ISCOM Matrix Research Articles

Refinement of the Equine Influenza model: the benefits of individual nebulisation for experimental infection

Equine influenza (EI) is a major respiratory disease of the horse induced by the highly contagious equine influenza virus (EIV). Worldwide, diverse EI vaccines are available commercially. The EIV strain, adjuvant composition and vaccine technology differs greatly. The use of the same EI vaccine throughout the life of a horse is unlikely, due to change of ownership, veterinary practitioner, location and/or vaccine availability. The compatibility of different EI vaccines has rarely been studied but is of great importance as immunisation failure could favour EIV dissemination. Study aims: To determine the impact of mixed EI vaccination on protective immunity to EIV in horses and to measure herd immunity in Hong Kong (HK). Methods: HK has approximately 1,250 race horses with around 450 new horses arriving each year. Imported racehorses must have been vaccinated with an approved EI vaccine, irrespective of their point of origin. All horses are given a new primary course on arrival with a unique ISCOM matrix adjuvanted EI vaccine and are vaccinated twice a year thereafter. EI-specific antibody titres were measured by single radial haemolysis (SRH) assay (A/eq/South Africa/4/03 EIV strain) in 1050+ archived sera collected between 2010 and 2014 from 129+ horses. Objectives: 1) to determine the SRH antibody titre in serums from HK resident horses (i.e. in HK for at least 1.5 year), 2) to evaluate the antibody titre on arrival at HK in relation to their country of origin, 3) to measure the effect of post-arrival mandatory EI primary vaccination and 4) to compare this response with antibody levels measured in HK resident horses. Results: Preliminary results indicates that the average yearly SRH antibody titre for HK resident horses was 113±34mm2 (n1⁄4467), with only 15% of horses below the clinical protection threshold (i.e. 85mm2) and less than 3% of horses below 50mm2. A significant increase was detectable (p-value<0.00004) after each immunisation boost (maximum average titre of 128±42mm2 in October, n1⁄455). The average SRH antibody titre in the week following importation to HK (and prior the mandatory EI vaccination in HK) was 180±40mm2 (n1⁄479), with results in horses originating in Australia significantly higher (207±36mm2, n1⁄410, p-value 1⁄4 0.0281) than horses from New Zealand (171±34mm2, n1⁄442), Great Britain (168±38mm2, n1⁄417) or Ireland (172±31mm2, n1⁄43). SRH antibody titres at the time of importation were maintained for around 75 days (Figure 1) due

Immune response and functional role of antibodies raised in heifers against a Staphylococcus aureus CP5 lysate and recombinant antigens vaccine formulated with Iscom Matrix adjuvant

Staphylococcus aureus is the most frequently isolated pathogen from bovine intramammary infections worldwide. Commercially available vaccines for mastitis control are composed either of S. aureus lysates or inactivated whole-cells formulated with traditional adjuvants. We recently showed the ability of a S. aureus CP5 lysate vaccine adjuvanted with Iscom Matrix to generate a longer lasting specific antibody response in blood and milk, with improved opsonic capacity, compared with a S. aureus CP5 whole-cell formulation. The aim of the present study was to obtain an experimental immunogen composed of lysed cells of a CP5 S. aureus strain supplemented with recombinant clumping factor A, fibronectin binding protein A and β-toxin formulated with Iscom Matrix, characterize the immune response generated when immunizing pregnant heifers and assess the functional role of antibodies raised against this immunogen in experimental models. Both a lysate vaccine and a lysate+recombinant antigens vaccine elicited antibodies that promoted neutrophil phagocytosis and inhibited internalization into mammary epithelial cells, in vitro. Incorporation of defined antigenic molecules to the lysate formulation elicited a strong specific humoral immune response against both lysate and recombinant antigens and was associated with higher expression of regulatory and pro-inflammatory cytokines. In addition, antibodies were efficient for blocking S. aureus binding to bovine fibrinogen and fibronectin, and neutralizing β-toxin effect in vitro, placing these antigens as candidates to be included in a formulation directed to prevent staphylococcal bovine mastitis.

Functional role of antibodies generated in heifers through immunization with Staphylococcus aureus vaccines in invasion and phagocytosis assays.

