Formulation of HBsAg in Montanide ISA 51VG adjuvant: Immunogenicity study and monitoring long-lived humoral immune responses.

Montanide ISA 51VG adjuvant has been approved for human clinical application and stimulates cellular and humoral immune responses. Here, HBsAg was formulated in Montanide ISA51VG adjuvant to compare its potency with the Fendrix and HBsAg-alum vaccines. In particular, the long-term humoral response was assessed up to 220 days after the final immunization. BALB/c mice were allocated into six groups. Treatment groups were injected with HBsAg-Montanide ISA51VG, the Fendrix and commercial HBsAg-alum, respectively. Montanide ISA51 VG, Alum and PBS injected mice were considered as control groups. Mice were immunized three times with 2-week intervals on days 0, 14 and 28 by subcutaneous injection. Lymphocyte proliferation was assessed with the BrdU method. IFN-γ, IL-2 and IL-4 cytokines, specific total IgG and IgG1/IgG2a isotypes were assessed using ELISA. The HBsAg-Montanide ISA51VG vaccine resulted in a significant increase in lymphocyte proliferation versus HBsAg-alum and higher IL-2 cytokine production versus the Fendrix. Comparable IL-4 and IFN-γ cytokines responses were observed for these vaccines. Following the first immunization, IgG increased more in HBs-Montanide 51VG group versus the HBs-alum group, while after the second and third shots comparable responses were observed in comparison to the HBs-alum group. Monitoring for 220 days after the final vaccination showed the superiority of HBsAg-Montanide ISA 51VG vaccine versus HBsAg-alum and even the Fendrix vaccine in the induction of long-term antibody responses. This study suggests that HBsAg-Montanide ISA51VG as a novel vaccine formulation can trigger both cellular and long-lasting humoral immune responses more efficiently than conventional HBsAg vaccines.

HAMLET a human milk protein-lipid complex induces a pro-inflammatory phenotype of myeloid cells.

HAMLET is a protein-lipid complex with a specific and broad bactericidal and tumoricidal activity, that lacks cytotoxic activity against healthy cells. In this study, we show that HAMLET also has general immune-stimulatory effects on primary human monocyte-derived dendritic cells and macrophages (Mo-DC and Mo-M) and murine RAW264.7 macrophages. HAMLET, but not its components alpha-lactalbumin or oleic acid, induces mature CD14low/- CD83+ Mo-DC and M1-like CD14+ CD86++ Mo-M surface phenotypes. Concomitantly, inflammatory mediators, including IL-2, IL-6, IL-10, IL-12 and MIP-1α, were released in the supernatant of HAMLET-stimulated cells, indicating a mainly pro-inflammatory phenotype. The HAMLET-induced phenotype was mediated by calcium, NFκB and p38 MAPK signaling in Mo-DCs and calcium, NFκB and ERK signaling in Mo-M as inhibitors of these pathways almost completely blocked the induction of mature Mo-DCs and M1-like Mo-M. Compared to unstimulated Mo-DCs, HAMLET-stimulated Mo-DCs were more potent in inducing T cell proliferation and HAMLET-stimulated macrophages were more efficient in phagocytosis of Streptococcus pneumoniae in vitro. This indicates a functionally activated phenotype of HAMLET-stimulated DCs and macrophages. Combined, we propose that HAMLET has a two-fold anti-bacterial activity; one inducing direct cytotoxic activity, the other indirectly mediating elimination of bacteria by activation of immune cells of the myeloid lineage.

EpOMEs act as immune suppressors in a lepidopteran insect, Spodoptera exigua.

