Th1 immune maturation effects of Nocardia rubra cell-wall skeleton via PI3K/Akt/PAX8 regulatory axis.

Nocardia rubra cell-wall skeleton (Nr-CWS) is reported as an external immunotherapeutic enhancer with the advantage of antitumor effect on human cancers. However, the immune regulatory role of Nr-CWS is not fully illustrated. We studied mouse CD4+ T lymphocytes isolated from mice spleen were induced by Nr-CWS and observed that the differentiation of Th1 CD4+ T cells and the cytokines of IL-2, TNF-α, IFN-γ were all enhanced by Nr-CWS. Furthermore, RNA sequencing was conducted to investigate the different mRNA profiling induced by Nr-CWS. We observed that paired box 8 (PAX8) was significantly up-regulated in Nr-CWS-treated Th1 cells compared to control. As a transcription factor, chromatin immunoprecipitation sequencing was carried out to study the genome-wide distribution of PAX8. Interestingly, we found that the binding domain of PAX8 was elevated by Nr-CWS, and the target genes associated with these binding sites showed a positive correlation between their transcription and PAX8 binding strength. Finally, we determined that Nr-CWS could enhance the activity of the PI3 K/Akt signaling pathway. Akt agonist could mimic the effect of Nr-CWS for PAX8 up-regulation, while Akt inhibitor compromised the expression of PAX8. Taken together, we determined a novel role of Nr-CWS in boosting the activity of Th1 maturation via the PI3 K/Akt/PAX8 axis.

Nocardia rubra cell-wall skeleton promotes CD4+ T cell activation and drives Th1 immune response.

Several lines of evidences have shown that Nocardia rubra cell wall skeleton (Nr-CWS) has immunoregulatory and anti-tumor activities. However, there is no information about the effect of Nr-CWS on CD4+ T cells. The aim of this study was to explore the effect of Nr-CWS on the phenotype and function of CD4+ T cells. Our results of in vitro experiments showed that Nr-CWS could significantly up-regulate the expression of CD69 and CD25 on CD4+ T cells, promote the proliferation of CD4+ T cells, increase the production of IFN-γ, TNF-α and IL-2 in the supernatants, but has no significant effect on the apoptosis and death of CD4+ T cells. Results of in vivo experiments showed that Nr-CWS could promote the proliferation of CD4+ T cells, and increase the production of IL-2, IFN-γ and TNF-α (Th1 type cytokines). These data suggest that Nr-CWS can enhance the activation of CD4+ T cells, promote the proliferation of CD4+ T cells and the differentiation of CD4+ T cells to Th1 cells.

Mechanism responsible for the antitumor effect of BCG-CWS using the LEEL method in a mouse bladder cancer model.

We previously reported on the development of a water soluble formulation of the cell wall skeleton of BCG (BCG-CWS), a major immune active center of BCG, by encapsulating it into a nanoparticle (CWS-NP). The CWS-NP allowed us to clarify the machinery associated with the BCG mediated anti-bladder tumor effect, especially the roles of bladder cancer cells and dendritic cells (DCs) in the initial step, which remains poorly understood. We show herein that the internalization of BCG-CWS by bladder cancer cells, but not DCs, is indispensable for the induction of an antitumor effect against bladder cancer. Tumor growth was significantly inhibited in mice that had been inoculated with mouse bladder cancer (MBT-2) cells containing internalized BCG-CWS. On the other hand, the internalization of BCG-CWS by DCs had only a minor effect on inducing an antitumor effect against MBT-2 tumors. This was clarified for the first time by using the CWS-NP. This finding provides insights into our understanding of the role of bladder cancer cells and DCs in BCG therapy against bladder cancer.

Optimization of cell-wall skeleton derived from Mycobacterium bovis BCG Tokyo 172 (SMP-105) emulsion in delayed-type hypersensitivity and antitumor models.

