Evaluation of the humoral immune response to a multicomponent recombinant vaccine against S. aureus in healthy pregnant heifers.

Staphylococcus aureus is a worldwide pathogen that causes mastitis in dairy herds. Shortcomings in control programs have encouraged the development of vaccines against this pathogen. This study evaluated the vaccine candidate VacR, which included recombinant S. aureus protein clumping factor A (rClf), fibronectin binding protein A (rFnBP) and hemolysin beta (rBt), formulated with a novel immune-stimulating complex. Comparisons were made between healthy pregnant heifers that received either VacR (n=8; VacR group) or phosphate buffered saline (PBS) plus adjuvant (control group) SC in the supramammary lymph node area on days 45 and 15 before the expected calving date. Blood and foremilk samples were collected from 7 to 60days post-calving. After calving, heifers in the VacR group produced higher total IgG (IgGtotal) titers against each component, in both serum (rBt, 3.4×105; rClf, 3.1×105; rFnBP, 2.3×105) and milk (rBt, 2.6×104; rClf, 1.3×104; rFnBP, 1.1×104), than control heifers (P<0.0001). There were increased concentrations of IgG1 and IgG2 in VacR group (P<0.05), in both serum and milk. Humoral responses remained high throughout the period most susceptible to intramammary infections (P<0.01). Antibodies produced against S. aureus rClf and rFnBP reduced bacterial adherence to fibronectin and fibrinogen by 73% and 67%, respectively (P<0.001). Milk antibodies against these adhesins inhibited S. aureus invasion of a mammary epithelial cell line (MAC-T), resulting in 15.7% of bacteria internalized (P<0.0001). There was an approximately 6-fold reduction in the hemolysis titer for the native hemolysin in the VacR group compared to the control group (P<0.0001) and a significantly increase in the proportion of positive neutrophils (VacR, 29.7%; PBS, 13.1%) and the mean fluorescent index (VacR, 217.4; PBS, 152.6; P<0.01) in the VacR group. The results suggest that VacR is a valuable vaccine candidate against S. aureus infections, and merits further field trials and experimental challenges.

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 ß-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 ß-toxin effect in vitro, placing these antigens as candidates to be included in a formulation directed to prevent staphylococcal bovine mastitis.

Immune responsiveness in the neonatal period.

The maintenance of pregnancy requires suppression of the maternal immune system which would naturally recognize the developing fetus as an allograft and seek to destroy it by mounting a Th1 regulated cytotoxic immune response. During pregnancy a range of soluble factors are produced by the placenta which switch maternal immune regulation towards a protective Th2 phenotype. These factors also influence the developing fetal immune system and all newborns initially have an immunological milieu skewed towards Th2 immunity. Vaccination during the neonatal period must therefore overcome the dual challenge of the inhibitory effect of maternally derived antibody and this natural Th2 regulatory environment. One means of overcoming these obstacles is by the use of adjuvant systems that can redirect the neonatal immune response towards an appropriate Th1 regulated reaction that affords protection from infectious disease. In this overview, experiments are described in which viral antigens incorporated into immune stimulatory complexes (ISCOMs) are able to induce immune responses with balanced Th1 and Th2 regulation in neonatal mice, as evidenced by the nature of the IgG subclass response and cytokine profile, and the induction of cytotoxic lymphocytes. ISCOM adjuvanted vaccines are able to induce similar protective immunity in the newborn of larger animal species including cattle, horses and dogs.

Comparison of intranasal with targeted lymph node immunization using PR8-Flu ISCOM adjuvanted HIV antigens in macaques.

The rapidly spreading HIV epidemic requires a vaccine that elicits potent mucosal immunity to halt or slow transmission. Induction of these responses will depend on the use of appropriate adjuvants and targeting of the mucosal immune system. Previously, immune stimulating complexes (ISCOM) have shown great potency as adjuvant in the induction of mucosal responses in mice and systemic responses in non-human primates. In this study, HIV formulated in PR8-Flu ISCOM adjuvant was applied to immunize rhesus macaques against HIV; targeting the mucosa either via intranasal (IN) application or via targeted lymph node immunization (TLNI). While, strong systemic, HIV specific, cytokine, lymphoproliferative, and antibody responses were induced via the TLNI route, the IN application generated only low responses. Furthermore, all four animals immunized via TLNI developed vaginal IgA antibodies against gp120. In conclusion, in contrast to what has been demonstrated in mice, the IN application of PR8-Flu ISCOM did not induce strong immune responses in rhesus macaques unlike those immunized by the TLNI route.

