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Vaccination is one of the most widely used strategies to protect horses against pathogens. However, available equine vaccines often have limitations, as they do not always provide effective, long-term protection and booster injections are often required. In addition, research efforts are needed to develop effective vaccines against emerging equine pathogens. In this review, we provide an inventory of approved adjuvants for equine vaccines worldwide, and discuss their composition and mode of action when available. A wide range of adjuvants are used in marketed vaccines for horses, the main families being aluminium salts, emulsions, polymers, saponins and ISCOMs. We also present veterinary adjuvants that are already used for vaccination in other species and are currently evaluated in horses to improve equine vaccination and to meet the expected level of protection against pathogens in the equine industry. Finally, we discuss new adjuvants such as liposomes, polylactic acid polymers, inulin, poly-ε-caprolactone nanoparticles and co-polymers that are in development. Our objective is to help professionals in the horse industry understand the composition of marketed equine vaccines in a context of mistrust towards vaccines. Besides, this review provides researchers with a list of adjuvants, either approved or at least evaluated in horses, that could be used either alone or in combination to develop new vaccines. [ABSTRACT FROM AUTHOR]

Quillaja saponins have an intrinsic capacity to interact with membrane lipids that self-assembles in nanoparticles (immunostimulating complexes or ISCOM-matrices) with outstanding immunoadjuvant activity and low toxicity profile. However, the expensive and laborious purification processes applied to purify Quillaja saponins used to assemble ISCOM-matrices show an important drawback in the large-scale use of this vaccine adjuvant. Thus, in this study, we describe a protocol to appropriately formulate ISCOM-matrices using the raw aqueous extract (AE) of Quillaja lancifolia leaves. In the presence of lipids, AE was able to self-assemble in nanostructures that resembles immunostimulating complexes (ISCOM). These negatively charged nanoparticles of approximately 40 nm were characterized by transmission electron microscopy and dynamic light scattering. In addition, well-known saponins with remarkable immunoadjuvant activity, as QS-21, were detected into nanoparticles. Thus, the easier, robust, cheaper, and environmentally friendly method developed here may be an alternative to the classical methods for ISCOM-matrices production that use high-purified saponins. [ABSTRACT FROM AUTHOR]

Saponin-based adjuvants (SBAs) are promising new adjuvants that stand out as they not only enforce CD4 + T cell-mediated immunity and antibody responses, but also induce an unprecedented level of antigen cross-presentation by dendritic cells (DC) and subsequent CD8 + T cell activation. We discovered that SBA’s ability to boost cross-presentation depends on the induction of lipid bodies (LBs). Moreover, the MHCIIloCD11bhi DC subset was identified to be most responsive to SBA-induced cross-presentation. The aim is to further unravel the mechanisms behind the induction of DC cross-presentation by SBAs. Here we show that SBAs specifically induce the PKR-like Endoplasmic Reticulum kinase (PERK) pathway and that SBA-induced DC cross-presentation is dependent on activation of the PERK pathway. PERK activation and LB formation are both crucial for SBA-induced cross-presentation and PERK inhibition has little or no effect on SBA-induced LB formation. SBA’s responsiveness, LB formation and PERK activation are specific for the MHCIIloCD11bhi DCs. These findings contribute to understanding the pathways involved in SBA-induced cross-presentation and immune activation which will ultimately lead to the development of vaccines with improved efficiency and safety. [ABSTRACT FROM AUTHOR]

Nanoadjuvants that combine immunostimulatory properties and delivery systems reportedly bestow major improvements on the efficacy of recombinant, protein-based vaccines. Among these, self-assembled micellar formulations named ISCOMs (immune stimulating complexes) show a great ability to trigger powerful immunological responses against infectious pathogens. Here, a nanoadjuvant preparation, based on saponins from Quillaja brasiliensis , was evaluated together with an experimental Zika virus (ZIKV) vaccine (IQB80-zEDIII) and compared to an equivalent vaccine with alum as the standard adjuvant. The preparations were administered to mice in two doses (on days zero and 14) and immune responses were evaluated on day 28 post-priming. Serum levels of anti-Zika virus IgG, IgG1, IgG2b, IgG2c, IgG3 were significantly increased by the nanoadjuvant vaccine, compared to the mice that received the alum-adjuvanted vaccine or the unadjuvanted vaccine. In addition, a robust production of neutralizing antibodies and in vitro splenocyte proliferative responses were observed in mice immunized with IQB80-zEDIII nanoformulated vaccine. Therefore, the IQB80-zEDIII recombinant preparation seems to be a suitable candidate vaccine for ZIKV. Overall, this study identified saponin-based delivery systems as an adequate adjuvant for recombinant ZIKV vaccines and has important implications for recombinant protein-based vaccine formulations against other flaviviruses and possibly enveloped viruses. [ABSTRACT FROM AUTHOR]