Cell-wall skeleton prepared from Mycobacterium bovis BCG (BCG-CWS) is known as a potent adjuvant and has been shown to possess antitumor activity in many non-clinical and clinical studies. As there are no approved BCG-CWS formulations for cancer therapy, we investigated the potential for cancer immunotherapy of SMP-105, our originally produced BCG-CWS. For optimizing SMP-105 emulsion, we compared the effects of drakeoland squalane-based SMP-105 emulsions on IFN-γ production in rats and evaluated their ability to induce skin reaction in guinea pigs. Both emulsions had the same activity in both experiments. We selected squalane as base material and produced two types of squalane-based formulations (vialed emulsion and pumped emulsion) that can easily be prepared as oil-in-water emulsions. Although the vialed emulsion showed the same pattern of distribution as a usual homogenized emulsion, the pumped emulsion showed more uniform distribution than the other two emulsions. Whereas both emulsions enhanced strong delayed type hypersensitivity (DTH) reaction in a mouse model, the pumped emulsion induced slightly smaller edema. Data on oil droplet size distribution suggest that few micrometer oil droplet size might be appropriate for oil-in-water microemulsion of SMP-105. The antitumor potency of SMP-105 emulsion was stronger than that of some of the launched toll-like receptor (TLR) agonists (Aldara cream, Picibanil, and Immunobladder). Aldara and Picibanil showed limited antitumor effectiveness, while Immunobladder had almost the same effect as SMP-105 at the highest dose, but needed about 10 times the amount of SMP-105. These findings first indicate that SMP-105 has great potential in cancer immunotherapy.

The liposome-incorporating cell wall skeleton of Mycobacterium bovis bacillus Calmette-Guéin can directly enhance the susceptibility of cancer cells to lymphokine-activated killer cells through up-regulation of natural-killer group 2, member D ligands.

OBJECTIVE: • To conduct a preclinical evaluation of the ability of natural killer cells to cytolyze bladder cancer cells that were modified to show enhanced expression of natural-killer group 2, member D (NKG2D) ligands by R8-liposome-bacillus Calmette-Guéin (BCG)-cell wall skeleton (CWS) treatment. MATERIALS AND METHODS: • The T24 cells and RT-112 cells were co-cultured with R8-liposome-BCG-CWS and BCG for 2, 4, or 6 h, and then the surface expression of NKG2D ligands was analyzed using TaqMan real-time quantitative RT-PCR. • Peripheral blood mononuclear cells were obtained with a conventional preparation kit, and then lymphokine-activated killer (LAK) cells were generated from these purified peripheral blood mononuclear cells via interleukin-2 stimulation. • The anti-tumour effect of LAK cells against untreated and R8-liposome-BCG-CWS co-cultured with cells of the human bladder cancer cell lines T24 and RT-112 was analyzed using the cytotoxic WST-8 assay method at 4 h of culture at various effector/target (E : T) ratios. RESULTS: • Major histocompatibility complex class I-related chain B (MICB) expression was increased ≈1.5-fold on T24 cells and RT-112 cells with BCG. • UL-16-binding protein (ULBP) 1 expression was also increased ≈1.5-fold on T24 cells and RT-112 cells with BCG. R8-liposome-BCG-CWS increased the surface expression of MICB 2.2-fold on T24 cells but did not increase it significantly on RT-112 cells. • ULBP1 expression was increased ≈2.2-fold on RT-112 cells, although no differences were observed between the expression of ULBP2 and 3 with R8-liposome-BCG-CWS. • T24 cells that were co-cultured with R8-liposome-BCG-CWS showed an ≈1.3-fold increase in sensitivity to cytolysis by LAK cells at an E : T ratio of 4 and RT-112 cells showed an ≈1.4-fold increase at an E : T ratio of 2. CONCLUSIONS: • In the present study, the induction of surface NKG2D ligands by R8-liposome-BCG-CWS rendered cancer cells more susceptible to cytolysis by LAK cells. • T24 cells and RT-112 cells, even when cultured singly in the absence of immune cells, can directly respond to R8-liposome-BCG-CWS. • The results obtained in the present study may therefore indicate a novel adoptive immunotherapy against bladder cancers.

Bacillus Calmette-Guerin and BCG cell wall skeleton suppressed viability of bladder cancer cells in vitro.