Antibody responses to human rotavirus (HRV) in gnotobiotic pigs following a new prime/boost vaccine strategy using oral attenuated HRV priming and intranasal VP2/6 rotavirus-like particle (VLP) boosting with ISCOM.

Safer and more effective human rotavirus (HRV) vaccines are needed. We evaluated oral priming with attenuated WaHRV (AttHRV) followed by boosting with two intranasal (IN) doses of VP2/6 virus-like particles (2/6 VLP) with immunostimulating complexes (ISCOM) to determine if this regimen induces protection against diarrhoea and viral shedding in the gnotobiotic pig model. IgM, IgA and IgG antibody titres in serum and intestinal contents were quantified by enzyme-linked immunosorbent assay (ELISA) and serum neutralizing antibody titres were measured by a virus neutralization (VN) test. Seven groups of neonatal gnotobiotic pigs were vaccinated at post-inoculation days (PID) 0, 10 and 21 and challenged with virulent WaHRV at PID 28. The vaccine groups included: (1, 2) oral priming with AttHRV and boosting with two IN immunizations with 2/6 VLP-ISCOM (Att + 2/6 VLP-ISCOM) at VLP concentrations of 250 micro g or 25 micro g; (3, 4) three IN immunizations with 2/6 VLP-ISCOM at VLP concentrations of 250 micro g or 25 micro g (2/6 VLP-ISCOM); (5) three oral immunizations with AttHRV (3xAttHRV); (6) one oral immunization with AttHRV (1xAttHRV); (7) controls (ISCOM matrix and/or diluent). The pigs that received 3xAttHRV or Att + 2/6 VLP250-ISCOM had the highest protection rates against diarrhoea upon challenge at PID 28 with virulent WaHRV. The IgA antibody titres to HRV in intestinal contents were significantly higher in the Att + 2/6 VLP250-ISCOM group than in all other groups prechallenge (PID 28). Serum VN antibody titres were statistically similar after the first inoculation among the groups given AttHRV, but at PID 28 VN antibody titres were significantly higher for the 3xAttHRV and Att + 2/6 VLP250-ISCOM groups than for the 1xAttHRV group suggesting that boosting with 2/6 VLP also boosted VN antibody responses. In humans, intestinal IgA antibodies have been correlated with protection against symptomatic reinfection. Thus the vaccine regimen of one oral dose of AttHRV and two IN immunizations with 2/6 VLP250-ISCOM may be an alternative to multiple-dose live oral vaccines in humans.

Experimental field trial with an immunostimulating complex (ISCOM) vaccine against contagious bovine pleuropneumonia.

The experimental field trial with an immunostimulating complex (ISCOM) vaccine has been an occasion to explore the role of a Th1 response in the pathogenesis caused by Mycoplasma mycoides subsp. mycoides small colony (MmmSC) and in immune protection. The ISCOM complex is known to promote Th1 response. Antibodies to MmmSC were detected by indirect enzyme-linked immunosorbent assay (ELISA) in the vaccinated cattle, although the levels were lower than in a previous study. No antibodies were detected by complement fixation test (CF). After the challenge infection, vaccinated animals developed CF antibody response. They showed significantly reduced mortality compared with controls. However, gross pathological and histopathological score for vaccinated animals was as high as for the non-vaccinated, characterized by a high inflammatory reaction with histopathology dominated by interlobular pneumonia with vasculitis.

Protection and antibody responses to oral priming by attenuated human rotavirus followed by oral boosting with 2/6-rotavirus-like particles with immunostimulating complexes in gnotobiotic pigs.

We evaluated antibody responses and protection induced by attenuated Wa human rotavirus (AttHRV) and VP2/6-rotavirus-like particles (VLP), 100 or 250 microg/dose, with immunostimulating complexes (ISCOM) (VLP/ISCOM) each given orally, alone or sequentially to gnotobiotic pigs. The AttHRV-VLP 250 microg/ISCOM and three-dose-AttHRV (AttHRV3x) groups had significantly higher serum IgA, IgG and intestinal IgA antibody titers to HRV pre-challenge than the three-dose-VLP 100 microg/ISCOM group (VLP/ISCOM3x) and controls (diluent/ISCOMmatrix). Protection rates against viral shedding and diarrhea were highest in the AttHRV-VLP250 microg/ISCOM and AttHRV3x groups, lower in the AttHRV-VLP 100 microg/ISCOM group, with no protection in the VLP/ISCOM3x group and controls. Thus, VLP/ISCOM boosted antibody titers and protection after priming with AttHRV.