Epoxyoctadecamonoenoic acids (EpOMEs) are epoxide derivatives of linoleic acid (9,12-octadecadienoic acid) and include 9,10-EpOME and 12,13-EpOME. They are synthesized by cytochrome P450 monooxygenases (CYPs) and degraded by soluble epoxide hydrolase (sEH). Although EpOMEs are well known to play crucial roles in mediating various physiological processes in mammals, their role is not well understood in insects. This study chemically identified their presence in insect tissues: 941.8 pg/g of 9,10-EpOME and 2,198.3 pg/g of 12,13-EpOME in fat body of a lepidopteran insect, Spodoptera exigua. Injection of 9,10-EpOME or 12,13-EpOME into larvae suppressed the cellular immune responses induced by bacterial challenge. EpOME treatment also suppressed the expression of antimicrobial peptide (AMP) genes. Among 139 S. exigua CYPs, an ortholog (SE51385) to human EpOME synthase was predicted and its expression was highly inducible upon bacterial challenge. RNA interference (RNAi) of SE51385 prevented down-regulation of immune responses at a late stage (> 24 h) following bacterial challenge. A soluble epoxide hydrolase (Se-sEH) of S. exigua was predicted and showed specific expression in all development stages and in different larval tissues. Furthermore, its expression levels were highly enhanced by bacterial challenge in different tissues. RNAi reduction of Se-sEH interfered with hemocyte-spreading behavior, nodule formation, and AMP expression. To support the immune association of EpOMEs, urea-based sEH inhibitors were screened to assess their inhibitory activities against cellular and humoral immune responses of S. exigua. 12-(3-adamantan-1-yl-ureido) dodecanoic acid (AUDA) was highly potent in suppressing the immune responses. The addition of AUDA to a pathogenic bacterium significantly increased bacterial pathogenicity by suppressing host immune defense. In sum, this study demonstrated that EpOMEs play a crucial role in facilitating anti-inflammatory responses in S. exigua.

Incomplete Freund's adjuvant reduces arginase and enhances Th1 dominance, TLR signaling and CD40 ligand expression in the vaccine site microenvironment.

BACKGROUND: Immunogenicity of cancer vaccines is impacted by adjuvants and schedule, but systematic assessments of their effects have not been performed. Montanide ISA-51, an incomplete Freund's adjuvant (IFA), is used in many vaccine trials, but concerns have been raised about negative effects in murine studies. We found in humans that IFA enhances systemic immune responses and that repeat vaccination at one site (same site vaccination (SSV)) creates tertiary lymphoid structures (TLS) in the vaccine site microenvironment (VSME). We hypothesized that vaccination with peptides+IFA+pICLC or SSV×3 with peptides in IFA would create an immunogenic milieu locally at the VSME, with activated dendritic cells (DC), TLS-associated chemokines and a Th1-dominant VSME. METHODS: Biopsies of the VSME were obtained from participants on two clinical trials who were immunized with multiple melanoma peptides (MELITAC 12.1) in adjuvants comprising IFA and/or the TLR3-agonist pICLC. Biopsies were obtained either a week after one vaccine or a week after SSV×3. Controls included normal skin and skin injected with IFA without peptides. Gene expression analysis was performed by RNAseq. RESULTS: VSME samples were evaluated from 27 patients. One vaccine with peptides in pICLC+IFA enhanced expression of CD80, CD83, CD86 (p<0.01), CD40 and CD40L (p<0.0001) over normal skin; these effects were significantly enhanced for SSV with peptides+IFA. Vaccines containing pICLC increased expression of TBX21 (T-bet) but did not decrease GATA3 over normal skin, whereas SSV with peptides in IFA dramatically enhanced TBX21 and decreased GATA3, with high expression of IFNγ and STAT1. SSV with peptides in IFA also reduced arginase-1 (ARG1) expression and enhanced expression of TLR adapter molecules TICAM-1 (TRIF) and MYD88. Furthermore, SSV with IFA and peptides also enhanced expression of chemokines associated with TLS formation. CONCLUSIONS: These findings suggest that SSV with peptides in IFA enhances CD40L expression by CD4 T cells, supports a Th1 microenvironment, with accumulation of activated and mature DC. Increased expression of TLR adaptor proteins after SSV with peptides in IFA might implicate effects of the skin microbiome. Reduced ARG1 may reflect diminished suppressive myeloid activity in the VSME. TRIAL REGISTRATION NUMBER: (NCT00705640, NCT01585350).

9t18:1 and 11t18:1 activate the MAPK pathway to regulate the expression of PLA2 and cause inflammation in HUVECs.

trans fatty acids (TFAs) have been reported to promote vascular diseases mainly by promoting apoptosis and inflammation of vascular endothelial cells. However, it has been reported in recent years that elaidic acid (9t18:1) and vaccenic acid (11t18:1) may have different effects on vascular health. This study investigated the effects of 9t18:1 and 11t18:1 on human umbilical vein endothelial cell (HUVEC) function and the possible mechanism of inflammation by analyzing the changes in the phospholipid composition and the relationship between phospholipase A2 (PLA2) and MAPK pathway. Here we found that the effect of 11t18:1 on cell viability, membrane damage and cellular inflammation was significantly lower than that of 9t18:1 (p < 0.05). And 9t18:1 and 11t18:1 had different effects on phospholipid composition. Both 9t18:1 and 11t18:1 significantly increased the protein expression of PLA2. Moreover, the MAPK pathway regulated the expression of PLA2, inflammatory cytokines and cyclooxygenase-2 (COX-2) and the secretion of prostaglandin E2 (PGE2) in HUVECs induced by 9t18:1 and 11t18:1. In conclusion, 9t18:1 and 11t18:1 activated the MAPK pathway which regulated the expression of PLA2 to cause inflammation in HUVECs.