AIM: Bacillus Calmette-Guerin (BCG) is one of therapeutic options for urothelial carcinoma (UC). The objectives of this study were to determine the direct effect of viable or heat-killed BCG and BCG cell wall skeleton (BCG-CWS) on UC cells in vitro. MATERIALS AND METHODS: UC cell lines were co-cultured with viable or heat-killed BCG Immunobladder® (Tokyo 172 strain) and BCG-CWS. Viability of the cells, apoptosis and BrdU incorporation were estimated. RESULTS: BCG induced cell growth retardation in highly malignant UC bearing integrin α5ß1 (VLA5). VLA5-blocking antibody partially abrogated this effect. BCG treatment induced a modest increase in the sub-G(1) fraction of cells and a decrease of BrdU incorporation. Cell growth retardation effect of viable BCG was reproduced by both heat-killed BCG and BCG-CWS. CONCLUSION: The results indicate that VLA5 may be a biomarker of UC with sensitivity to BCG. Moreover, BCG-CWS is a promising substance which might replace BCG, preventing life-threatening complications of viable BCG treatment.

Mycobacterial cell-wall skeleton as a universal vaccine vehicle for antigen conjugation.

Mycobacterial cell-wall skeleton (CWS) is an immunoactive and biodegradable particulate adjuvant and has been used for immunotherapy in patients with cancer. The CWS of Mycobacterium bovis bacillus Calmette-Guérin (BCG-CWS) was studied as a universal vaccine vehicle for antigen conjugation, to develop potentially effective and safe vaccines. Here, we describe experiments in which protein antigens, such as keyhole limpet haemocyanin (KLH), ovalbumin (OVA) and bovine serum albumin (BSA) were highly efficiently coupled to 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide (EDC/NHS)-activated carboxyl groups of BCG-CWS, and tested the immunogenicity of OVA-conjugated BCG-CWS vaccine. We found that a strong immune response was induced in mice immunised with OVA-conjugated BCG-CWS, which was similar to the enhancement of the immune responses in mice immunised with OVA and complete Freund's adjuvant. Covalent conjugation of OVA to BCG-CWS was essential for Th1-skewed immune responses, with prominent expression of IFN-γ. Furthermore, antigen-conjugated BCG-CWS vaccine is simple to manufacture, safe, and easy to use. Our results suggest that mycobacterial CWS as a universal vaccine vehicle for conjugation of a wide variety of antigens constitutes a breakthrough for development of the most promising vaccines for infections, allergic diseases, and cancer.

Bacillus calmette-guerin cell wall cytoskeleton enhances colon cancer radiosensitivity through autophagy.

The cell wall skeleton of Mycobacterium bovis Bacillus Calmette-Guerin (BCG/CWS) is an effective antitumor immunotherapy agent. Here, we demonstrate that BCG/CWS has a radiosensitizing effect on colon cancer cells through the induction of autophagic cell death. Exposure of HCT116 colon cancer cells to BCG/CWS before ionizing radiation (IR) resulted in increased cell death in a caspase-independent manner. Treatment with BCG/CWS plus IR resulted in the induction of autophagy in colon cancer cells. Either the autophagy inhibitor 3-methyladenine or knockdown of beclin 1 or Atg7 significantly reduced tumor cell death induced by BCG/CWS plus IR, whereas the caspase inhibitor z-VAD-fmk failed to do so. BCG/CWS plus IR-mediated autophagy and cell death was mediated predominantly by the generation of reactive oxygen species (ROS). The c-Jun NH(2)-terminal kinase pathway functioned upstream of ROS generation in the induction of autophagy and cell death in HCT116 cells after co-treatment with BCG/CWS and IR. Furthermore, toll-like receptor (TLR) 2, and in part, TLR4, were responsible for BCG/CWS-induced radiosensitization. In vivo studies revealed that BCG/CWS-mediated radiosensitization of HCT116 xenograft growth is accompanied predominantly by autophagy. Our data suggest that BCG/CWS in combination with IR is a promising therapeutic strategy for enhancing radiation therapy in colon cancer cells through the induction of autophagy.

Bacillus Calmette-Guérin cell-wall skeleton enhances the killing activity of cytotoxic lymphocyte-activated human dendritic cells transduced with the prostate-specific antigen gene.

