary perspective

Summary The retinal projections of two species of flatfish (Scophthalmus maximus, Scophthalmidae; Platichthys flesus, Pleuronectidae) were investigated by autoradiography and by a HRP technique. Contralateral projections to five hypothalamic centres (area optica preoptica ventralis, nucleus opticus preopticus parvocellularis posterior pars lateralis, n. suprachiasmaticus, n. opticus hypothalami ventromedialis and area optica hypothalami posterior), thirteen thalamo-pretectal centres (nucleus opticus dorsolateralis (partes medialis, ventralis and lateralis), n. opticus ventrolateralis, n. opticus commissurae posterioris (partes dorsalis and ventralis), n. opticus accessorius, n. geniculatus lateralis mesencephali, nn. opticus pretectalis dorsalis, medialis and ventralis and n. corticalis), three layers of the optic tectum (stratum opticum pars externa, stratum fibrosum et griseum superficiale, stratum album centrale), and a single target in the tegmentum (n. opticus tegmenti mesencephali dorsalis), were identified in both species. Interspecific variation of the contralateral visual projections is relatively small. Ipsilateral visual projections of fibres which recross the midline in the minor and transverse commissures were also identified; in S. maximus this ipsilateral contingent is poorly developed and concerns principally hypothalamic structures, while in P. flesus the ipsilateral projections are considerably more extensive and involve both hypothalamic and thalamo-pretectal primary visual centres. No differences in the projections from the fixed and from the migrated eye were observed in either species. The findings are discussed in the general context of the existing literature on the visual projections of teleosts, in an attempt to characterize the primary visual system of the Pleuronectiformes in an evolutionary context.List of Abbreviations AOHp Area optica hypothalami posterior - AOPv Area optica preoptica ventralis - CER Cerebellum - Com. H Commissura horizontalis - Com. M Commissura minor - Com. T Commissura transversalis - Cp Commissura posterioris - FDtro Fasciculus dorsalis tractus optici - FHtro Fasciculus hypothalami tractus optici - FOCM Fasciculus opticus commissurae minor - FOCT Fasciculus opticus commissurae transversalis - FOHpv Fasciculus opticus hypothalami posterior pars ventralis - FVLtro Fasciculus ventrolateralis tractus optici - FVLtroi Fasciculus ventrolateralis optici ipsilateralis - FVMtro Fasciculus ventromedialis tractus optici - FVMtroi Fasciculus ventromedialis tractus optici ipsilateralis - FVtro Fasciculus ventralis tractus optici - Hyp Hypophysis cerebri - IS Interlobular sulcus - LO Lobus opticus - LOd Lobus opticus dorsalis - LOv Lobus opticus ventralis - LON left optic nerve - NC Nucleus corticalis - NDLi Nucleus diffusus lobi inferioris - NDM Nucleus dorsomedialis - NE Nucleus entopeduncularis - NG Nucleus glomerulosus - NGL Nucleus geniculatus lateralis - NGLM Nucleus geniculatus lateralis mesencephali - NOA Nucleus opticus accessorius - NOCPpd Nucleus opticus commissurae posterions pars dorsalis - NOCPpv Nucleus opticus commissurae posterioris pars ventralis - NODL Nucleus opticus dorsolateralis - NODLpl Nucleus opticus dorsolateralis pars lateralis - NODLpm Nucleus opticus dorsolateralis pars medialis - NODLpv Nucleus opticus dorsolateralis pars ventralis - NOHvl Nucleus opticus hypothalamicus ventrolateralis - NOPd Nucleus opticus pretectalis dorsalis - NOPL Nucleus opticus pretectalis lateralis - NOPm Nucleus opticus pretectalis medialis - NOPPpl Nucleus opticus preopticus parvocellularis posterior pars lateralis - NOPv Nucleus opticus pretectalis ventralis - NOTMd Nucleus opticus tegmenti mesencephali dorsalis - NOTMdl Nucleus opticus tegmenti mesencephali dorsalis pars lateralis - NOTMdm Nucleus opticus tegmenti mesencephali dorsalis pars medialis - NOVL Nucleus opticus ventrolateralis - NPG Nucleus preglomerulosus - NPMg Nucleus preopticus magnocellularis - NPP Nucleus preopticus parvocellularis posterior - NPPa Nucleus preopticus parvocellularis anterior - NPs Nucleus pretectalis superficialis - NRL Nucleus recessus lateralis - NSC Nucleus suprachiasmaticus - NVM Nucleus ventromedialis - RON right optic nerve - sac stratum album centrale - sfgs stratum fibrosum et griseum superficiale - sfpv stratum fibrosum periventriculare - sgc stratum griseum centrale - sgpv stratum griseum periventriculare - sm stratum marginale - soe stratum opticum pars externa - soi stratum opticum pars interna - SV saccus vascularis - Tel telencephalon - TL Torus longitudinalis - TM Tegmentum mesencephali - TO Tectum opticum - TROA Tractus opticus accessorius - TROdm Tractus opticus dorsomedialis - TROdmd Tractus opticus dorsomedialis dorsalis - TROdme Tractus opticus dorsomedialis pars externa - TROdmi Tractus opticus dorsomedialis pars interna - TROdmv Tractus opticus dorsomedialis ventralis - TROdmvd Tractus opticus dorsomedialis ventralis pars dorsalis - TROdmvv Tractus opticus dorsomedialis ventralis pars ventralis - TROM Tractus opticus marginalis - TROvl Tractus opticus ventrolateralis - TROvld Tractus opticus ventrolateralis dorsalis - TROvle Tractus opticus ventrolateralis pars externa - TROvli Tractus opticus ventrolateralis pars interna - TROvldd Tractus opticus ventrolateralis dorsalis pars dorsalis - TROvldv Tractus opticus ventrolateralis dorsalis pars ventralis - TROvlv Tractus opticus ventrolateralis pars ventralis - TS Torus semicircularis - v ventricle - VC Valvula cerebelli - I Nervus olfactorius - II Nervus opticus - V Nervus trigeminus - VII Nervus facialis - VIII Nervus octavolateralis - IX Nervus glossopharyngeus - X Nervus vagus     