Protective efficacy of inactivated reverse genetics based equine influenza vaccine candidate adjuvanted with MontanideTM Pet Gel in murine model.

Equine influenza is a leading cause for respiratory illness in equines. Major control measures involve vaccination which requires continuous harmonization owing to antigenic drift. The present study focused on assessing the protective efficacy of an inactivated recombinant equine influenza virus (rgEIV) vaccine candidate adjuvanted with MontanideTM Pet Gel in murine model. The rgEIV was generated using reverse genetics by incorporating HA and NA segments from EIV/H3N8, clade 2-Florida sublineage in an A/WSN/33 /H1N1 backbone and inactivated by formalin. The vaccine was prepared by mixing inactivated rgEIV with MontanideTM Pet Gel adjuvant followed by intranasal inoculation into BALB/c mice intranasally. The immune responses and protective efficacy of the vaccine was evaluated by measurement of antibody titer, immunoglobulin subtyping, cytokines, clinical signs and pathological lesions after immunization and challenge with wild EIV. Serology and cytokine expression pattern indicated that the vaccine activated mixed Th1- and Th2-like responses of vaccine. Booster immunization stimulated strong antibody responses (HAI titre: 192 ± 28.6) at 42 days post immunization and the predominant antibody subtype was IgG1. Upregulation of interferon (IFN)-gamma, interleukin (IL)-12 and IL-2 levels indicates effective induction of Th1 type response. We found that vaccination has protected mice against equine influenza virus challenge as adjudged through a lack of nonappearance of visible clinical signs of disease, no loss of body weight loss, reduced pathology in the lungs and markedly reduced virus shedding from the respiratory tract. Therefore, we conclude that recombinant EIV vaccine candidate adjuvanted with MontanideTM Pet Gel could aid in quick harmonization of the vaccines through replacement of HA and NA genes for control of EIV outbreaks.

Raising antibodies against epoxyscillirosidine, the toxic principle contained in Moraea pallida Bak. (Iridaceae), in rabbits.

Moraea pallida Bak. (yellow tulp) poisoning is the most important plant cardiac glycoside toxicosis in South Africa. The toxic principle, a bufadienolide, is 1α, 2α-epoxyscillirosidine. The aim was to investigate the potential to develop a vaccine against epoxyscillirosidine. Epoxyscillirosidine, proscillaridin and bufalin, were successfully conjugated to hen ovalbumin (OVA), bovine serum albumin (BSA) and keyhole limpet haemocyanin (KLH). There was a low immune response following vaccination of adult male New Zealand White rabbits with epoxyscillirosidine-OVA (n = 3) and OVA (n = 3) using Freund's adjuvant in Trial (T) 1. The immune response improved significantly in T2 following doubling of the dose to 0.8 mg/rabbit and changing the adjuvant to Montanide. In T3, the rabbits (n = 15), allocated into 5 equal groups, vaccinated with proscillaridin-BSA, bufalin-BSA, epoxyscillirosidine-KLH, epoxyscillirosidine-BSA and BSA respectively, using Montanide adjuvant, developed antibodies against the administered immunogens, with epoxyscillirosidine-KLH inducing the highest immune response. Proscillaridin and bufalin antibodies cross-reacted with epoxyscillirosidine in an enzyme linked immunosorbent assay. The conjugation methodology will be adjusted in the future to target optimal conjugation efficiency. Additional vaccination will be conducted in search of neutralizing antibodies against the yellow tulp toxin. The cross-reactivity of proscillaridin and bufalin antibodies with epoxyscillirosidine could be studied in future to explore the potential to prevent yellow tulp poisoning.

The fatty acid oleate is required for innate immune activation and pathogen defense in Caenorhabditis elegans.

Fatty acids affect a number of physiological processes, in addition to forming the building blocks of membranes and body fat stores. In this study, we uncover a role for the monounsaturated fatty acid oleate in the innate immune response of the nematode Caenorhabditis elegans. From an RNAi screen for regulators of innate immune defense genes, we identified the two stearoyl-coenzyme A desaturases that synthesize oleate in C. elegans. We show that the synthesis of oleate is necessary for the pathogen-mediated induction of immune defense genes. Accordingly, C. elegans deficient in oleate production are hypersusceptible to infection with diverse human pathogens, which can be rescued by the addition of exogenous oleate. However, oleate is not sufficient to drive protective immune activation. Together, these data add to the known health-promoting effects of monounsaturated fatty acids, and suggest an ancient link between nutrient stores, metabolism, and host susceptibility to bacterial infection.