A successful Staphylococcus aureus vaccine should elicit a long-term antibody response that prevents establishment of the infection. The aim of the present study was to evaluate the functional role of antibodies raised against different S. aureus CP5 vaccines in invasion to bovine mammary epithelial cells (MAC-T) and phagocytosis by bovine milk macrophages in vitro. Sera and whey from cows immunized with a whole-cell S. aureus CP5 vaccine adjuvanted with Al(OH)3 or with ISCOM Matrix, significantly reduced internalization of S. aureus in MAC-T cells without significant differences between both groups. The effect of antibodies generated by a S. aureus whole-cell and a lysate vaccine formulated with ISCOM Matrix was also evaluated. Sera and whey from both immunized groups significantly reduced S. aureus internalization in MAC-T cells without significant differences between both groups. Whey antibodies against whole-cell and lysate vaccines were also able to inhibit internalization in MAC-T cells of a heterologous S. aureus strain. In addition, sera from animals vaccinated with S. aureus lysate or bacterin promoted milk macrophage phagocytosis. These results provide an insight into the potential mechanisms by which these vaccines can afford protection to the mammary gland against S. aureus intramammary infection.

Comparison of multiple adjuvants on the stability and immunogenicity of a clade C HIV-1 gp140 trimer

Immunogens based on the human immunodeficiency virus type-1 (HIV-1) Envelope (Env) glycoprotein have to date failed to elicit potent and broadly neutralizing antibodies against diverse HIV-1 strains. An understudied area in the development of HIV-1 Env-based vaccines is the impact of various adjuvants on the stability of the Env immunogen and the magnitude of the induced humoral immune response. We hypothesize that optimal adjuvants for HIV-1 gp140 Env trimers will be those with high potency but also those that preserve structural integrity of the immunogen and those that have a straightforward path to clinical testing. In this report, we systematically evaluate the impact of 12 adjuvants on the stability and immunogenicity of a clade C (CZA97.012) HIV-1 gp140 trimer in guinea pigs and a subset in non-human primates. Oil-in-water emulsions (GLA-emulsion, Ribi, Emulsigen) resulted in partial aggregation and loss of structural integrity of the gp140 trimer. In contrast, alum (GLA-alum, Adju-Phos, Alhydrogel), TLR (GLA-aqueous, CpG, MPLA), ISCOM (Matrix M) and liposomal (GLA-liposomes, virosomes) adjuvants appeared to preserve trimer integrity as measured by size exclusion chromatography. However, multiple classes of adjuvants similarly augmented Env-specific binding and neutralizing antibody responses in guinea pigs and non-human primates.

Immune response of heifers against a Staphylococcus aureus CP5 whole cell and lysate vaccine formulated with ISCOM Matrix adjuvant

Staphylococcus aureus is the most frequently isolated pathogen from bovine intramammary infections worldwide. Commercially available vaccines for mastitis control are composed either of S. aureus lysates or whole-cells formulated with traditional adjuvants. We recently showed the ability of a S. aureus CP5 whole-cell vaccine adjuvanted with ISCOM Matrix to increase specific antibodies production in blood and milk, improving opsonic capacity, compared with the same vaccine formulated with Al(OH)3. However, there is no information about the use of ISCOM Matrix for the formulation of bacterial lysates. The aim of this study was to characterize the innate and humoral immune responses induced by a S. aureus CP5 whole-cell or lysate vaccine, formulated with ISCOM Matrix after immunization of pregnant heifers. Both immunogens stimulated strong humoral immune responses in blood and milk, raising antibodies that increased opsonic capacity. Lysate formulation generated a higher and longer lasting antibody titer and stimulated a higher expression of regulatory and pro-inflammatory cytokines compared with the whole-cell vaccine.

Iscom matrix enhances the immunogenicity of hepatitis B virus surface antigen containing Pres epitopes

