Immune-stimulating Complexes Research Articles

Induction of cell death by saponin and antigen delivery.

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 ELA mouse lymphoma cells, expected to be a possible mechanism of the cytotoxic T-lymphocyte (CTL) effect elicited by the ISCOM. 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. 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. Results obtained in this study demonstrated that saponin induced both apoptosis and necrosis in ELA cells. These events are critical for antigen processing and presentation.

From toxin to adjuvant: the rational design of a vaccine adjuvant vector, CTA1-DD/ISCOM.

Mucosally active vaccine adjuvants which will prime a full range of local and systemic immune responses against defined antigenic epitopes are much needed. Cholera toxin (CT) and lipophilic immune stimulating complexes (ISCOMs) containing Quil A can both act as adjuvants for orally administered antigens, but through separate pathways, as evidenced by the dependence on IL-12 for the effect of ISCOMs, whereas CT is independent of this cytokine. Unfortunately the toxicity of CT and recent findings of accumulation of CT in the olfactory nerve and bulb after intranasal administration precludes the clinical use of CT. However, we have been successful in separating the adjuvant and toxic effects of CT, by constructing a gene fusion protein, CTA1-DD, that combines the enzymatically active CTA1-subunit with a B cell targeting moiety, D, derived from Staphylococcus aureus protein A. The present review gives a background to mucosal immunization and the use of -adjuvants in general, followed by a description of a strategy to rationally design a vaccine adjuvant vector that fulfils the criteria of targeting and immunomodulating innate immunity in order to boost a strong adaptive immune response. We have combined CTA1-DD and ISCOMs into a new highly promising vaccine adjuvant vector, CTA1-DD/ISCOMs. The combined vector is immunogenic when given by the subcutaneous, oral or nasal routes, inducing strong cell--mediated and humoral immune responses, including local mucosal IgA. It requires the ADP ribosylating property of the CTA1-enzyme and the effect of the combined vector greatly exceeded the effect of either ISCOMs or CT used alone. Antigens could be incorporated into or just admixed with the new vector. Thus, we have demonstrated that rationally designed vectors consisting of CTA1-DD and ISCOMS may provide a novel strategy for the generation of potent and safe mucosal vaccines.

The role of antigen-presenting cells and interleukin-12 in the priming of antigen-specific CD4+ T cells by immune stimulating complexes.

Immune stimulating complexes (ISCOMs) containing the saponin adjuvant Quil A are vaccine adjuvants that promote a wide range of immune responses in vivo, including delayed-type hypersensitivity (DTH) and the secretion of both T helper 1 (Th1) and Th2 cytokines. However, the antigen-presenting cell (APC) responsible for the induction of these responses has not been characterized. Here we have investigated the role of dendritic cells (DC), macrophages (Mphi) and B cells in the priming of antigen-specific CD4+ T cells in vitro by ISCOMs containing ovalbumin (OVA). OVA ISCOMs pulsed bone marrow (BM)-derived DC but not BM Mphi, nor naïve B cells prime resting antigen-specific CD4+ T cells, and this response is greatly enhanced if DC are activated with lipopolysaccharide (LPS). Of the APC found in the spleen, only DC had the capacity to prime resting antigen specific CD4+ T cells following exposure to OVA ISCOMs in vitro, while Mphi and B cells were ineffective. DC, but not B cells purified from the draining lymph nodes of mice immunized with OVA ISCOMs also primed resting antigen-specific CD4+ T cells in vitro, suggesting that DC are also critical in vivo. Using DC and T cells from interleukin (IL)-12 p40-/- mice, we also identified a crucial role for IL-12 in the priming of optimal CD4+ T cell responses by OVA ISCOMs. We suggest that DC are the principal APC responsible for the priming of CD4+ T cells by ISCOMs in vivo and that directed targeting of these vectors to DC may enhance their efficancy as vaccine adjuvants.

