Immune-stimulating Complexes Research Articles

Serum antibody profile and reproductive performance during two consecutive pregnancies of cows naturally infected with Neospora caninum

The objective of this study was to record how the antibody levels change over time during pregnancy in dairy cows naturally infected with the protozoan parasite Neospora caninum, and relate this to the reproductive performance. Eighteen cows with antibodies to N. caninum were serum sampled monthly during their first pregnancy and 13 of them were also followed for a second pregnancy. In all, five pregnancies ended in abortion and two in stillbirth. Antibodies to N. caninum in serum were demonstrated by immune stimulating complex enzyme-linked immunosorbent assay (iscom ELISA). The N. caninum antibody titres remained well above the 1 : 100 cut-off limit for the test used during 2 years in all cows. In the non-aborting cows, mean values of antibody titres to N. caninum rose 1.5–2.5 dilution steps to reach a plateau 4–5 months before parturition, and thereafter decreased from 2 months before parturition. These changes were statistically significant ( p < 0.001). The same pattern was seen in the aborting cows. The consistent pattern of rise in antibody titres observed during both pregnancies in all cows indicated a reactivation rather than a reinfection of the parasite at mid-gestation.

Efforts to broaden HIV-1-specific immunity by boosting with heterologous peptides or envelope protein and the influence of prior exposure to virus.

In two previous studies, we have demonstrated the successful protection of human immunodeficiency virus type 1 (HIV-1)-vaccinated rhesus macaques from challenge with SHIV(SF13) with envelop immunogens derived from the closely related HIV-1(SF2) strain. Here we report on two follow-up studies in which we aimed to broaden immunity in order to elicit protection from a more diverse heterologous challenge with SHIV(SF33). In the first study, animals were boosted once with HIV-1(SF33) V2 and V3 peptides that were cross-linked to influenza immune-stimulating complexes (ISCOMs). In the second study, monkeys were boosted twice at 12-week intervals, using a heterologous recombinant gp120 derived from HIV-1(SF33) that was either incorporated into ISCOMs or mixed with the MF59 adjuvant. In both studies, the animals were challenged with 50 monkey infectious doses of SHIV(SF33) 4 weeks after the final boost. All controls became readily infected with the heterologous challenge virus SHIV(SF33). Neither boosting with heterologous SF33 peptides or gp120 afforded protection from infection to SF2-vaccinated animals that had previously resisted SHIV(SF13) challenge. These results demonstrate the importance of developing vaccine strategies that are capable of generating broad immune responses early in the immunization protocol. Furthermore, these findings may illustrate the potential pitfalls of early antigenic sin.

Immunogenicity of various presentation forms of PorA outer membrane protein of Neisseria meningitidis in mice

In this study we compare different vaccine formulations containing meningococcal PorA outer membrane protein; purified PorA, outer membrane vesicles (OMV) and immune-stimulating complexes (iscom). Bactericidal antibodies could be generated by the OMV and iscom formulation but not with purified PorA using either AlPO 4 or Quil-A as adjuvant. OMV and iscom formulations revealed similar immunogenicity when tested in a dose response manner, with respect to bactericidal as well as OMV-binding antibodies. The anti-OMV IgG subclass response induced by PorA in OMV formulation was found in all subclasses IgG1, IgG2a, IgG2b, IgG3. OMP–iscoms induced very high IgG1 anti-OMV antibodies but almost no IgG3 response. Also, OMP–iscoms appeared to be a potent inducer of antibodies directed against linear peptides corresponding to surface exposed loops of PorA. In addition, iscoms as well as purified PorA with Quil-A as adjuvant (but not with AlPO 4) induced high levels of antibodies against purified PorA. In summary, in addition to the OMV formulation, only iscoms containing PorA are able to generate an anamnestic and bactericidal antibody response.