Objective To investigate the effect of Iscom matrix on the immunogenicity of recombinant hepatitis B surface antigen containing PreS epitopes(SS1S2). Methods SS1S2+ Al(OH)3 and SS1S2+Iscom vaccines were made by combining purified SS1S2 antigen with Al( OH)3 adjuvant or Iscom matrix.Groups of BALB/c mice were injected i. m or s. c by either of the two vaccines at day 0 and day 14. Half of the mice were sacrificed and sera were taken and spleen cells separated from the mice 14 days after each injection. Anti-S, anti-PreS1, and anti-PreS2 antibody titers were measured, and total IgG1 and IgG2a titers were further detected for each serum sample. IFN-γ ELISPOT assay was performed to detect IFN-γsecreting cells from the pooled spleen cells for each vaccine group. Results The seroconversion rates and geometric mean titers(GMTs) and the numbers of IFN-γ secreting cells were approximately at the same level for the differently formulated vaccines after the first injection except that the ratio of IgG2a to IgG1 in the Iscom group was higher than the Al(OH)3 group. After boost injection, the GMTs of total IgG rise slightly in the Al(OH)3 group but significantly in the Iscom group. The IgG2a to IgG1 ratio in the Iscom group kept balanced while dropped further in the Al(OH)3 group. The number of specific IFN-γsecreting cells triggered by the Iscom vaccine exceeded significantly the number of Al( OH)3 vaccine, showinga stronger cellular response. Conclusion The results in this study shows that Iscom matrix is more potent in enhancing the immunogenicity of recombinant hepatitis B virus surface antigen containing PreS epitopes than Al( OH)3 adjuvant. Key words: Iscom; PreS; Epitope; Hepatitis B virus surface antigen; Immunogenicity

A novel guinea pig model of Chlamydia trachomatis genital tract infection

Genital Chlamydia trachomatis infections often result in pelvic inflammatory disease and sequelae including infertility and ectopic pregnancies. In addition to the already established murine models, the development of other animal models is necessary to study the safety and efficacy of prototype vaccine candidates. The intravaginal infection of guinea pigs with C. trachomatis has been tested in three independent studies. The first two studies investigated the effect of hormonal treatment of the animals prior to infection with serovars D and E. The results showed that estradiol treatment was required for sustained infection. The third study conducted an immunization-challenge experiment to explore the feasibility of measuring protection in this guinea pig model. C. trachomatis bacteria were sampled using vaginal swabs and measured by qPCR. Using immunohistochemistry the bacteria were detected in the oviducts 19 days post-infection, indicating that the estradiol treatment resulted in ascending infection. Furthermore, immunization of guinea pigs with live EB formulated with ISCOM matrix led to reduction of cervico-vaginal shedding and diminished the severity of pathology. In this study we have developed a new guinea pig model of C. trachomatis female genital tract infection for the purpose of evaluating potential vaccine candidates.

Immunization of Syrian golden hamsters with F subunit vaccine of human metapneumovirus induces protection against challenge with homologous or heterologous strains

Human metapneumovirus (hMPV), a newly discovered paramyxovirus, is associated with acute respiratory-tract illness, primarily in young children, individuals with underlying disease and the elderly. Two genetic lineages of hMPV circulate around the world, and viruses from these two lineages demonstrate antigenic differences. The clinical impact of hMPV warrants the development of vaccines. Recombinant soluble fusion (F) proteins of prototype viruses of the two main lineages of hMPV that can be produced in high yields have been constructed. In this study, the antigenicity, immunogenicity and protective efficacy of these soluble F subunit vaccines were evaluated in Syrian golden hamsters (Mesocricetus auratus). Immunization of hamsters with the soluble F proteins, adjuvanted with Specol or iscom matrix, induced high virus-neutralization titres, with higher titres against the homologous than the heterologous virus. The neutralizing antibodies protected from subsequent infection of the lungs with both homologous and heterologous virus. Upon challenge, viral titres in the nasal turbinates of immunized animals were reduced significantly compared with those of PBS-immunized animals. In conclusion, a soluble F subunit vaccine for hMPV that induces cross-protective immunity for infection of the lower respiratory tract in Syrian golden hamsters has been generated.

Prevention of naturally occurring infectious bovine keratoconjunctivitis with a recombinant Moraxella bovis pilin– Moraxella bovis cytotoxin–ISCOM matrix adjuvanted vaccine