Dendritic cell maturation enhances CD8+ T-cell responses to exogenous antigen via a proteasome-independent mechanism of major histocompatibility complex class I loading.

Immune stimulating complexes (ISCOMS) containing the saponin adjuvant Quil A are vaccine adjuvants that induce a wide range of immune responses in vivo, including strong class I major histocompatibility complex (MHC)-restricted cytotoxic T-lymphocyte activity. However, the antigen-presenting cell responsible for the induction of these responses has not been characterized. Here we have investigated the role of dendritic cells (DC) in the priming of antigen-specific CD8+ T cells in vitro by ISCOMS containing ovalbumin. Resting bone marrow DC pulsed with ovalbumin ISCOMS efficiently prime resting CD8+ T cells through a mechanism that is transporter associated with antigen processing (TAP) dependent, but independent of CD40 ligation and CD4+ T-cell help. Lipopolysaccharide-induced maturation of DC markedly enhances their ability to prime CD8+ T cells through a mechanism which is also independent of CD4+ T-cell help, but is dependent on CD40 ligation. Furthermore, DC maturation revealed a TAP-independent mechanism of CD8+ T-cell priming. Our results also show that class I MHC-restricted presentation of ovalbumin in ISCOMS by DC is sensitive to chloroquine and brefeldin A but insensitive to lactacystin. We suggest that DC may be the principal antigen-presenting cells responsible for the priming of CD8+ T cells by ISCOMS in vivo and that targeting these vectors to activated DC may enhance their presentation via a novel pathway of class I antigen processing.

A role for dendritic cells in the priming of antigen-specific CD4+ and CD8+ T lymphocytes by immune-stimulating complexes in vivo.

Immune-stimulating complexes (ISCOMS) are adjuvant vectors which are unusual in being able to prime both CD4(+) and CD8(+) T cells by parenteral and mucosal routes. However, their mode of action is unclear and to define better the cellular interactions involved we have studied the ability of ISCOMS containing ovalbumin (OVA) to prime TCR transgenic CD4(+) or CD8(+) T cells in vivo. Immunization with OVA ISCOMS caused activation and clonal expansion of CD4(+) and CD8(+) T cells in the T cell areas of the draining lymph nodes, followed by the migration of both CD4(+) and CD8(+) T cells into the B cell follicle. The T cells were primed to proliferate and secrete IFN-gamma after re-stimulation in vitro with the appropriate OVA peptide and CD8(+) T cell priming occurred in the absence of CD4(+) T cells. Increasing the number of dendritic cells (DC) in vivo with flt3 ligand augmented the expansion and activation of the OVA-specific T cells, particularly CD8(+) T cells. These studies indicate DC play a central role in the priming of both CD4(+) and CD8(+) T cells in vivo, and suggest that an ability to target DC may allow ISCOMS to be powerful vaccine vectors for stimulating protective immunity.

Adjuvants in veterinary vaccines: modes of action and adverse effects.

Vaccine adjuvants are chemicals, microbial components, or mammalian proteins that enhance the immune response to vaccine antigens. Interest in reducing vaccine-related adverse effects and inducing specific types of immunity has led to the development of numerous new adjuvants. Adjuvants in development or in experimental and commercial vaccines include aluminum salts (alum), oil emulsions, saponins, immune-stimulating complexes (ISCOMs), liposomes, microparticles, nonionic block copolymers, derivatized polysaccharides, cytokines, and a wide variety of bacterial derivatives. The mechanisms of action of these diverse compounds vary, as does their induction of cell-mediated and antibody responses. Factors influencing the selection of an adjuvant include animal species, specific pathogen, vaccine antigen, route of immunization, and type of immunity needed.

Vaccination with recombinant alphavirus or immune-stimulating complex antigen against respiratory syncytial virus.