The present study was conducted to develop and evaluate an experimental ISCOM-based infectious bursal disease (IBD) vaccine. The indigenous very virulent infectious bursal disease virus (vvIBDV) already attenuated and adapted to Vero cell line was used. After denaturation of viral proteins with sodium dodecyl sulphate (SDS), an IBD-ISCOM was constructed. The non-incorporated viral components were separated from ISCOM by centrifugation of dialysate. The pathogenicity and immunogenicity trials were conducted in 3-week-old broiler chicken. A commercial oil-emulsified vaccine (CEVAC IBD K) was used for comparison. There were no clinical signs of disease, gross or microscopic lesions in bursa of Fabricius in group G1 vaccinated with ISCOM-based vaccine and bursa to body weight ratio were comparable to un-vaccinated control group (G3). The virus-neutralizing antibody titers were significantly (P<0.05) higher in group G1 as compared with group G2 which was vaccinated with commercial vaccine. On challenge with vvIBDV, 100%, 75% and 0.00% protection was achieved in G1, G2 and G3, respectively. The results indicated that ISCOM-based IBD vaccine is safe and immunogenic. [ABSTRACT FROM AUTHOR]

Immunostimulating complexes (ISCOM) have been modified by replacement of structural components of ISCOM; phosphatidylcholine has been replaced, by glycolipid of monogalactosyldiacylglycerol from sea macrophytes, saponin QuilA has been replaced by triterpene glycoside, Cucumarioside A2-2. The resultant complex includes morphological structures of two types: the ISCOM-like structures with the characteristic morphology and sizes and also the tubular structures with diameter of about 40 nm and length of 150–400 nm. We have defined these structures as TI-complexes. These TI-complexes exhibit considerably lower toxicity than ISCOM. They may include an amphiphilic protein antigen and provide immunoadjuvant effect during experimental vaccination. Under conditions of the experimental immunization of mice with a weak immunogen (subunit membrane pore protein from Y. pseudotuberculosis), TI-complexes with antigen provided stronger humoral immune response to antigen than the complexes of porin with classical ISCOM, liposomes and Freund’s adjuvant. Thus, the TI-complexes represent a new type of adjuvant carriers for antigens. [ABSTRACT FROM AUTHOR]

The immunostimulating complex or‘iscom’ was first described 20 years ago as an antigen delivery system with powerful immunostimulating activity. Iscoms are cage-like structures, typically 40 nm in diameter, that are comprised of antigen, cholesterol, phospholipid and saponin. ISCOMTM-based vaccines have been shown to promote both antibody and cellular immune responses in a variety of experimental animal models. This review focuses on the evaluation of ISCOMTM-based vaccines in animals over the past 10 years, as well as examining the progress that has been achieved in the development of human vaccines based on ISCOMTM adjuvant technology. [ABSTRACT FROM AUTHOR]

Purpose. Saponin is the major component in the formation of immune stimulating complex (ISCOM), a potent adjuvant able to induce both humoral and cellular immune reactions. The immunogenicity induced by saponin, however, has been unclear. The objective of this study was to investigate the apoptotic and necrotic effects induced by saponin in EL4 mouse lymphoma cells, expected to be a possible mechanism of the cytotoxic T-lymphocyte (CTL) effect elicited by the ISCOM. Methods. EL4 cells were treated with saponin, and viability of the cells was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase release assays. Fluorescence microscopy was used to detect the morphological changes by staining saponin-treated cells with Hoechst 33342. Extent of apoptosis and necrosis was determined by Annexin V-FITC/ propidium iodide staining, followed by flow cytometric analysis. Dendritic cells were cultured with either saponin-protein complexes or saponin-treated cells and analyzed by flow cytometry. Results. Treatment of EL4 cells with saponin resulted in concentration-dependent cytotoxicity and the appearance of the hypodiploid DNA peak. Cells treated with saponin showed highly condensed chromatin when stained with fluorescent DNA-binding dye Hoechst 33342. Analysis of EL4 cells by flow cytometry after Annexin V/propidium iodide staining demonstrated that saponin induced both apoptosis and necrosis. Pretreatment of EL4 cells with zVAD-fmk, a broad-range caspase inhibitor, did not prevent cell death induced by saponin, indicating the non-caspase-dependent cell death. Dendritic cells were shown to phagocytose both the antigen-saponin complexes and the saponin-induced dead cells. Conclusions. Results obtained in this study demonstrated that saponin induced both apoptosis and necrosis in EL4 cells. These events are critical for antigen processing and presentation. [ABSTRACT FROM AUTHOR]