Immune-Stimulating Complexes Induce an IL-12-Dependent Cascade of Innate Immune Responses

The development of subunit vaccines requires the use of adjuvants that act by stimulating components of the innate immune response. Immune-stimulating complexes (ISCOMS) containing the saponin adjuvant Quil A are potential vaccine vectors that induce a wide range of Ag-specific responses in vivo encompassing both humoral and CD4 and CD8 cell-mediated immune responses. ISCOMS are active by both parenteral and mucosal routes, but the basis for their adjuvant properties is unknown. Here we have investigated the ability of ISCOMS to recruit and activate innate immune responses as measured in peritoneal exudate cells. The i.p. injection of ISCOMS induced intense local inflammation, with early recruitment of neutrophils and mast cells followed by macrophages, dendritic cells, and lymphocytes. Many of the recruited cells had phenotypic evidence of activation and secreted a number of inflammatory mediators, including nitric oxide, reactive oxygen intermediates, IL-1, IL-6, IL-12, and IFN-gamma. Of the factors that we investigated further only IL-12 appeared to be essential for the immunogenicity of ISCOMS, as IL-6- and inducible nitric oxide synthase knockout (KO) mice developed normal immune responses to OVA in ISCOMS, whereas these responses were markedly reduced in IL-12KO mice. The recruitment of peritoneal exudate cells following an injection of ISCOMS was impaired in IL-12KO mice, indicating a role for IL-12 in establishing the proinflammatory cascade. Thus, ISCOMS prime Ag-specific immune responses at least in part by activating IL-12-dependent aspects of the innate immune system.

ISCOM vaccine induced protection against a lethal challenge with a human H5N1 influenza virus

Recently avian influenza A viruses of the H5N1 subtype were shown to infect humans in the Hong Kong area, resulting in the death of six people. Although these viruses did not efficiently spread amongst humans, these events illustrated that influenza viruses of subtypes not previously detected in humans could be at the basis of a new pandemic. In the light of this pandemic threat we evaluated and compared the efficacy of a classical non-adjuvanted subunit vaccine and a vaccine based on immune stimulating complexes (ISCOM) prepared with the membrane glycoproteins of the human influenza virus A/Hong Kong/156/97 (H5N1) to protect roosters against a lethal challenge with this virus. The ISCOM vaccine induced protective immunity against the challenge infection whereas the non-adjuvanted subunit vaccine proved to be poorly immunogenic and failed to induce protection in this model.

Production of a highly immunogenic subunit ISCOM vaccine against Bovine Viral Diarrhea Virus

Bovine Viral Diarrhea Virus (BVDV) is a major pathogen of cattle in most countries. The main reservoir of virus in herds are BVDV persistently infected animals, which arise as a result of infection of the bovine fetus early in gestation. The spread of virus to the unborn fetus may be prevented by vaccination of the dam. We describe in this report the production and initial testing of an inactivated subunit vaccine against BVDV. The vaccine is based on production of antigen in primary bovine cell cultures, extraction of antigens from infected cells with detergent, chromatographic purification, concentration, and insertion of antigens into immune stimulating complexes (ISCOMs). Vaccines based on two different Danish strains of BVDV were injected into calves and the antisera produced were tested for neutralising activity against a panel of Danish BVDV strains. The two vaccines induced different neutralisation responses, which seem to partly complement each other. The implication of these observations for successful vaccination against BVDV is discussed.

Oral vaccination with immune stimulating complexes

There is a need for non-living adjuvant vectors which will induce a full range of local and systemic immune responses to orally administered purified antigens. Here we describe our experience with lipophilic immune stimulating complexes (ISCOMS) containing the saponin adjuvant Quil A. When given orally, ISCOMS containing the model protein antigen ovalbumin (OVA) induce a wide range of systemic immune responses, including Th1 and Th2 CD4 dependent activity, class I MHC restricted cytotoxic T-cell responses and local production of secretory IgA antibodies. More recent results indicate that ISCOMS may act partly by enhancing the uptake of protein from the gut. In addition, intraperitoneal injection of ISCOMS recruits and activates many components of the innate immune system, including neutrophils, macrophages, and dendritic cells. In parallel, there is increased production of nitric oxide (NO), reactive oxygen intermediates (ROI), interleukins (IL) 1, 6, 12, and γ interferon ( γIFN). Of these factors, only IL12 is essential for the immunogenicity of ISCOMS in vivo, as mucosal and systemic responses to ISCOMS are reduced in IL12KO mice, but not in IL4KO, IL6KO, inducible NO synthase (iNOS) KO, or γIFN receptor KO mice. We propose that ISCOMS act by targetting antigen and adjuvant to macrophages and/or dendritic cells. This pathway may be amenable to exploitation for vaccine development, especially if combined with another vector with a different mucosal adjuvant profile, such as cholera toxin.