Respiratory syncytial virus (RSV) causes severe respiratory diseases in infants and young children. Inappropriate immunity to the virus can lead to disease enhancement upon subsequent infection. In this study, we have characterized the antiviral immunity elicited by the recombinant Semliki Forest virus (SFV) encoding the RSV fusion (F) and attachment (G) protein, and compared with that induced by the immune-stimulating complex (ISCOM)-incorporated FG proteins. Antiviral immunity against RSV elicited nasally or parentally by either of the immunogen having divergent profiles could reduce lung RSV titers upon challenge. However, resistance to RSV without disease enhancement was only observed in those vaccinated with SFV recombinants via nasal route. Presence of postvaccination pulmonary IFN-gamma response to the H-2K(d)-restricted T cell epitope (F(85-93); KYKNAVTEL) was found to be associated with absence of enhanced pulmonary disease and goblet cell hyperplasia as well as reduced Th2-cytokine expression. This result demonstrates that the SFV recombinants can result in enhanced clearance of RSV without enhancing the RSV-associated disease, and underlines the importance in priming pulmonary MHC class I-restricted T cells when RSV FG-based vaccines are used.

A method for the incorporation of ovalbumin into immune stimulating complexes prepared by the hydration method

This study describes the development of a method for the incorporation of fluorescently labelled ovalbumin (FITC-OVA) into immune stimulating complexes (ISCOMs) prepared by the hydration method. Conjugation of palmitic acid was performed to fluorescently labelled OVA (pFITC-OVA) or to non-labelled OVA, with subsequent conjugation of FITC to the resulting palmitified OVA (FITC-pOVA). Both pFITC-OVA and FITC-pOVA, but not FITC-OVA, could be incorporated into ISCOMs and other non-liposomal colloidal structures. The degree of incorporation of pFITC-OVA or FITC-pOVA in non-liposomal colloidal particles reaches a maximum, if ISCOMs are the predominant colloids in the system.

Induction of local innate immune responses and modulation of antigen uptake as mechanisms underlying the mucosal adjuvant properties of immune stimulating complexes (ISCOMS)

Lipophilic immune stimulating complexes (ISCOMS) containing the Quil A adjuvant can induce local and systemic immune responses against orally delivered protein antigens. Here, we have examined the possibility that ISCOMS act by modulating local innate immune responses and antigen uptake in the intestine. Feeding ISCOMS to mice induced recruitment of dendritic cells (DCs), activated macrophages (mφ) and lymphocytes into the mesenteric lymph node (MLN), as well as recruitment of macrophages and B cells into the PP. Feeding ISCOMS also enhanced the absorption into circulation of a test feed of ovalbumin (OVA), with accelerated kinetics and increased peak levels. Serum taken 1 h after feeding a tolerogenic dose of OVA to mice given ISCOMS orally did not induce the tolerance of systemic delayed type hypersensitivity (DTH) and antibody responses found in mice receiving serum from donors fed OVA alone. Thus, ISCOMS may increase the immunogenicity of intestinal antigen via a combination of effects on antigen uptake and on local accessory cells.

Potential applications of recent advances in vaccine research and development in overcoming constraints to animal diseases' vaccination programmes in developing countries – a review

Immunization remains the most cost-effective and sustainable health intervention in developing countries. Vaccination programmes for animals had faced the major constraints of inadequacy in the quantity and quality of vaccines obtainable, difficulties in maintaining a cold chain in storage/delivery of vaccines and shortages of qualified veterinary manpower/personnel. Recent advances in vaccine research and development offer ample opportunities for overcoming these constraints. These include “sugar-glass” drying of vaccines to enhance their thermostability, the discovery of more effective adjuvants called immune-stimulating complexes (ISCOMs), and the application of the principle of micro-encapsulation in vaccine production to overcome the difficulties associated with multiple visits by a veterinary personnel as required in routine vaccination schedules. Also nucleic acid vaccines now offer opportunities to vaccinate animals without using the infectious agent/its parts, and to produce one single vaccine for all major diseases. Further, the possibilities of producing plant/edible vaccines, combination vaccines, and the recognition of the great value of the use of mucosal route for vaccination stand out as great promises for more viable animal health and husbandry worldwide. KEY WORDS: Vaccine research & development, Animal diseases prevention/control. Nigerian Veterinary Journal Vol.23(1) 2002: 27-32