To evaluate the efficacy of a recombinant Moraxella bovis pilin– M. bovis cytotoxin subunit vaccine to prevent naturally occurring infectious bovine keratoconjunctivitis (IBK; pinkeye), a randomized, blinded, controlled field trial was conducted during summer 2005 in a northern California herd of beef cattle. One hundred and one steers were vaccinated with ISCOM matrix (adjuvant control), recombinant M. bovis cytotoxin carboxy terminus + ISCOM matrix (MbxA), or recombinant M. bovis pilin–cytotoxin carboxy terminus + ISCOM matrix (pilin–MbxA); calves received secondary vaccinations 21 days later. Calves were examined once weekly for 18 weeks for the development of corneal ulcers associated with IBK. Overall, the pilin–MbxA vaccinated group had the lowest overall cumulative proportion of ulcerated calves. Calves that received MbxA, whether alone or with pilin had significantly higher M. bovis cytotoxin serum neutralizing titers as compared to control calves. Results of ocular cultures suggested that vaccination with an M. bovis antigen affected organism type isolated from an ulcer: M. bovis was cultured more often from the eyes of control calves than from the eyes of calves vaccinated with MbxA and pilin–MbxA. In addition, vaccination of calves with MbxA and pilin–MbxA resulted in a higher prevalence of Moraxella bovoculi sp. nov. in ocular cultures. While no significant difference was observed between a cytotoxin versus pilin + cytotoxin vaccine against IBK, the reduced cumulative proportion of IBK in the pilin–cytotoxin vaccinated calves suggests it may provide an advantage over a cytotoxin vaccine alone. Efficacy of an M. bovis vaccine may be reduced in herds where IBK is associated with M. bovoculi sp. nov.

Immunogenicity and protective effect against murine cerebral neosporosis of recombinant NcSRS2 in different iscom formulations

Recombinant NcSRS2, a major immunodominant surface antigen of the intracellular protozoan parasite Neospora caninum, was used as a model antigen to compare the immunogenicity of iscoms prepared according to three different methods. Two NcSRS2 fusion proteins were used, one that was biotinylated upon expression in Escherichia coli and linked to Ni 2+-loaded iscom matrix (iscom without any protein) via a hexahistidyl (His 6)-tagged streptavidin fusion protein, and another that contained both a His 6-tag and streptavidin (His 6-SA-SRS2′) and was coupled to either Ni 2+-loaded or biotinylated matrix. While all three iscom preparations induced N. caninum specific antibodies at similar levels, His 6-SA-SRS2′ coupled to biotinylated matrix generated the strongest cellular responses measured as in vitro proliferation and production of interferon-gamma and interleukin-4 after antigen stimulation of spleen cells. However, the relationship between the levels of these cytokines as well as between IgG1 and IgG2a titres in serum induced by the three iscom preparations were similar, indicating that the balance between Th1 and Th2 responses did not differ. After challenge infection, mice immunised with His 6-SA-SRS2′ coupled to biotinylated matrix had significantly lower amounts of parasite DNA in their brains compared to the other immunised groups. Possible reasons for the performance of the different iscom formulations are discussed.

Applying biotin-streptavidin binding for iscom (immunostimulating complex) association of recombinant immunogens.

We have previously reported strategies for Escherichia coli production of recombinant immunogens fused to hydrophobic peptide or lipid tags to improve their capacity to be incorporated into an adjuvant formulation. In the present study, we have explored the strong interaction between biotin and SA (streptavidin) (K(D) approximately 10(-15) M) to couple recombinant immunogens to iscoms (immunostimulating complexes). Two different concepts were evaluated. In the first concept, a His(6)-tagged SA fusion protein (His(6)-SA) was bound to Ni(2+)-loaded iscom matrix (iscom without associated protein), and biotinylated immunogens were thereafter associated with the SA-coated iscoms. The immunogens were either biotinylated in vivo on E. coli expression or double biotinylated in vivo and in vitro. In the second concept, the recombinant immunogens were expressed as SA fusion proteins, which were directly bound to a biotinylated iscom matrix. A 53-amino-acid malaria peptide (M5), derived from the central repeat region of the Plasmodium falciparum blood-stage antigen Pf155/RESA, and a 232-amino-acid segment (SRS2') from the central region (from Pro-97 to Lys-328) of the major surface antigen NcSRS2 of the protozoan parasite Neospora caninum, served as model immunogens in the present study. All fusion proteins generated were found to be efficiently expressed and could be recovered to high purity using affinity chromatography. The association between the different immunogen-containing fusion proteins and the corresponding iscom matrix was demonstrated by analytical ultracentrifugation in a sucrose density gradient. However, some fusion proteins were, to a certain extent, also found to associate unspecifically with a regular iscom matrix. Furthermore, selected iscom fractions were demonstrated to induce high-titre antigen-specific antibody responses on immunization of mice. For the particular target immunogen SRS2', the induced antibodies demonstrated reactivity to the native antigen NcSRS2. We believe that the presented concepts offer convenient methods to achieve efficient adjuvant association of recombinant immunogens, and the advantages and disadvantages of the two concepts are discussed.