ISCOM: A delivery system for neonates and for mucosal administration

This chapter describes Immune stimulating complex (ISCOM). ISCOM is a delivery system for antigen and adjuvant together in the same particle. The unique component of ISCOM is a mixture of Quillaja saponins extracted from the bark of the tree Quillaja saponaria Molina. The ISCOM is created to make antigens optimally immunogenic by (1) presenting the antigens in a physically immunogenic form—that is, several copies in a submicroscopic particle to resemble that of an infectious agent, (2) optimizing the targeting of antigen and adjuvant after both mucosal and parenteral modes of administration to lymphatic organs and cells by enclosure of adjuvant and antigen in a stable, uniform particle, and (3) optimizing the immunomodulatory capacity both for neonate and adult immune system by presenting the adjuvant and antigen components in the same particle, reducing the amount of antigen and adjuvant required for efficiently enhancing the immune response.

Effects of maternal antibodies on protection and development of antibody responses to human rotavirus in gnotobiotic pigs.

Although maternal antibodies can protect against infectious disease in infancy, they can also suppress active immune responses. The effects of circulating maternal antibodies, with and without colostrum and milk antibodies, on passive protection and active immunity to human rotavirus (HRV) were examined in gnotobiotic pigs. Pigs received intraperitoneal injections of high-titer serum (immune pigs [groups 1 and 2]) from immunized sows, low-titer serum from naturally infected sows (control pigs [groups 3 and 4]), or no serum (group 5). Immune or control colostrum and milk were added to the diet of groups 2 and 4, respectively. After inoculation (3 to 5 days of age) and challenge (postinoculation day [PID] 21) with virulent HRV, the effects of maternal antibodies on protection (from diarrhea and virus shedding), and on active antibody responses (measured by quantitation of antibody-secreting cells [ASC] in intestinal and systemic lymphoid tissues by ELISPOT) were evaluated. Groups 1 and 2 had significantly less diarrhea and virus shedding after inoculation but higher rates of diarrhea and virus shedding after challenge than did groups 3 and 5. Group 1 and 2 pigs had significantly fewer immunoglobulin A (IgA) ASC in intestinal tissues at PID 21 and at postchallenge day (PCD) 7 compared to group 5. Significantly fewer IgG ASC were present in the intestines of group 2 pigs at PID 21 and PCD 7 compared to group 5. There was a trend towards fewer ASC in intestinal tissues of group 2 than group 1, from PID 21 on, with significantly fewer IgA ASC at PCD 7. IgG ASC in the duodenum and mesenteric lymph nodes of group 3 and 4 pigs were significantly fewer than in group 5 at PCD 7. These decreases in ASC emphasize the role of passive antibodies in impairing induction of ASC rather than in merely suppressing the function of differentiated B cells. To be successful, vaccines intended for populations with high titers of maternal antibodies (infants in developing countries) may require higher titers of virus, multiple doses, or improved delivery systems, such as the use of microencapsulation or immune stimulating complexes, to overcome the suppressive effects of maternal antibodies.

Enteric viral infections of pigs and strategies for induction of mucosal immunity

This chapter discusses the enteric viral infections of pigs and strategies for the induction of mucosal immunity. Enteropathogenic viruses—such as transmissible gastroenteritis virus (TGEV) and rotavirus—replicate and induce lesions only in the gastrointestinal tract. The susceptible target cell is the villous enterocyte. Thus, active immunity against enteropathogenic viral infections depends on stimulation of local immune responses within the intestine. To date, only limited success has been achieved in the development of oral vaccines to prevent neonatal viral diarrheas, and commercial vaccines show limited efficacy in the field. Currently, only oral vaccines containing live replicating organisms have been highly effective in inducing mucosal immune responses, especially secretory, (S)IgA antibodies. The oral administration of soluble or killed antigens generally induces immunity of short duration or even systemic tolerance. Whether the problems encountered with oral administration of soluble protein antigens can be overcome by the use of improved mucosal adjuvants (such as muranyl dipeptide, immune stimulating complexes [ISCOMs], cholera or Escherichia coli enterotoxins, avridine, proteosomes, and cytokines] or new and novel delivery systems (liposomes, live recombinant vectors, microspheres, DNA plasmids, virus-like particles) requires further investigation, and specific examples are given in this chapter.