A gentle method for BHV-1 purification - allowing the preservation of viral envelope - was used by applying a moderate centrifugal force and metrizamide as gradient. The virus purified by this procedure was used for the preparation of iscoms. A total recovery of 25% of viral proteins was achieved in iscoms as the final product. The iscoms were characterized physico-chemically. With the aid of biotinylated lectins, glycosylated viral proteins were shown to be present, incorporated into the iscoms. Furthermore, a high degree of immunogenicity of the prepared iscoms was demonstrated on mice. Zusammenfassung Charakterisierung eines immunstimulierenden Komplexes, der die Glykoproteine der Hülle des bovinen Herpesvirus Typ 1 (BHV-1) enthält Eine Methode zur schonenden Reinigung von Partikeln des BHV-1, bei der die Hüllstrukturen erhalten bleiben, wird beschrieben. Eine Hüllpräparation solch gereinigter Viruspartikel wurde zur Herstellung vom immunstimulierenden Komplex (ISCOM) verwendet. Dabei konnte ein Viertel des Virusproteingehaltes in den Komplex eingelagert werden. Mit Hilfe biotinylierter Lektine ließen sich in diesem Komplex virale Glykoproteine nachweisen. In Immunisierungsversuchen an Mäusen erwies sich der immunstimulierende Komplex als besonders immunogen. [ABSTRACT FROM AUTHOR]

A selective Toxoplasma gondii antigen preparation consisting of extracted T. gondii tachyzoite cell membrane proteins arranged into submicroscopical particles (iscoms), was used as coating antigen in an ELISA (iscom ELISA). By means of sodium-dodecyl-sulphate polyacrylamid gel electrophoresis (SDS-PAGE), the Toxoplasma iscoms were found to consist of 4 major proteins with estimated molecular weights of 50 kD, 44 kD, 32 kD and 20 kD. By immunoblot analysis using T. gondii positive sheep sera, 3 major antigenic components with estimated molecular weights of 50 kD, 44 kD and 32 kD were detected. 85 sheep sera submitted for routine T. gondii serology were used to compare the results in the iscom ELISA, the Toxoplasma IFAT (indirect fluorescent antibody test) and an ELISA (FT-S ELISA) employing a soluble (cytoplasmatic) T. gondii antigen. The iscoms ELISA was found to be as sensitive as the other tests in detecting low levels of T. gondii antibodies and in discriminating positive and negative sera. In strongly positive sera, most likely also containing antibodies to intracellular components, the FT-S ELISA gave considerably higher titres than did the iscom ELISA and the IFAT. Since positive FT-S ELISA results have been recorded during recent monoinfection with Sarcocystis cruzi in calves, whereas no such cross-reaction was observed in the iscom ELISA of IFAT (U ggla et al., 1987), it was concluded that the iscom ELISA combines the specificity of the IFAT with the objectivity and simplicity of the ELISA. Zusammenfassung Eine selektive Toxoplasma gondii-Antigen-Präparation aus extrahierten Proteinen aus der Zellmembran von T. gondii-Tachyzoiten, angeordnet in submikroskopischen Partikeln (iscoms, immuno stimulating complexes), wurde als Schicht-Antigen in einem ELISA (iscom ELISA) verwendet. Mit Hilfe der Sodium-Dodecyl-Sulfat Polyacrylamid-Gel-Elektrophorese (SDS-PAGE) wurde nachgewiesen, daß die Partikel aus vier Hauptproteinen mit Molekulargewichten von schätzungsweise 50 kD, 44 kD, 32 kD und 20 kD bestehen. Bei Verwendung von T. gondii-positiven Schafseren in der Immunoblot-Analyse wurden drei antigene Hauptkomponenten mit Molekulargewichten von schätzungsweise 50 kD, 44 kD und 32 kD ermittelt. 85 Schafseren, die für routinemäßige T. gondii-Serologie vorgelegt waren, wurden unter Verwendung eines löslichen (cytoplasmatischen) T. gondii-Antigens zu einem Vergleich der Ergebnisse im iscom ELISA, im Toxoplasma IFAT ( indirekter Fluoreszenz- Antikörper- Test) und im konventionellen ELISA (FT-S ELISA) herangezogen. Der iscom-ELISA erwies sich als ebenso empfindlich wie die anderen Tests bei der Erfassung niedriger Antikörpertiter gegen T. gondii und bei der Unterscheidung positiver und negativer Seren. In stark positiven Seren, die höchstwahrscheinlich auch Antikörper gegen intrazelluläre Komponenten enthielten, ergab der FT-S ELISA bedeutend höhere Titer als der iscom ELISA und der IFAT. Nachdem im FT-S ELISA auch positive Ergebnisse bei einer frischen Monoinfektion mit Sarcocystis cruzi in Kälbern registriert wurden, während im iscom ELISA und IFAT keine derartige Kreuzreaktion zu beobachten war, wurde der Schluß gezogen, und der iscom ELISA die Spezifität des IFAT mit der Objektivität und Einfachheit des ELISA vereint. [ABSTRACT FROM AUTHOR]