UNLABELLED: To determine whether dendritic cells (DC) transduced with the prostate-specific antigen (PSA) gene can induce PSA-specific cytotoxic lymphocytes (CTL) against prostate cancer cells, and whether bacillus Calmette-Guérin (BCG) cell-wall skeleton (CWS) can enhance the maturation of DC-PSA and the killing activity of subsequently induced PSA-specific CTL. MATERIALS AND METHODS; We generated an adenovirus encoding the PSA gene (AxCA-PSA) using the cosmid-terminal protein complex method. DC were infected with AxCA-PSA using the centrifugal method. The ability of CTL to lyse target cells expressing PSA, i.e the PSA-positive prostate cancer cell line, LNCap, and PSA-transduced autologous phytohaemagglutinin (PHA) blasts expressing PSA, was assessed using the 51Cr-release assay. The maturation of DC-PSA stimulated by BCG-CWS was assayed by flow cytometry. The cytotoxic activity enhanced by BCG-CWS was assessed by the 51Cr-release assay. RESULTS: DC-PSA induced PSA-specific CTL with 85% cytotoxic activity against LNCaP (effector: target ratio, E:T, of 50:1). However, the cytotoxic activity against PSA-negative cells was very low. Anti-CD8 and anti-major histocompatibility (MHC) class I antibodies blocked PSA-specific cytotoxicity. The PSA-specific killing was reproducible against autologous PHA blast cells expressing PSA, independently of human leukocyte antigen haplotype. Furthermore, the combination of DC-PSA with BCG-CWS remarkably enhanced the PSA-specific cytotoxicity against PHA blasts expressing PSA (15-30% at an E:T ratio of 50:1). CONCLUSION: These findings suggest that DC-PSA can induce MHC class I-restricted PSA-specific CD8+ CTL responses and that DC-PSA matured by BCG-CWS enhance PSA-specific cytotoxicity. The combination of DC-PSA with BCG-CWS might be a useful approach for treating advanced prostate cancer.

[Effect of Nocardia rubra cell wall skeleton on the growth of HeLa cell line infected with HPV].

OBJECTIVE: To investigate the effects of Nocardia rubra cell wall skeleton (Nr-CWS) on the HeLa cell line, one of the cell lines of human cervical cancer, infected with HPV. METHODS: HPV-infected HeLa (HPV 18-positive cells) cultured in vitro were divided into two groups: the experiment group and control group. Nr-CWS was added to the experiment group and PBS to the control. The growth and proliferation of HeLa cells were detected with MTT and flow cytometry technology. Inhibitive effect of HeLa transplanted tumor was investigated in Scid mice. RESULTS: The growth of HeLa cells in the experimental group was apparently decreased compared with that of the control. The results of flow cytometry demonstrated that more HeLa cells were transferred into quiescent phase in the experimental group than that in the control. While less in the proliferative phase, both of the volume and weight of HeLa transplanted tumor with drug-added group were less than those of control group. CONCLUSION: The Nocardia rubra cell wall skeleton is a potiental growth inhibitor and inducer of apoptosis of cervical cancer cells in vitro and may provide a new way in prevention or supplementary management of anti-human papilloma virus.

Gamma interferon and monophosphoryl lipid A-trehalose dicorynomycolate are efficient adjuvants for Mycobacterium tuberculosis multivalent acellular vaccine.

In this study, we examined the immunogenicity and protective efficacy of six immunodominant Mycobacterium tuberculosis recombinant antigens (85B, 38kDa, ESAT-6, CFP21, Mtb8.4, and 16kDa) in a multivalent vaccine preparation (6Ag). Gamma interferon (IFN-gamma) and monophosphoryl lipid A-trehalose dicorynomycolate (Ribi) adjuvant systems were used separately or in combination for immunization with the recombinant antigens. Our results demonstrate that immunization of mice with Ribi emulsified antigens in the presence of IFN-gamma (Ribi+6Ag+IFN-gamma) resulted after challenge with a virulent M. tuberculosis strain in a significant reduction in the CFU counts that was comparable to that achieved with the BCG vaccine ( approximately 0.9-log protection). Antigen-specific immunoglobulin G (IgG) titers in the Ribi+6Ag+IFN-gamma-immunized mice were lower than in mice immunized with Ribi+6Ag and were oriented toward a Th1-type response, as confirmed by elevated IgG2a levels. In addition, splenocyte proliferation, IFN-gamma secretion, and NO production were significantly higher in splenocytes derived from Ribi+6Ag+IFN-gamma-immunized mice, whereas IL-10 secretion was decreased. These findings confirm the induction of a strong cellular immunity in the vaccinated mice that correlates well with their enhanced resistance to M. tuberculosis. The adjuvant effect of IFN-gamma was dose dependent. A dose of 5 mug of IFN-gamma per mouse per immunization gave optimal protection, whereas lower or higher amounts (0.5 or 50 mug/ mouse) of IFN-gamma failed to enhance protection.