Role of mucosal adjuvants in mucosal immunization

Although the bacterial enterotoxins cholera toxin (CT) and Eschericia coli heat labile toxin (LT) are the most potent mucosal adjuvants currently known, their enterotoxicity may preclude their use in humans. Studies conducted with genetically engineered mutant molecules demonstrate that target cell binding is necessary but not sufficient for adjuvant activity and suggest that some degree of toxin activity is required for oral immunization. Although toxin activity may not be required for intranasal adjuvanticity, further studies are needed. Several studies have demonstrated that CT and LT can enhance the expression of costimulatory molecules on antigen-presenting cells and thus indirectly influence subsequent T-cell responses. Direct effects on B and T cells may also be important. Although they have not been as extensively studied as CT and LT, compounds such as liposomes, immune-stimulating complexes, microparticles and bacterial membrane proteins have also been shown to have mucosal adjuvant activity.

Antigen detection in vivo after immunization with different presentation forms of rabies virus antigen, II. Cellular, but not humoral, systemic immune responses against rabies virus immune-stimulating complexes are macrophage dependent.

In this paper we describe the effect of depletion of splenic macrophages on the uptake, and immune response against, different formulations of rabies virus antigen. Splenic macrophages were removed by intravenous injection with clodronate liposomes. beta-propiolacton inactivated rabies virus (RV-BPL) and immune-stimulating complexes (iscom) containing these antigens were given to macrophage-depleted and control mice. In the absence of phagocytic cells in the spleen, antigen is still trapped in the red pulp and to a lesser extent in the peri-arteriolar lymphocyte sheaths (PALS) for both antigen formulations. The localization pattern in the main area of immune response induction, namely the follicles, was unaltered after macrophage depletion. Functionally, the depletion of splenic and liver macrophages had no influence on the induction of specific antibody responses in both RV-BPL or RV-iscom immunized mice, even though the latter presentation form was clearly associated with specific localization in the marginal metallophillic macrophages. In RV-BPL immunized mice, macrophage depletion had no influence on proliferative T-cell responses. However, macrophage-depleted mice that were immunized with RV-iscom showed a significant decrease in proliferative T-cell responses. These results confirm existing ideas on the spleen as a physical filter rather than an induction site for humoral responses and shed new light on the efficient role of iscoms as antigen-presenting moieties in relation to their specific in vivo localization patterns and partial macrophage dependency.

Studies on experimental adjuvanted influenza vaccines: comparison of immune stimulating complexes (Iscoms™) and oil-in-water vaccines

Detergent-disrupted influenza virus vaccines, formulated as Iscoms™, or oil-in-water (o/w) emulsions, were administered parenterally to mice and evaluated for immunogenicity and protective efficacy. Both formulations enhanced both primary and secondary serum antibody responses. The magnitude of these responses with o/w emulsions was further enhanced by the addition of the non-ionic block copolymer L121 in the emulsion. Four weeks after primary immunization, mice were challenged by exposure to an aerosol containing infectious virus. Resistance to challenge in terms of survival rate and weight change correlated well with serum antibody titre for all formulations. Two major differences were observed between the adjuvant formulations. Iscom™ vaccines, formulated with Quil-A or the less toxic Quillaia saponin preparation Iscoprep™ 703, induced specific cytotoxic T-lymphocyte responses, whereas the o/w-based vaccines did not. In addition, dose-site reactivity studies in sheep showed that Iscom™ vaccines were less reactive than o/w-based vaccines, the degree of reactivity of the latter increasing sharply with increasing L121 concentration. On the basis of these studies, Iscoms™ were chosen for development as a potential adjuvant for human influenza vaccines.

Respiratory syncytial virus vaccines.