Different respiratory syncytial virus and Quillaja saponin formulations induce murine peritoneal cells to express different proinflammatory cytokine profiles.

The recognition of a pathogen or a vaccine antigen formulation by cells in the innate immune system leads to production of proinflammatory cytokines, which will determine the ensuing acquired immune response quantitatively and qualitatively. Tumour necrosis factor (TNF)-alpha, interleukin (IL)-1 and IL-6 are the first set of cytokines produced upon such an encounter, which have roles both in protective immunity and immunopathogenesis evident with respiratory syncytial virus (RSV). RSV antigens in different physical adjuvant-vaccine formulations were analysed for their capacity to provoke cultured murine peritoneal cells to produce these three proinflammatory cytokines. RSV immunostimulating complex (ISCOM), i.e. both antigen and adjuvant are incorporated in the same particle, induced high levels of IL-1alpha being of the same magnitude or higher than those of live RSV and lipopolysaccharide (LPS). Live virus and LPS induced higher levels of IL-6 and TNF-alpha than ISCOM and so did non-adjuvanted UV-inactivated RSV but only at high doses. ISCOM-Matrix, i.e. ISCOM without antigens, admixed as a separate entity to inactivated RSV, downregulated or blocked the cytokine response to the inactivated RSV in contrast to ISCOM. Kinetic studies showed that ISCOM induced cytokine production first detected at hours 1, 2, 4 for TNF-alpha, IL-6 and IL-1alpha respectively, which was earlier than for the other antigen formulations containing corresponding doses of antigen and/or Quillaja adjuvant. Peak values for production of TNF-alpha and IL-6 were at 8 h and for IL-1alpha at 72 h following stimulation with ISCOM. The delayed appearance of IL-1alpha may reflect the cell-bound nature of this cytokine.

Immunostimulating complexes (ISCOMs) for nasal vaccination.

The immunostimulating complex (ISCOM) is documented as a strong adjuvant and delivery system for parenteral immunization. Its effectiveness for mucosal immunization has also been proven with various incorporated antigens. Lövgren et al. were the first to demonstrate the capacity of influenza virus ISCOMs to induce mucosal immune response and protection after one comparatively low nasal dose. Further studies show that similar to Cholera toxin (CT) and Escherichia coli heat-labile toxin (LT), ISCOMs break immunological tolerance and exert strong mucosal adjuvant activity, resulting in secretory IgA and systemic immune responses. Striking is the capacity of ISCOMs to induce CTL response also after nasal administration. In contrast to CT, ISCOMs initiate mucosal as well as systemic immune responses in an IL-12 dependent manner but independently of IL-4. The recombinant B subunit of cholera toxin (rCTB) was incorporated in the same ISCOM particle to explore symbiotic effects. The IgA response to rCTB in lungs was increased 100-fold when rCTB was administered nasally in ISCOMs and more than 10-fold in the remote mucosa of the genital tract. An enhanced IgA response to a passenger antigen OVA was recorded in the remote genital tract. After i.n. administration of the envelope proteins of respiratory syncytial virus in ISCOMs, high serum antibodies were induced, almost at the same levels as those following parenteral immunization and potent IgA responses were also evoked both at the local respiratory mucosa, and in the cases tested at the distant mucosae of the genital and intestinal tracts. Similar results have also been recorded with ISCOMs containing envelope proteins from Herpes simplex virus, Influenza virus and Mycoplasma mycoides. The mucosal targeting property of envelope proteins of RSV was utilized in an HIV-gp120 RSV ISCOM formulation. After nasal administration an enhanced mucosal IgA response to gp120 was observed in the female reproductive tract. In general, antigens derived from envelope viruses or cell membranes incorporated into ISCOMs retain their biological activity and conformation, encompassing the mucosal targeting and virus neutralizing properties.

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.

Effect of oral rotavirus/iscom vaccines on immune responses in gnotobiotic lambs.

A comparison of the effect on the immune responses in gnotobiotic lambs was made between an iscom vaccine prepared from recombinant rotavirus VP6 protein, an inactivated rotavirus/iscom-matrix vaccine and a vaccine comprising inactivated rotavirus alone. All three vaccines induced immunological priming and some degree of protection was observed after a single oral dose. However, different immune responses were induced in response to a virulent infection. The group vaccinated with the rotavirus/iscom-matrix vaccine showed a Th2-like response characterised by rotavirus-specific antibodies and a down-regulation of IFNgamma in jejunal Peyer's patches. Both Th1-like and Th2-like immune responses were induced in the group receiving the VP6 vaccine as seen by significantly increased expressions of IFNgamma and IL-6 in the jejunal Peyer's patch together with an increased percentage of CD8+ T cells in the intestine and rotavirus-specific antibodies at mucosal surfaces. Iscom vaccines given orally have the ability to induce both Th1-like and Th2-like immune responses in a ruminant model.