A single dose of an ISCOM influenza vaccine induces long-lasting protective immunity against homologous challenge infection but fails to protect Cynomolgus macaques against distant drift variants of influenza A (H3N2) viruses

Since the production of influenza vaccines is complicated by the continuous variation of these viruses, it would be desirable to develop vaccines that induce cross-protective immunity against influenza virus strains that circulate in subsequent winter epidemics. We have recently demonstrated that antibodies induced after vaccination with an immune stimulating complex (ISCOM)-based vaccine exhibited a certain degree of cross-reactivity with other influenza virus strains. In the present study, ISCOM-based vaccines were evaluated retrospectively by testing the protective immunity induced by ISCOM prepared with the membrane glycoproteins of A/Philippines/2/82 against the more recent strain A/Netherlands/18/94 in monkeys with or without a history of prior infection with an A/Philippines/2/82-like virus. It was found that the monkeys immunized with the A/Philippines/2/82 ISCOM were not protected from challenge infection with A/Netherlands/18/94. On the other hand, vaccination of monkeys which experienced a prior infection with an influenza A/Philippines/2/82-like virus, with a single dose of ISCOM vaccine induced long-lasting protective immunity against challenge infection with the homologous virus A/Netherlands/18/94.

Rinderpest virus (RPV) ISCOM vaccine induces protection in cattle against virulent RPV challenge

Rinderpest virus (RPV), a member of genus Morbillivirus in the family Paramyxoviridae, causes an acute and often fatal disease in cattle and other large ruminants. A subunit rinderpest vaccine consisting of an immune-stimulating complex (ISCOM) incorporating the RPV haemaggulutinin (H) protein, was examined for its ability to induce protective immunity in cattle, the natural host of RPV. All of four cattle vaccinated with the ISCOM vaccine survived challenge with virulent virus. Three were solidly protected, showing no clinical signs of infection, while the fourth animal developed only mild and transient symptoms. Virus neutralizing antibodies were produced at a significant level in all vaccinated cattle. These results indicate that this ISCOM vaccine is effective in producing protective immunity in cattle and should be a suitable means of delivering glycoprotein antigens from other morbilliviruses.

Field Studies on Equine Influenza Vaccination Regimes in Thoroughbred Foals and Yearlings

SUMMARYThe purpose of these studies was to examine the response of Thoroughbred foals and yearlings to different influenza vaccines and vaccination regimes. The horses’ antibody levels against haemagglutinin, an established correlate of protection were measured by haemagglutination inhibition. The first study investigated the extent to which maternal antibodies interfered with the humoral response to a subunit vaccine. The findings suggest that repeat vaccination in the face of maternal antibodies may induce tolerance as defined by serological testing. The second study compared the immune response elicited by a subunit immune stimulating complex (ISCOM) vaccine, an inactivated whole virus vaccine and the same product containing equine herpesviruses and equine reoviruses in addition to equine influenza virus. The monovalent vaccine induced a significantly better response than the ISCOM or the multivalent vaccine. The final study demonstrated that the inclusion of an additional booster vaccination, between the second and third vaccination recommended by the vaccine manufacturers and required under the rules of racing in certain countries, is of benefit to young horses. Since these studies were performed, several of the vaccines have been updated with more recent virus strains in line with WHO/OIE recommendations. However, the general principles investigated in the studies remain relevant to these vaccines.

Phage display of epitopes from HIV-1 elicits strong cytolytic responses in vitro and in vivo.