Prevention of naturally occurring infectious bovine keratoconjunctivitis with a recombinant Moraxella bovis cytotoxin-ISCOM matrix adjuvanted vaccine

The efficacy of a recombinant Moraxella bovis cytotoxin subunit vaccine to prevent naturally occurring infectious bovine keratoconjunctivitis (IBK) was evaluated in a randomized, blinded, controlled field trial. Ninety-three cross bred beef calves were vaccinated with either saline, ISCOM matrix (adjuvant control), or a recombinant M. bovis cytotoxin carboxy terminus peptide plus ISCOM matrix and boostered 21 days later. Ocular examinations were performed once weekly for 20 weeks. At week 12, the cumulative proportion of calves with ulcerated eyes in the recombinant vaccine group was significantly lower than in the saline control group. Throughout the 20 week trial, the cumulative proportion of ulcerated calves remained lowest in the recombinant vaccine group. By week 7, nonulcerated calves in the recombinant vaccine group had significantly higher changes in serum neutralizing titers and cytotoxin specific to total IgG ratios in serum and tears as compared to calves in the control groups. The trend for a reduced cumulative proportion of IBK in the vaccinated calves over the 20 week trial suggests that a recombinant M. bovis cytotoxin vaccine may be beneficial in helping to prevent naturally occurring IBK.

Pseudo-ternary phase diagrams of aqueous mixtures of Quil A, cholesterol and phospholipid prepared by the lipid-film hydration method

Pseudo-ternary phase diagrams of the polar lipids Quil A, cholesterol (Chol) and phosphatidylcholine (PC) in aqueous mixtures prepared by the lipid film hydration method (where dried lipid film of phospholipids and cholesterol are hydrated by an aqueous solution of Quil A) were investigated in terms of the types of particulate structures formed therein. Negative staining transmission electron microscopy and polarized light microscopy were used to characterize the colloidal and coarse dispersed particles present in the systems. Pseudo-ternary phase diagrams were established for lipid mixtures hydrated in water and in Tris buffer (pH 7.4). The effect of equilibration time was also studied with respect to systems hydrated in water where the samples were stored for 2 months at 4 °C. Depending on the mass ratio of Quil A, Chol and PC in the systems, various colloidal particles including ISCOM matrices, liposomes, ring-like micelles and worm-like micelles were observed. Other colloidal particles were also observed as minor structures in the presence of these predominant colloids including helices, layered structures and lamellae (hexagonal pattern of ring-like micelles). In terms of the conditions which appeared to promote the formation of ISCOM matrices, the area of the phase diagrams associated with systems containing these structures increased in the order: hydrated in water/short equilibration period<hydrated in buffer/short equilibration period<hydrated in water/prolonged equilibration period. ISCOM matrices appeared to form over time from samples, which initially contained a high concentration of ring-like micelles suggesting that these colloidal structures may be precursors to ISCOM matrix formation. Helices were also frequently found in samples containing ISCOM matrices as a minor colloidal structure. Equilibration time and presence of buffer salts also promoted the formation of liposomes in systems not containing Quil A. These parameters however, did not appear to significantly affect the occurrence and predominance of other structures present in the pseudo-binary systems containing Quil A. Pseudo-ternary phase diagrams of PC, Chol and Quil A are important to identify combinations which will produce different colloidal structures, particularly ISCOM matrices, by the method of lipid film hydration. Colloidal structures comprising these three components are readily prepared by hydration of dried lipid films and may have application in vaccine delivery where the functionality of ISCOMs has clearly been demonstrated.

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 His 6-ABP affinity tag, in which the hexahistidyl tag was utilized to obtain lipidation via a Cu 2+-chelating lipid. A 238 amino acid segment ΔSAG1, 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-His 6-ABP–ΔSAG1 and His 6-ABP–ΔSAG1, 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 His 6-ABP–ΔSAG1 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, ΔSAG1, the induced antibodies demonstrated poor reactivity to the native antigen, although slightly better for the preparation employing the in vitro lipidation strategy, indicating that ΔSAG1 was suboptimally folded or presented. Nevertheless, we believe that the presented strategies offer convenient alternative ways to achieve efficient adjuvant incorporation for recombinant immunogens.