Therapeutic anti-tumor immunity triggered by injections of immunostimulating single-stranded RNA

Stabilized synthetic RNA oligonucleotides (ORN) and protected messenger RNA (mRNA) were recently discovered to possess an immunostimulatory capacity through their recognition by TLR 7 and 8. We wanted to find out whether this danger signal is capable of triggering anti-tumor immunity when injected locally into an established tumor. Using the mouse glioma tumor cell line SMA-560 in syngenic VM/Dk mice, we were able to show that intra-tumor injections of protamine-stabilized mRNA do indeed induce tumor regression and long-term anti-tumor immunity. Residual RNA-injected tumors show CD8 infiltration. Distant injections of protamine-protected mRNA and intra-tumor injection of naked mRNA also result in anti-tumor immunity. Although they are strong danger signals, RNA are labile molecules with a short half-life: they do not trigger side effects such as long-term, uncontrolled immunostimulation evidenced by splenomegaly in CpG DNA-treated mice. In conclusion, RNA molecules are potent and safe danger signals that are relevant for active immunotherapy strategies aimed at the eradication of solid tumors.     

Dopaminergic mechanisms in the immunostimulating effect of μ-opioid receptors

Immunogenesis was appreciably stimulated in CBA mice injected the μ-opioid receptor agonist DAGO. This stimulation was prevented by transecting the pituitary peduncle. Immunoactivation induced by opioid peptide was due to interaction with the dopaminergic system, because the effect is canceled by preliminary haloperidol blocking of D₂-postsynaptic receptors. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 121, No. 4, pp. 373–375, April, 1996 Presented by V. A. Trufakin, Member of the Russian Academy of Medical Sciences     

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 Freund's 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 Freund's 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.     

Protection of mice against an influenza virus infection by oral vaccination with viral nucleoprotein incorporated into immunostimulating complexes

Influenza A virus nucleoprotein (NP) was integrated into immunostimulating complexes (ISCOMs) after attachment of bacterial lipopolysaccharide to the antigen. Oral immunization with these NP-ISCOMs protected mice fully against an otherwise lethal challenge infection with an unrelated influenza virus subtype without the appearance of severe clinical signs or extensive pathological lesions in the lungs. Mice immunized with analogous bovine serum albumine-incorporated ISCOMs all died. After oral immunization, high titers of NP-specific antibodies, particularly IgA, could be detected in the bronchoalveolar fluid and in the blood serum. No cytotoxic lymphocytes could be demonstrated in the spleens or the lungs of vaccinated mice, and no anti-NP antibody-dependent cytolysis of infected host cells was mediated by complement or in the form of an antibody-dependent cell cytotoxicity. However, a vigorous delayed-type hypersensitivity reaction was produced after probing vaccinated animals with purified NP. No comparable protective immunity or antibody response was induced by a strictly intragastric administration of NP-ISCOMs. It appears, therefore, that the general and local immune response in the lungs was primarily stimulated through contact of NP-ISCOMs with the mucous membrane of the oro-pharyngeal cavity and that cytotoxic effects did not play a major role for the establishment of the protective immunity. Partial protection against a lethal challenge was observed in chickens immunized with NP-ISCOMs in the drinking water.