Respiratory syncytial virus (RSV) is the most important cause of viral lower respiratory tract illness (LRI) in infants and children worldwide and causes significant LRI in the elderly and in immunocompromised patients. The goal of RSV vaccination is to prevent serious RSV-associated LRI. There are several obstacles to the development of successful RSV vaccines, including the need to immunize very young infants, who may respond inadequately to vaccination; the existence of two antigenically distinct RSV groups, A and B; and the history of disease enhancement following administration of a formalin-inactivated vaccine. It is likely that more than one type of vaccine will be needed to prevent RSV LRI in the various populations at risk. Although vector delivery systems, synthetic peptide, and immune-stimulating complex vaccines have been evaluated in animal models, only the purified F protein (PFP) subunit vaccines and live attenuated vaccines have been evaluated in recent clinical trials. PFP-2 appears to be a promising vaccine for the elderly and for RSV-seropositive children with underlying pulmonary disease, whereas live cold-passaged (cp), temperature-sensitive (ts) RSV vaccines (denoted cpts vaccines) would most probably be useful in young infants. The availability of cDNA technology should allow further refinement of existing live attenuated cpts candidate vaccines to produce engineered vaccines that are satisfactorily attenuated, immunogenic, and phenotypically stable.

Immune stimulating complexes as mucosal vaccines.

There is a need for non-living adjuvant vectors that will allow a full range of local and systemic immune responses to orally administered purified antigens. Here we describe our experience with lipophilic immune-stimulating complexes (ISCOMs) containing the saponin adjuvant Quil A. When given orally, ISCOMs containing the model protein antigen ovalbumin (OVA) induce a wide range of systemic immune responses, including Th1 and Th2 CD4-dependent activity, serum IgG antibodies and class I MHC-restricted cytotoxic T cell responses. In addition, there is local production of secretory IgA antibodies in the intestine itself, as well as priming of CD4 and CD8 T cell responses in the draining lymphoid tissues. Preliminary results indicate that the mucosal adjuvant properties of ISCOMs may reflect their ability to deliver antigen combined with the pro-inflammatory properties of Quil A in a particulate form. Of the many inflammatory mediators induced, interleukin-12, derived from dendritic cells and/or macrophages, appears to be of central importance. These results indicate that ISCOMs may prove to be useful mucosal vaccine vectors with functions which are distinct from existing vectors of this type.

Induction of a Th1 Immune Response and Simultaneous Lack of Activation of a Th2 Response Are Required for Generation of Immunity to Leishmaniasis

Experimental systems based on immunization with plasmid DNA or immune-stimulating complexes were used to delineate the requirements for generation of protective immunity against murine leishmaniasis. Vaccination with plasmid DNA encoding the host-protective Leishmania major parasite surface Ag-2 primed for an essentially exclusive Th1 response that protected mice against L. major infection. In contrast, parasite surface Ag-2 in immune-stimulating complexes generated an immune response with mixed Th1-like and Th2-like properties that was not protective despite the activation of large numbers of CD4+ T cells secreting IFN-gamma. These results indicate that a Th1 response is sufficient to protect against cutaneous leishmaniasis, but the induction of a simultaneous Th2 response abrogates the Th1 effector function. DNA vaccines may therefore have an advantage for diseases in which protection depends on the induction of Th1 responses.

The role of type-1 and type-2 T-helper immune responses in HIV-1 vaccine protection.

The dichotomy of type-1 and type-2 T-helper (Th) immune responses is thought to be an obstacle to develop Human immunodeficiency virus-type- (HIV-1) vaccines capable of inducing effective cellular as well as humoral immune responses. Macaca mulatta were immunized using two different HIV-1sf2 envelope vaccine strategies, based on either immune-stimulating complexes (ISCOM) or chimeric Fowlpox (FP) vaccines. One month following the third immunization all animals were heterologously challenged with simian/human immunodeficiency virus (SHIVsf13). Vaccinated monkeys, which were protected had the highest levels of both type-1 and type-2 HIV-1 specific T-helper cell (Th) responses in addition to the highest homologous and heterogenous virus neutralizing antibodies. To determine how long Th responses persisted and if they correlated with protection, animals were rechallenged after waiting for four months without re-boosting. Macaques which maintained the highest gp120-specific type-1 (IFN-gamma) responses were protected, while there was evidence of viral clearance in two others. These findings demonstrate the importance of both or mixed type-1 and type-2 Th responses in HIV-1 vaccine induced immunity while suggesting a possible role of persistent type-1 responses in maintaining protective immunity over time.

Preservation of mucosal and systemic adjuvant properties of ISCOMS in the absence of functional interleukin-4 or interferon-gamma.