Preparation and characterisation of quillaja saponin with less heterogeneity than Quil-A

Immunisation against pathogens remains one of the most effective ways of preventing or reducing losses due to infectious diseases in animal husbandry. When inactivated vaccines are used, adjuvants are most often required to obtain satisfactory immune responses. One such type of adjuvant is saponin derived from the bark of Quillaja saponaria Molina, a tree of the rose family. A few different commercial sources exist, but due to the structural complexity and heterogeneity of these saponin preparations, it has been difficult to establish exactly which components are responsible for the adjuvant activity. By carefully selecting the bark source, we have succeeded in preparing a much less heterogeneous preparation of quillaja saponin. In this report we describe the preparation, in terms of structural complexity, hemolytic activity, adjuvant activity, and its ability to form ISCOM matrix. This new preparation could have implications for use per se, or as starting material for more effective preparation of pure substances.

Immunomodulation by Iscoms, Immune Stimulating Complexes

The iscom is a uniform stable complex consisting of cholesterol, phospholipid, adjuvant-active saponin, and antigen. The iscom matrix is a particulate complex with identical composition, shape, and morphology, but lacking the incorporated antigen. The assembly of the complex is based on hydrophobic interactions, but antigens that are not hydrophobic can be conjugated with a hydrophobic tail or hidden hydrophobic regions can be exposed, e.g., by acid treatment, to facilitate the incorporation into iscoms. The functional aspects of iscoms are described emphasizing immunomodulation in mouse models. Iscoms prominently enhance the antigen targeting, uptake, and activity of antigen presenting cells including dendritic and B cells and macrophages resulting in the production of proinflammatory cytokines, above all interleukin (IL)-1, IL-6, and IL-12. The expression of costimulatory molecules major histocompatibility complex (MHC) class II, B7.1 and B7.2, is also enhanced. The latter partly explains why the iscom is an efficient adjuvant for elderly mice. Iscoms enhance the Th1 type of response with increased production of IL-2 and interferon γ. However, with some antigens and particularly in monkeys immunized with HIV iscoms, the production of IL-4 was enhanced. IL-4, IL-2, and interferon γ (IFNγ) together with the β chemokines MIP-1α and MIP-1β correlated with protection against challenge infection with a chimeric virus (simian immunodeficiency virus–human immunodeficiency virus). Iscoms were also shown to induce a potent immune response in the newborn and to be an efficient delivery system for mucosal administration. Technical information is given about formulation of iscoms and about handling of antigens to optimize their incorporation into iscoms.

Chimeric plant virus particles administered nasally or orally induce systemic and mucosal immune responses in mice.

The humoral immune responses to the D2 peptide of fibronectin-binding protein B (FnBP) of Staphylococcus aureus, expressed on the plant virus cowpea mosaic virus (CPMV), were evaluated after mucosal delivery to mice. Intranasal immunization of these chimeric virus particles (CVPs), either alone or in the presence of ISCOM matrix, primed CPMV-specific T cells and generated high titers of CPMV- and FnBP-specific immunoglobulin G (IgG) in sera. Furthermore, CPMV- and FnBP-specific IgA and IgG could also be detected in the bronchial, intestinal, and vaginal lavage fluids, highlighting the ability of CVPs to generate antibody at distant mucosal sites. IgG2a and IgG2b were the dominant IgG subclasses in sera to both CPMV and FnBP, demonstrating a bias in the response toward the T helper 1 type. The sera completely inhibited the binding of human fibronectin to the S. aureus FnBP. Oral immunization of the CVPs also generated CPMV- and FnBP-specific serum IgG; however, these titers were significantly lower and more variable than those generated by the intranasal route, and FnBP-specific intestinal IgA was undetectable. Neither the ISCOM matrix nor cholera toxin enhanced these responses. These studies demonstrate for the first time that recombinant plant viruses have potential as mucosal vaccines without the requirement for adjuvant and that the nasal route is most effective for the delivery of these nonreplicating particles.

Characterization of immune responses to experimental polyvalent subunit vaccines assembled in iscoms

Immune responses to experimental polyvalent subunit vaccines assembled in a particulate adjuvant\\delivery system, iscoms, are described. The fusion protein ZZ-M5 comprises structures of staphylococcal protein A (ZZ) and the Plasmodium falciparum malaria antigen Pf155\\RESA (M5). MHC congenic mice were immunized with ZZ-M5 conjugated to iscoms containing human influenza virus antigen (flu ag, M5-flu-isc) or to iscom matrix (iscom particles without flu ag, M5-isc). Comparison of antibody and T-cell responses to M5-isc and M5-flu-isc demonstrated that the flu ag in M5-flu-isc exhibits carrier-related helper functions and that the assembly of immunogens in M5-flu-isc did not result in any apparent antigenic competition. In addition, assembly of ZZ-M5 and flu ag in iscoms induced an alteration of the IgG subclass profile of the antibody response to M5. The results suggest that assembly of immunogens in iscoms may be a useful approach to the design of subunit vaccines but that both quantitative and qualitative aspects of the immunogenic properties of such constructs should be scrutinized.