Mycobacterium bovis BCG cell wall-specific differentially expressed genes identified by differential display and cDNA subtraction in human macrophages.

We have analyzed the gene expression profile of monocytes in response to a highly purified cell wall fraction of Mycobacterium bovis BCG, a clinically approved adjuvant known as BCG cell wall skeleton (BCG-CWS). It is composed of mycolic acid, arabinogalactan, and peptidoglycan and confers Toll-like receptor 2 (TLR2)- and TLR4-dependent signaling that induces monocytes to differentiate into antigen-presenting cells (APCs). Here we report differential gene expression analysis with BCG-CWS-stimulated versus nonstimulated monocytes. BCG-CWS exerted massive induction of genes regulated by TLR signaling. Marked gene regulatory characteristics in BCG-CWS-stimulated cells compared to lipopolysaccharide (LPS)-stimulated cells follow. (i) Spliced mRNAs encoding soluble forms of TREM-1 and TREM-2 (recently discovered inflammatory-signal-amplifying receptors) were regulated by BCG-CWS, resulting in their differential expression. (ii) The genes for zinc-iron transporter protein (ZIP)-like family proteins HKE-1 and LIV-1 were induced exclusively by BCG-CWS. (iii) Interleukin-23 (IL-23), rather than IL-12p70, was induced by BCG-CWS, while interferon-inducible genes were induced only by LPS. By Northern and reverse transcription-PCR analyses, we confirmed the differential expression of more than 30 BCG-CWS regulatory genes, and their expression was compared with that of LPS and other known TLR ligands. A battery of genes responded rapidly and for a short time to LPS but for a long time to BCG-CWS. Structural analysis of the identified novel or hypothetical proteins revealed that some are potential candidates as signaling mediators or transcriptional regulators. Hence, BCG-CWS may profoundly modulate APC responses in a way distinct from that of LPS, leading to possible advantages for its adjuvant-active therapeutic potential.

Mycobacterium bovis BCG cell wall and lipopolysaccharide induce a novel gene, BIGM103, encoding a 7-TM protein: identification of a new protein family having Zn-transporter and Zn-metalloprotease signatures.

To identify novel genes induced during innate immune activation, we screened a cDNA library prepared from monocytes stimulated with Mycobacterium bovis BCG cell wall. A novel transcript with three-protein coding potential was identified, and the expressed proteins from individual frames showed distinct intracellular localization. Live and heat-killed Mycobacterium, bacterial cell wall, and inflammatory cytokines like TNFalpha were found to be potent inducers of the transcript. Expression of this gene is very low or undetectable in unstimulated monocytes, while a steady expression level was observed during differentiation of monocytes to dendritic cells and macrophages. The entire gene consisted of eight major exons and was localized on chromosome 4q22-q24, spanning approximately 84 kb. The main open reading frame of the transcript encoded a putative seven-transmembrane (TM) protein that showed homology with a number of functionally unknown proteins in the database. Further analysis revealed that all of these proteins have detectable similarity with the ZIP family of metal transporters. In fact, increased accumulation of intracellular Zn(2+) was observed due to the expression of BIGM103 in CHO cells. However, the identified proteins are structurally unique compared to known ZIP members and they also possess the hallmark of Zn-metalloproteases, suggesting a new class of multi-TM protein with dual features. Here we present a collection of these proteins and discuss the functional aspects of BIGM103, based on our results and current findings on two members of the family, Drosophila Catsup and Arabidopsis IAR1.

Two receptor theory in innate immune activation: studies on the receptors for bacillus Culmet Guillen-cell wall skeleton.