Vaccines based on synthetic peptides representing epitopes recognized by T-helper or cytolytic T-cells have been widely investigated as the basis for effective vaccines. However, their efficacy suffers from a number of important limitations. First, they are poor immunogens and exhibit short life-spans both in serum and within the cell (1). Secondly, the amount of peptide required to bind a threshold number of MHC class II molecules on the surface of an APC is reportedly higher than the minimal amount needed to bind to class II MHC molecules in the intracellular compartment in which the processing of the corresponding parent antigen takes place after internalization (2). Soluble antigens carrying CTL epitopes not only have the same drawbacks, but normally are unable to enter the appropriate intracellular compartment to undergo processing and presentation on class I MHC molecules (3). Thus, delivery systems based on immune-stimulating complexes, liposomes and synthetic lipopeptides have been designed in attempts to circumvent these limitations (1).

A randomized, double blind study in young healthy adults comparing cell mediated and humoral immune responses induced by influenza ISCOM™ vaccines and conventional vaccines

Although current influenza vaccines have been shown to reduce influenza-related morbidity and mortality, there is a desire to develop more efficacious products. Vaccines which can induce CD8 + cytotoxic T cell (CTL) responses in addition to strong antibody responses may be more effective in preventing disease since it has been demonstrated that CTL contribute to protective immunity, even against drift variants of influenza A viruses. The immunogenicity of two types of experimental influenza vaccines, which were based on immune stimulating complexes (ISCOM™), were evaluated and compared with a conventional non-adjuvanted inactivated split virion vaccine, after immunization of human volunteers. In this randomized, double blind study, it was shown that the ISCOM™ vaccines altered the kinetics of the serum antibody response, resulting in more rapid titer rises against the vaccine strains. This accelerated antibody response coincided with enhanced in vitro proliferative T cell responses, which were observed shortly after vaccination. In addition, CTL responses were observed in a higher proportion of the vaccinees receiving an ISCOM™ vaccine, than in vaccinees receiving the conventional influenza vaccine.

Introduction of the haemagglutinin transmembrane region in the influenza virus matrix protein facilitates its incorporation into ISCOM and activation of specific CD8 + cytotoxic T lymphocytes

The gene encoding the influenza virus A matrix (MA) protein was cloned into the bacterial expression vector pMalC with and without the sequence encoding the transmembrane region of the haemagglutinin (HA). With the resulting recombinant proteins, immune stimulating complexes (ISCOM) were prepared. The MA protein with the hydrophobic anchor region (rMAHA) associated more efficiently with ISCOM than the unmodified MA protein (rMA). A B-lymphoblastoid cell line (B-LCL) was lysed by an autologous CD8 + cytotoxic T lymphocyte (CTL) clone specific for the MA protein after incubation with rMAHA–ISCOM but not after incubation with rMA, rMAHA, rMA–ISCOM or empty ISCOM. The B-LCL was also lysed by the CTL clone after incubation with empty ISCOM mixed with the respective MA proteins. Incubation of ISCOM with the rMAHA protein proved to be the most efficient in this respect. Addition of the proteasome inhibitors lactacystin or clasto-lactacystin β-lactone to the B-LCL incubated with rMAHA–ISCOM or the MA proteins mixed with empty ISCOM dramatically decreased the lysis by the CD8 + CTL clone. These results indicate that the addition of a hydrophobic anchor to hydrophilic proteins in combination with ISCOM facilitates their entry in the MHC class I processing and presentation pathway. This may be an attractive approach for the development of subunit vaccines aiming at the induction of CTL-mediated immunity.

Mucosal immunization with experimental feline immunodeficiency virus (FIV) vaccines induces both antibody and T cell responses but does not protect against rectal FIV challenge

Feline immunodeficiency virus (FIV) is a natural lentiviral pathogen of cats which can be experimentally transmitted via rectal and vaginal routes — the major routes of human immunodeficiency virus type 1 transmission in man. An important objective for lentiviral research is the development of vaccine strategies which generate good mucosal immune responses capable of giving protection from a mucosal virus challenge. The experimental vaccines employed in this study were based on (a) a peptide from the third variable region of the FIV envelope glycoprotein and (b) fixed whole FIV, Glasgow-8 strain. Adjuvants used were Quil A and cholera toxin for mucosal administration and incomplete Freund’s adjuvant and immune stimulating complexes for subcutaneous injection. Mucosal immunization was given by rectal and intranasal routes. Both antibody and proliferative responses were elicited by mucosal immunization and cholera toxin was found to be a good mucosal adjuvant. The addition of a lipo thioester to the FIV peptide improved IgG and IgA responses upon parenteral administration. However, no protection from a rectal FIV challenge was achieved.