IMMUNOMODULATION BYQUILLAJA SAPONARIAADJUVANT FORMULATIONS: IN VIVO STIMULATION OF INTERLEUKIN 12 AND ITS EFFECTS ON THE ANTIBODY RESPONSE

The capacity of adjuvants to activate Ag-presenting cells during the induction of the primary immune response is of critical importance for the development of protective immunity to a number of pathogens. In this context, interleukin 12 (IL-12) has a key role by controlling the differentiation of T helper cells and favouring the expansion of Th1cells. The capacity of iscoms with influenza virus Ag (flu-iscoms) and iscom matrix with EBV gp340 Ag to induce IL-12 was analysed in mice. The flu-iscom drives the immune response towards a Th1type subsequent to IL-12 induction as measured in the serum of H2b, H2dand H2kmice. The iscom presenting the Ag and adjuvant in the same particle was considerably more efficient than the formulation of matrix and Ag in separate particles. Inhibition experiments with mAb neutralizing IL-12, interferon gamma (IFN-γ) or IL-4, the latter two cytokines representing the Th1and Th2type of responses, showed that iscoms induce a broader immune response than that involving IL-12. This was shown by the additional effect that IL-4 neutralization had on the immune response to iscoms. Anti-IL 12 reduced the specific total Ab as well as IgG1, IgG2a and IgG2b while anti-IL 4 influenced the response to iscom by decreasing IgG2a and increasing IgG1. Further, the neutralization experiments indicate that IL-12 has a broader effect than IFN-γ on the Ab response by influencing the production of IgG1, IgG2a and IgG2b.

Efficacy trials with tissue-culture grown, inactivated vaccines against chlamydial abortion in sheep

Vaccines containing inactivated, semi-purified antigen of ovine abortifacient Chlamydia psittaci were prepared from tissue-culture grown harvests of two strains. These were evaluated for efficacy against experimentally reproduced enzootic abortion of ewes (EAE), the protection index (PI) of each vaccine being calculated from three variables. Trial 1 tested the adjuvants Marcol 52/Arlacel A, ISCOM matrix, Novasomes and Alhydrogel, each administered with a total antigen dose of 4 μg chlamydial protein (cp), against challenge with the two homologous vaccine strains. The first three formulations yielded PIs of 39–42%, while that of Alhydrogel was 23%. Marked granuloma and/or abscess formation occurred in the Marcol 52/Arlacel A group but in none of the others, and only this group seroconverted following vaccination. In Trial 2, ISCOM matrix containing 16 μg cp per dose was administered once or twice. Two challenges were used, one involving the two vaccine strains and the other these two strains plus five others, but no significant differences were seen between them. The double dose schedule, which was better than the single dose and also several Novasomes formulations tested concurrently, gave PIs of 64 and 77% against the two challenges (combined value 71.5%). Trial 3 studied duration of immunity over two lambing seasons using three different administration schedules of 16 μg cp in ISCOM matrix, namely single dose in year 1, single dose in year 2 (Group 1), double dose then booster (Group 2), and double dose without booster (Group 3). PIs against the homologous bivalent challenge were 79–80% for Groups 1 and 3 and 91% for Group 2. Combining Trials 2 and 3 data, double injection of 16 μg cp in ISCOM matrix reduced the proportions of ewes aborting from 31.5 to 4.8% and of those excreting chlamydiae at parturition from 74 to 27%, while the proportion of dead lambs fell from 29.9 to 6.5%, producing an overall PI of 74%. This vaccine appeared virtually innocuous, but seroconversion following its use was detectable only by ELISA. The findings suggested the efficacy of inactivated EAE vaccines to be antigen dose-dependent, and showed that good protection against heterologous challenge was achieved when 16 μg cp (containing approximately 30% major outer membrane protein) was combined with ISCOM matrix and administered twice or more.