Activation of the innate immune system is a prerequisite for the maturation of dendritic cells (DC) and macrophages (Mphi) followed by clonal expansion of the lymphocytes, targeting cells expressing "non-self' antigens. Microbes usually have a component competent to activate DC/Mphi for antigen presentation. This component has been called adjuvant, but recently renamed "pathogen-associated molecular pattern" (PAMP) or modulin based on its molecular identification. Here, we propose the hypothesis that DC/Mphi express two sorts of receptors for PAMP, whose signaling pathways lead to a sufficient antigen (Ag)-presenting state. In bacterial infection, a Toll-like receptor (TLR) and an uptake receptor participate in DC maturation and Mphi activation. Likewise, with a number of viruses, two of the receptors, with short consensus repeats (SCR), immunoglobulin-like domains or chemokine receptor-like motifs etc. induce functional modulation of DC/Mphi. In immune therapy for cancer, primary activation of the innate system would be essential for tumor Ag-specific T cell augmentation. Cancer cells express tumor-associated Ag but barely co-express PAMP, which situation does not allow for the activation of innate immune responses. Supplementing tumor-associated Ag with PAMP may be an effective therapy for patients with cancer. Here, we discuss the possibility of an innate immune therapy for cancer with reference to bacillus Culmet Guillen cell-wall skeleton (BCG-CWS).

Interferon gamma-producing ability in blood lymphocytes of patients with lung cancer through activation of the innate immune system by BCG cell wall skeleton.

An in vitro assay system was developed to assess the potency of the human innate immune system by measurement of IL-12, IL-18, IL-10 and IFNgamma in the supernatants of bacillus Calmette-Guerin cell wall skeleton (BCG-CWS)-stimulated blood samples. BCG-CWS is a ligand for Toll-like receptor (TLR) 2 and 4, and activates monocytes to macrophages (Mphi), and immature dendritic cells to mature antigen-presenting cells (APC). This system was found to allow the discrimination of immune suppressive states in patients with lung cancer from normal immune states in light of the cytokine profile. The following results were deduced from analyses of BCG-CWS-stimulated blood samples of lung cancer patients with reference to normal subjects. (1) The levels of production of IFNgamma and IL-10 by lymphocytes were decreased. (2) IL-12 p40 production by monocytes/Mphi was upregulated, while that of IL-10 was downregulated. (3) IL-18 was detected in all patients in a range similar to normal subjects. (4) Responses of lymphocytes to IL-2 and IL- 18 in terms of IFNgamma production were diminished. (5) The upregulated IL-12 levels were recovered to within the normal range in most patients after tumor resection. (6) Male patients showed more severe suppression of IL-12/IL-18-mediated IFNgamma production than female patients. Thus, the lesser IFNgamma production observed in patients' blood with high IL-12 p40 levels in response to BCG-CWS may reflect the production of p40 dimers or IL-23 instead of p70, or the presence of some unknown pathways to prohibit the interface between the innate and acquired immune systems. BCG-CWS-mediated Toll signaling may participate in IFNgamma induction for lymphocytes through Mphi/APC IL-12/I-18 modulation.

Salivary immunoglobulin A production against a synthetic oligopeptide antigen of Actinobacillus actinomycetemcomitans fimbriae.

Actinobacillus actinomycetemcomitans is an important pathogen in human periodontal diseases. We attempted to produce effective antibodies against A. actinomycetemcomitans fimbrial antigen, which was shown to be an attachment factor. An oligopeptide based on the amino acid sequence of A. actinomycetemcomitans fimbriae was synthesized and conjugated with branched lysine polymer resin beads. Mice were immunized with the synthetic antigen together with one or more of Freund's incomplete adjuvant, the Ribi adjuvant system, liposomes, mouse interleukin 4 expression plasmids, and cholera toxin. After immunization, salivary immunoglobulin A (IgA) and serum IgG levels against the synthetic oligopeptide antigen were determined by enzyme-linked immunosorbent assay. The serum IgG responses against the fimbrial antigen were significantly higher in animals immunized intramuscularly with the Ribi adjuvant system. A high salivary IgA response to the fimbriae was induced by intranasal mucosal immunization with cholera toxin and mouse interleukin 4 expression plasmids.

Human MD-1 homologue is a BCG-regulated gene product in monocytes: its identification by differential display.

BCG-CWS is a therapeutically potent immune activator which improves the prognosis of cancer patients. However, the targeting effector cells and molecules for BCG-CWS in the human immune system have not been determined. Here, we found that BCG-CWS activates human monocytes and concomitantly down-regulates expression of a human homologue of chicken MD-1 in the activated monocytes by differential display. According to a previous study, MD-1 forms a complex with the Toll family protein RP-105 on murine B cell lines to facilitate its stable expression. Thus, MD-1 may participate in regulation of innate immune activation on human monocytes. Our results, taken together with these recent findings regarding Toll family proteins, suggest that BCG-CWS acts on monocytes to modulate the human innate immune system via regulation of Toll family proteins.