Quillaja saponin formulations that stimulate proinflammatory cytokines elicit a potent acquired cell-mediated immunity.

We examined the ability of various Quillaja saponins in iscom-matrix formulations to induce proinflammatory cytokines, such as interleukin (IL)-1alpha and IL-6, and to stimulate acquired immune responses to influenza virus envelope proteins. The A-fraction of Quillaja saponins (QH-A) was shown to stimulate antigen-presenting cells (APC) to produce proinflammatory cytokines, and elicited a high primary antigen-specific antibody response and potent cell-mediated responses, as measured by T-cell proliferation, production of cytokines and cytotoxic T-lymphocyte (CTL) activity. The C-fraction of Quillaja saponins (QH-C) was shown to have a low capacity to stimulate proinflammatory cytokines and elicited low primary antibody and T-cell responses. However, the QH-C iscom-matrix mediated a potent booster effect, resulting in a high secondary antibody response. The ability of APC to discriminate and to respond to QH-A formulations more efficiently than to QH-C with release of proinflammatory cytokines, which precedes a potent acquired immune response, identifies an important mechanism through which some adjuvants may exert their immunoenhancing activities.

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 gamma. 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 gamma (IFNgamma) together with the beta chemokines MIP-1alpha and MIP-1beta 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.

Iscom and iscom-matrix enhance by intranasal route the IgA responses to OVA and rCTB in local and remote mucosal secretions.

Iscoms, with rCTB incorporated via the GM1 receptor, enhanced in mice the mucosal immunogenicity of rCTB as antigen after intranasal (i.n.) administration both by inducing IgA response in the remote intestinal tract mucosa and by a 100-fold increase of the specific IgA locally in the lungs. Iscom-matrix as a separate entity mixed with rCTB enhanced the rCTB-IgA response similarly. While OVA in iscoms induced high mucosal IgA responses, iscom-matrix co-administered with OVA induced low or no mucosal IgA response to OVA. A synergism between iscoms and rCTB could only be seen as an adjuvant targeting effect enhancing the IgA response to OVA in the remote genital tract mucosa. In serum, the immunomodulatory effect of iscoms after i.n. administration was seen as an enhanced serum IgG2a response.

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

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

The major Epstein-Barr virus (EBV) envelope glycoprotein gp340 when incorporated into Iscoms primes cytotoxic T-cell responses directed against EBV lymphoblastoid cell lines.

A recombinant form of the EBV envelope glycoprotein and vaccine candidate gp340, lacking its hydrophobic transmembrane region, was incorporated into Iscoms after coupling to phosphatidyl ethanolamine via carbohydrate residues. Coupling by partial oxidation of gp340 carbohydrate with sodium periodate partly denatured the incorporated gp340 as indicated by its reduced reactivity with monoclonal antibodies that recognise the major neutralising epitope. Immunisation of cottontop tamarins with these Iscoms elicited antibody responses to gp340, but these antibodies only poorly recognised the major neutralising epitope in a competition ELISA and were unable to neutralise EBV in vitro. Despite the lack of neutralising antibody, immunisation with these Iscoms primed significant in vitro proliferative responses to soluble gp340 in lymphocytes from the draining lymph nodes and spleen. T-cell lines were raised from both immunised and control animals by in vitro stimulation of peripheral blood lymphocytes or spleen cells with autologous EBV-transformed lymphoblastoid cell lines. The T-cell lines from control animals had higher numbers of CD4+ T-cells than CD8+ T-cells and were not cytotoxic for autologous lymphoblastoid cell lines (LCL). In contrast the lines from immunised animals contained more CD8+ T-cells than CD4+ T-cells and had marked cytotoxicity for autologous LCL.

HIV-1 vaccine-induced immune responses which correlate with protection from SHIV infection: compiled preclinical efficacy data from trials with ten different HIV-1 vaccine candidates.