Bioactive lipids ALIAmides differentially modulate inflammatory responses of distinct subsets of primary human T lymphocytes.

Autacoid local injury antagonist amides (ALIAmides) are a family of endogenous bioactive acyl ethanolamides that include the renowned palmitoyl ethanolamide (PEA), oleoyl ethanolamide (OEA), and stearoyl ethanolamide (SEA), and that are involved in several biologic processes such as nociception, lipid metabolism, and inflammation. The role of ALIAmides in the control of inflammatory processes has recently gained much attention and prompted the use of these molecules or their analogs, and the pharmacologic manipulation of their endogenous levels, as plausible therapeutic strategies in the treatment of several chronic inflammatory conditions. Since chronic inflammation is mainly driven by cells of adaptive immunity, particularly T lymphocytes, we aimed at investigating whether such bioactive lipids could directly modulate T-cell responses. We found that OEA, PEA, and eicosatrienoyl ethanolamide (ETEA) could directly inhibit both T-cell responses by reducing their production of TNF-α and IFN-γ from CD8 T cells and TNF-α, IFN-γ and IL-17 from CD4 T cells. Furthermore, neither SEA nor docosatrienoyl ethanolamide (DTEA) could affect cytokine production from both T cell subsets. Interestingly, unlike OEA and ETEA, PEA was also able to enhance de novo generation of forkhead box P3 (FoxP3)-expressing regulatory T cells from CD4-naive T cells. Our findings show for the first time that specific ALIAmides can directly affect different T-cell subsets, and provide proof of their anti-inflammatory role in chronic inflammation, ultimately suggesting that these bioactive lipids could offer novel tools for the management of T-cell dependent chronic inflammatory diseases.-Chiurchiù, V., Leuti, A., Smoum, R., Mechoulam, R., Maccarrone, M. Bioactive lipids ALIAmides differentially modulate inflammatory responses of distinct subsets of primary human T lymphocytes.

Topical electrophilic nitro-fatty acids potentiate cutaneous inflammation.

Endogenous electrophilic fatty acids mediate anti-inflammatory responses by modulating metabolic and inflammatory signal transduction and gene expression. Nitro-fatty acids and other electrophilic fatty acids may thus be useful for the prevention and treatment of immune-mediated diseases, including inflammatory skin disorders. In this regard, subcutaneous (SC) injections of nitro oleic acid (OA-NO2), an exemplary nitro-fatty acid, inhibit skin inflammation in a model of allergic contact dermatitis (ACD). Given the nitration of unsaturated fatty acids during metabolic and inflammatory processes and the growing use of fatty acids in topical formulations, we sought to further study the effect of nitro-fatty acids on cutaneous inflammation. To accomplish this, the effect of topically applied OA-NO2 on skin inflammation was evaluated using established murine models of contact hypersensitivity (CHS). In contrast to the effects of subcutaneously injected OA-NO2, topical OA-NO2 potentiated hapten-dependent inflammation inducing a sustained neutrophil-dependent inflammatory response characterized by psoriasiform histological features, increased angiogenesis, and an inflammatory infiltrate that included neutrophils, inflammatory monocytes, and γδ T cells. Consistent with these results, HPLC-MS/MS analysis of skin from psoriasis patients displayed a 56% increase in nitro-conjugated linoleic acid (CLA-NO2) levels in lesional skin compared to non-lesional skin. These results suggest that nitro-fatty acids in the skin microenvironment are products of cutaneous inflammatory responses and, in high local concentrations, may exacerbate inflammatory skin diseases.

Analysis of adipose tissue immune gene expression after vaccination of rainbow trout with adjuvanted bacterins reveals an association with side effects.