Immune responses and side effects of five different oil-based adjuvants in mice.

In this study, five different oil based adjuvants were compared to assess efficacy and side effects. Mice were injected subcutaneously (s.c.) or intraperitoneally (i.p.) with a weak immunogen (synthetic peptide) emulsified in Freund's adjuvant (FA), Specol, RIBI, TiterMax or Montanide ISA50. Efficacy of adjuvants was evaluated based on their properties to induce peptide specific IgG1, IgG2a and total IgG antibodies, native protein cross-reactive antibodies and cytokine production. Side effects were evaluated based on clinical and behavioural abnormalities, and (histo)pathological changes. Although marked differences in isotype profile and height of titre are observed among the different adjuvants used, we found that FA, Montanide ISA50 and Specol worked equally well in the s.c. and i.p. route, TiterMax functioned only when given i.p. and RIBI also did not perform up to par. The number of cytokine (interferon-gamma and interleukin-4) producing spleen cells was significantly higher after injection of RIBI compared with other adjuvants. Injection of FA or TiterMax resulted in severe pathological changes while after RIBI injection minimal changes were observed. In conclusion, high peptide specific antibody levels with limited side effects can be obtained by s.c. injection of peptide combined with Montanide ISA50 or Specol as alternatives to FA.

Protective efficacy of a recombinant protective antigen against Bacillus anthracis challenge and assessment of immunological markers.

The efficacy of recombinant Bacillus anthracis Protective Antigen (rPA) produced in Bacillus subtilis and formulated in Alhydrogel or MPL-TDM-CWS (Ribi adjuvant) has been tested and compared to the licensed UK human vaccine in guinea pigs challenged by the aerosol route with the Ames strain of B. anthracis. rPA combined with the Ribi adjuvant was found to be the only formulation to provide 100% protection from challenge. Analysis of immunological parameters in the individual animals revealed significant differences between the rPA/Ribi vaccine group and rPA/Alhydrogel and human vaccine groups for antigen specific lymphocyte proliferation, PA neutralisation and antigen specific IgG2 levels, but indicated no significant differences in PA-specific IgG1 levels. rPA formulated in Alhydrogel induced a mainly IgG1 response whilst the rPA/Ribi vaccine produced a predominantly IgG2 response.

Protective efficacy against malaria of a combination sporozoite and erythrocytic stage vaccine.

Most malariologists believe that optimal malaria vaccines will induce protective immune responses against different stages of the parasite's life cycle. A multiple antigen peptide (MAP) vaccine based on the Plasmodium yoelii circumsporozoite protein (PyCSP) protects mice against sporozoite challenge by inducing antibodies that prevent sporozoites from invading hepatocytes. A purified recombinant protein vaccine based on the P. yoelii merozoite surface protein-1 (PyMSP-1) protects mice against challenge with infected erythrocytes, presumably by inducing antibodies against the erythrocytic stage of the parasite. We now report studies designed to determine if the PyMSP-1 vaccine protects against challenge with sporozoites, the stage encountered in the field, and if immunization with a combination of the PyCSP and PyMSP-1 vaccines provides additive or synergistic protection against sporozoite challenge. In two experiments, using TiterMax or Ribi R-700 as adjuvant, 3 of 19 mice immunized with the PyMSP-1 vaccine were completely protected against sporozoite challenge. The remaining mice had significantly delayed onset and lower levels of peak parasitemia than did control mice (11.1 +/- 2.8% vs. 36.7 +/- 1.6% in experiment #2, P < 0.01). Immunization with the combination vaccine reduced by approximately 50% the level of antibodies induced to PyCSP and PyMSP-1, as compared to that induced by the individual components. However, in two experiments, there was evidence of additive protection. Six of 19 (31.6%) immunized with the PyCSP vaccine, 3 of 19 (15.8%) immunized with the PyMSP-1 vaccine, and 10 of 19 (52.6%) immunized with the combination were completely protected against sporozoit challenge. This modest increase in protection in the combination group may be a reflection of additive anti-PyCSP and anti-PyMSP-1 immunity, since mice in the combination group had diminished levels of antibodies to each components. These studies indicate that considerable work may be required to optimize the construction, delivery, and assessment of multi-stage malaria vaccines.