Priming with Chlamydia trachomatis major outer membrane protein (MOMP) DNA followed by MOMP ISCOM boosting enhances protection and is associated with increased immunoglobulin A and Th1 cellular immune responses.

We previously reported that DNA vaccination was able to elicit cellular immune responses and partial protection against Chlamydia trachomatis infection. However, DNA immunization alone did not generate immune responses or protection as great as that induced by using live organisms. In this study, we evaluated the immunologic effects of a combinational vaccination approach using C. trachomatis mouse pneumonitis (MoPn) major outer membrane protein (MOMP) DNA priming followed by boosting with immune-stimulating complexes (ISCOM) of MOMP protein (MOMP ISCOM) for protection of BALB/c mice against MoPn lung infection. Substantially better protection to challenge infection was observed in mice given combinational vaccination compared with mice given MOMP ISCOM immunization alone, and the protection approximated that induced by live organisms. Enhanced protection was correlated with stronger delayed-type hypersensitivity, higher levels of gamma interferon production, and increased immunoglobulin A antibody responses in lung homogenates. The results indicate that DNA priming followed by ISCOM protein boosting may be useful in designing a fully protective chlamydial vaccine.

Hydration of lipid films with an aqueous solution of Quil A: a simple method for the preparation of immune-stimulating complexes

Immune-stimulating complexes (ISCOMs) are stable colloidal complexes of the adjuvant Quil A, cholesterol and phospholipid, which are effective carriers for subunit vaccines. The techniques currently available for the preparation of ISCOMs from the constituent components are rather complex and are based on either centrifugation or dialysis. This note reports a new simple procedure for the preparation of ISCOM matrices based on hydration of a cholesterol/phospholipid film with an aqueous solution of Quil A. It is demonstrated that ISCOM matrices do not form in the absence of phospholipid when prepared by this method. Further, the ratio by weight of phospholipid to either cholesterol or Quil A must be greater than that required for preparation by either dialysis or centrifugation. Photon correlation spectroscopy, negative stain transmission electron microscopy and centrifugation through a sucrose gradient demonstrate that ISCOM matrices can be prepared from cholesterol/lipid films by hydration with an aqueous solution of Quil A when the ratio of phospholipid:cholesterol:Quil A by weight is 6:1:4, respectively. Lower ratios of phospholipid:cholesterol reduce the efficiency of ISCOM formation while higher ratios produce systems containing a mixture of ISCOMs together with liposomes.

Advances in vaccine adjuvants

Currently, aluminum salts and MF59 are the only vaccine adjuvants approved for human use. With the development of new-generation vaccines (including recombinant subunit and mucosal vaccines) that are less immunogenic, the search for more potent vaccine adjuvants has intensified. Of the novel compounds recently evaluated in human trials, immunostimulatory molecules such as the lipopolysaccharide derived MPL and the saponin derivative QS21 appear most promising, although doubts have been raised as to their safety in humans. Preclinical work with particulate adjuvants, such as the MF59 microemulsion and lipid-particle immune-stimulating complexes (Iscoms), suggest that these molecules are also potent elicitors of humoral and cellular immune responses. In addition, preclinical data on CpG oligonucleotides appear to be encouraging, particularly with respect to their ability to selectively manipulate immune responses. While all these adjuvants show promise, further work is needed to better define the mechanisms of adjuvant action. Ultimately, the development of more potent adjuvants may allow vaccines to be used as therapeutic, rather than prophylactic, agents.