The specific immune mechanisms necessary and/or sufficient to elicit HIV-vaccine protection remain undefined. Utilising the SHIV rhesus macaque model the immunogenicity as well as the efficacy of ten different HIV-1 vaccine candidates was evaluated. Comparison of the immune responses induced, with the ability of the vaccine to protect from SHIV infection provided a means to determine which type of immune responses were necessary for protection. Vaccine candidates included VLPs, DNA, subunit protein with novel adjuvant formulations, ISCOMs and pox-virus vectors. Protection from SHIV infection was achieved in approximately half of the animals which received a primary intravenous cell-free challenge. The presence of CTL in the absence of other effector responses did not correlate with protection from this route and type of challenge. Virus neutralising antibodies (Nab) appeared to be necessary but alone were insufficient for protection. If Ag-specific IFN-gamma and/or IL-4 as well as lymphoproliferative (LP) responses were found with the lack of a detectable IL-2 response, then protection was not observed. Immunity correlated with the magnitude of Nab responses, beta-chemokines and as well as balanced, qualitative T-helper responses.

Comparison of immunity generated by nucleic acid-, MF59-, and ISCOM-formulated human immunodeficiency virus type 1 vaccines in Rhesus macaques: evidence for viral clearance.

The kinetics of T-helper immune responses generated in 16 mature outbred rhesus monkeys (Macaca mulatta) within a 10-month period by three different human immunodeficiency virus type 1 (HIV-1) vaccine strategies were compared. Immune responses to monomeric recombinant gp120SF2 (rgp120) when the protein was expressed in vivo by DNA immunization or when it was delivered as a subunit protein vaccine formulated either with the MF59 adjuvant or by incorporation into immune-stimulating complexes (ISCOMs) were compared. Virus-neutralizing antibodies (NA) against HIV-1SF2 reached similar titers in the two rgp120SF2 protein-immunized groups, but the responses showed different kinetics, while NA were delayed and their levels were low in the DNA-immunized animals. Antigen-specific gamma interferon (IFN-gamma) T-helper (type 1-like) responses were detected in the DNA-immunized group, but only after the fourth immunization, and the rgp120/MF59 group generated both IFN-gamma and interleukin-4 (IL-4) (type 2-like) responses that appeared after the third immunization. In contrast, rgp120/ISCOM-immunized animals rapidly developed marked IL-2, IFN-gamma (type 1-like), and IL-4 responses that peaked after the second immunization. To determine which type of immune responses correlated with protection from infection, all animals were challenged intravenously with 50 50% infective doses of a rhesus cell-propagated, in vivo-titrated stock of a chimeric simian immunodeficiency virus-HIVSF13 construct. Protection was observed in the two groups receiving the rgp120 subunit vaccines. Half of the animals in the ISCOM group were completely protected from infection. In other subunit vaccinees there was evidence by multiple assays that virus detected at 2 weeks postchallenge was effectively cleared. Early induction of potent type 1- as well as type 2-like T-helper responses induced the most-effective immunity.

Iscom is an efficient mucosal delivery system for Mycoplasma mycoides subsp. mycoides (MmmSC) antigens inducing high mucosal and systemic antibody responses.

The purpose of this study was to explore the iscom as a mucosal delivery system for Mycoplasma mycoides subsp. mycoides small colony (MmmSC) antigens. BALB/c female mice were immunised intranasally (i.n.) twice, 8 weeks apart with three different doses (3, 10 and 20 microg) or subcutaneously (s.c.) with 3 microg of M. mycoides antigens incorporated into iscoms. Mycoplasma cells were administered s.c. twice, 8 weeks apart at a dose of 3 microg or i.n. at 10 microg as for iscoms. Both i.n. and s.c. modes of immunisation with iscoms induced prominent primary serum antibody responses in a dose-dependent manner, which were efficiently boosted. Compared to whole mycoplasma cells, iscoms enhanced the total Ig and IgG subclass (IgG1, IgG2a and IgG2b) responses in serum and in lungs greatly, and this enhancement was more prominent after i.n. than after s.c. immunisation. By the i.n. mode of immunisation iscoms containing mycoplasma antigens induced a 60-fold higher IgA response in lungs than the whole cell antigen. Iscoms also induced substantially higher total Ig and IgG subclass responses in the lungs. By Western blot a reduced number of bands (7) were detected in lung secretion after both i.n. and s.c. immunisations with iscoms compared to a high number of bands (more than 30) detected by serum antibodies. Interestingly i.n. immunisation with iscoms induced antibodies in lungs as well as in serum to mycoplasma cell antigens which differed from those induced by s.c. immunisation as revealed by the Western blot patterns.