The iscom is a delivery system, designed for both parenteral and mucosal modes of administration, for both antigens and adjuvants, components which are interchangeable. By the parenteral route a prominent systemic Th1 type of response is evoked, but the mucosal immunoglobulin A (IgA) response was insignificant. Intranasal (i.n.) immunization with iscoms evoked potent mucosal IgA response and serum IgG which was much higher than that induced by i.n. administration of the B subunit of cholera toxin (rCTB), both to rCTB itself as well as to co-administered antigen. The immunomodulatory effect on rCTB or co-administered antigens imposed by the iscom was demonstrated by a potent mucosal IgA switch and an enhanced IgG2a serum response. The incorporation of a targeting molecule in the iscom enhanced the remote IgA response in the genital tract mucosa. The capacity to induce CD8-restricted cytotoxic T lymphocytes (CTL) is unique for the iscom as a non-replicating system, which is facilitated by the delivery of antigens to the cytosol. The immunomodulatory capacity of iscoms also paved the way to override the inhibitory effect of maternally derived antibodies and the relative unresponsiveness of an immature neonatal immune system. [ABSTRACT FROM AUTHOR]

The iscom is a delivery system, designed for both parenteral and mucosal modes of administration, for both antigens and adjuvants, components which are interchangeable. By the parenteral route a prominent systemic Th1 type of response is evoked, but the mucosal immunoglobulin A (IgA) response was insignificant. Intranasal (i.n.) immunization with iscoms evoked potent mucosal IgA response and serum IgG which was much higher than that induced by i.n. administration of the B subunit of cholera toxin (rCTB), both to rCTB itself as well as to co-administered antigen. The immunomodulatory effect on rCTB or co-administered antigens imposed by the iscom was demonstrated by a potent mucosal IgA switch and an enhanced IgG2a serum response. The incorporation of a targeting molecule in the iscom enhanced the remote IgA response in the genital tract mucosa. The capacity to induce CD8-restricted cytotoxic T lymphocytes (CTL) is unique for the iscom as a non-replicating system, which is facilitated by the delivery of antigens to the cytosol. The immunomodulatory capacity of iscoms also paved the way to override the inhibitory effect of maternally derived antibodies and the relative unresponsiveness of an immature neonatal immune system. [ABSTRACT FROM AUTHOR]

There is now compelling evidence that intradermal vaccination with an efficacious adjuvanted antigen triggers a series of coordinated responses characterized initially by the rapid mobilization and recruitment of granulocytes to the lung. Activation of effector cells of the innate immune system is intended to provide surveillance and temporary protective cover at vulnerable mucosal sites while both T and B cell precursors, as well as haematopoietic progenitor cells, are undergoing dramatic reductions in numbers during the first 2–4 days post-vaccination. Some of these events recapitulate those seen after infection with a pathogen. Initial decreases in cell numbers in the thymus and bone marrow (BM) are followed by rapid increases in cellular proliferation in these organs, probably in response to peripheral signals. Vaccine-induced cell death (by apoptosis) in the thymus may provide one of many stimuli needed to up-regulate BM production of progenitor cells, and cells of the B, myeloid and monocytic lineages so that depleted peripheral compartments are replenished. Reconstitution of the latter cell population is critical in ensuring sufficient numbers of APC are generated to deal with extraneous antigen resulting from either vaccination or proliferation of a pathogen. Ultimately, these APC, as effector cells of the innate immune system, must provide pattern recognition of dangerous pathogens and serve to activate appropriate T cell responses. Vaccination not only educates both the innate and adaptive arms of the immune response but also more interestingly, appears to regulate subsequent innate immune responses following exposure to a lethal challenge dose of bacteria. Under these conditions, the rate of loss of BM precursors is greatly attenuated in mice previously vaccinated with adjuvanted antigen compared to unvaccinated controls or mice that had received only antigen. Mice intradermally vaccinated with adjuvanted antigen also displayed increased rates... [ABSTRACT FROM AUTHOR]

An experimental influenza virus (A/PR/8/34(HINI)) vaccine was tested and evaluated in mice. The mice were inoculated once or twice intranasally or subcutaneously with 1 or 10 μg of iscoms prior to challenge with high doses of live virus. It was demonstrated that two intranasal administrations were as efficient as two s.e. administrations, both routes inducing high levels of antibody and protection against challenge infection. With a one-dose regimen, the s.c. route induced a somewhat higher antibody response than the intranasal route; this might be explained by technical difficulties connected with an intranasal administration. [ABSTRACT FROM AUTHOR]