Adjuvants are a critical component of non-viable vaccine vectors, particularly for those to be used via mucosal routes. Although most adjuvants act by inducing local inflammatory responses, the molecular basis of many of these effects is unclear. Here we have investigated whether interleukin-4 (IL-4) and interferon-gamma (IFN-gamma) are required for the induction of local and systemic immune responses by oral and parenteral administration of ovalbumin (OVA) in immune stimulating complexes (ISCOMS), a potent mucosal adjuvant vector. Our results show that after oral or systemic immunization with OVA ISCOMS, IL-4 knockout (IL4KO) and IFN-gamma receptor knockout (IFN-gamma RKO) mice develop an entirely normal range of immune responses including delayed-type hypersensitivity (DTH), serum immunoglobulin G (IgG) antibodies, T-cell proliferation and cytokine production, class I major histocompatibility complex (MHC)-restricted cytotoxic T lymphocyte (CTL) activity and intestinal IgA antibodies. These responses were of a similar magnitude to those found in the wild-type mice, indicating that the immunogenicity of ISCOMS is not influenced by the presence of IL-4 or IFN-gamma and emphasizing the potential of ISCOMS as widely applicable mucosal adjuvants.

Intranasal immunization of mice with herpes simplex virus type 2 recombinant gD2: the effect of adjuvants on mucosal and serum antibody responses

Mucosal immunization offers the potential for inducing IgA antibody responses in the vagina, the site of infection for many viruses, including herpes simplex type 2 (HSV-2). To investigate this possibility, mice were immunized intranasally with 10 micrograms glycoprotein D2 (gD2) from HSV combined with a series of adjuvants of proven efficacy; the oil in water emulsion MF59, poly(D,L-lactide-co-glycolide) microparticles (PLG) (encapsulated or co-administered), immune-stimulating complexes (iscoms) (incorporated or co-administered with iscomatrix) and the genetically detoxified enterotoxin from Escherichia coli, LT-K63. Encapsulation of gD2 into PLG microparticles, incorporation of gD2 into iscoms and co-administration of gD2 with LT-K63 induced mucosal IgA antibody responses (nasal wash, saliva and vaginal wash) which were greater than those induced by intramuscular administration of gD2 with MF59. Intranasal immunization with these formulations also induced substantial levels of serum IgG and neutralizing antibodies. These studies demonstrated that intranasal immunization with potent adjuvants is an effective means to induce mucosal antibody responses, even in the lower genital tract.

Feline immunodeficiency virus subunit vaccines that induce virus neutralising antibodies but no protection against challenge infection

Three experimental vaccines against feline immunodeficiency virus (FIV), all based on viral antigens presented via immune stimulating complexes (iscoms), were tested for their capacity to induce protection in cats from FIV infection. The respective vaccines consisted of FIV propagated in Crandell feline kidney (CrFK) cells (FIV-iscoms); FIV-iscoms spiked with recombinant vaccinia virus expressed FIV envelope glycoprotein incorporated into iscoms (FIV-iscoms+vGR657x15-iscoms) and vGR657x15-iscoms spiked with recombinant FIV Gag protein incorporated into iscoms (vGR657x15-iscoms+FIV-Gag-iscoms). Simian immunodeficiency virus envelope glycoprotein incorporated into iscoms, iscoms prepared with uninfected CrFK cells, and PBS served as controls. All cats vaccinated with vGR657x15-iscoms combined with FIV-iscoms or FIV-Gag-iscoms developed Env-specific plasma antibody responses. These antibodies neutralised FIV infection in CrFK cells, but failed to neutralise FIV infection in primary feline thymocytes. FIV-iscoms induced poor Env-specific responses and only one out of six cats developed antibodies that neutralised FIV in the CrFK cell based assay. Four weeks after challenge all cats proved to be infected, showing that none of the vaccine preparations provided protection. In contrast, 2 weeks after infection, virus infected peripheral blood mononuclear cells were only observed in cats vaccinated with FIV-iscoms+vGR657x15-iscoms or CrFK-iscoms and to a lesser extent in cats vaccinated with FIV-iscoms and vGR657x15-iscoms+FIV-Gag-iscoms, but not in cats vaccinated with SIV-iscoms or PBS. The differences found in cell associated virus loads amongst the respective groups are discussed in the light of antibody mediated enhancement of infectivity and protective effects provided by Gag-specific T cell responses.