Most existing fish vaccines are presented in the form of oil-based emulsions delivered by intraperitoneal injection. Whilst very effective they are frequently associated with inflammatory responses that can result in clinically significant side-effects often involving the adipose tissue that is in direct contact with the vaccine. To explore the potential of immune gene expression changes in the adipose tissue of fish to be markers of vaccination efficacy or development of side-effects we have studied the response to a bacterial (Aeromonas salmonicida) vaccine administered with two different adjuvants. The first adjuvant was Montanide™ ISA 763A VG, thought to induce a mostly humoral response, and the second was Montanide™ ISA 761 VG that gives a more balanced humoral and cell mediated response. Following vaccination tissue samples were collected at days 3, 14 and 28 for RTqPCR analysis. Fifty immune genes were studied with a focus on a) pro-inflammatory associated molecules and b) adaptive immune response related molecules linked with possible Th1, Th2, Th17 and T-regulatory pathways, with the expression data analysed for associations with Speilberg post-vaccination side effect scores. The results showed that the adipose tissue is a particularly sensitive and discriminatory tissue for studying adjuvant effects. A clear upregulation of many immune genes occurred in response to both vaccine groups, which persisted over time and overlapped with the appearance of visible adhesions. Our analysis revealed a relationship between adipose tissue immune function and the development of vaccine-induced adhesions giving the potential to use immune gene expression profiling in this tissue to predict the side-effects seen.

Montanide ISA 71 VG is Advantageous to Freund's Adjuvant in Immunization Against S. aureus Infection of Mice.

The enormous capacity of Staphylococcus aureus to acquire antibiotic resistance makes it a permanent task to search for and to develop new anti-infectives. One of the possible approaches is the early active immunization of risk patients and animal stocks to prevent S. aureus infections. Based on a S. aureus proteome screen with S. aureus-specific human antiserum, we have previously identified several anchorless cell wall proteins to be used as novel vaccine candidates. To develop an efficient anti-S. aureus vaccine, the supplemented adjuvants Montanide(™) ISA 71 VG and ISA 206 were compared to Freund's adjuvant in terms of handling, induction of cytokine profile, triggering antigen-specific immunoglobulin production of different IgG subclasses and provision of increased survival rates in our S. aureus sepsis mouse model. Immunization with ISA 71 VG in comparison with Freund's adjuvant induced slightly delayed but comparably strong increase of antigen-specific antibody titres and conferred protective effect against S. aureus challenge. In contrast using ISA 206 as adjuvant, significantly lower IgG titres and consequently, no protective effect against S. aureus infection were observed. Handling and tolerability of the Montanide is superior to Freund's adjuvant. Montanide(™) ISA 71 VG can serve as an effective adjuvant replacement for Freund's adjuvant in research with a prospective usage in animal and human vaccines against bacterial pathogens.

Evidence for induction of humoral and cytotoxic immune responses against devil facial tumor disease cells in Tasmanian devils (Sarcophilus harrisii) immunized with killed cell preparations.

Tasmanian devils (Sarcophilus harrisii) risk extinction from a contagious cancer, devil facial tumour disease (DFTD) in which the infectious agent is the tumor cell itself. Because devils are unable to produce an immune response against the tumor cells no devil has survived 'infection'. To promote an immune response we immunized healthy devils with killed DFTD tumor cells in the presence of adjuvants. Immune responses, including cytotoxicity and antibody production, were detected in five of the six devils. The incorporation of adjuvants that act via toll like receptors may provide additional signals to break 'immunological ignorance'. One of these devils was protected against a challenge with viable DFTD cells. This was a short-term protection as re-challenge one year later resulted in tumor growth. These results suggest that Tasmanian devils can generate immune responses against DFTD cells. With further optimization of immune stimulation it should be possible to protect Tasmanian devils against DFTD with an injectable vaccine.

Phase I clinical trial of vaccination with LY6K-derived peptide in patients with advanced gastric cancer.

BACKGROUND: Lymphocyte antigen 6 complex locus K (LY6K) has been identified as a tumor-associated antigen in lung cancers and esophageal squamous cell carcinomas. The immunogenicity of LY6K-177 peptide vaccine therapy has been demonstrated in patients with advanced esophageal cancer. This study extends this treatment to gastric cancer. METHODS: LY6K expression in clinical samples obtained from gastric cancer patients was examined by immunochemistry. As a phase I clinical trial, the safety and immunogenicity of LY6K-177 peptide vaccine emulsified with Montanide ISA 51 was evaluated in six patients with unresectable advanced gastric cancer. LY6K-177 peptide (1 mg in 1 ml sterile saline) was emulsified with incomplete Freund's adjuvant (1 ml) and intracutaneously administered to the inguinal region or axilla. One treatment course comprised four vaccinations, performed weekly for the first and second treatment courses and biweekly for the third treatment course. RESULTS: LY6K expression was confirmed in 85 % of gastric cancer tissues. Induration and redness at the vaccination site (grade I), possibly a delayed-type hypersensitivity reaction, was observed in all patients; however, no systemic toxicology was identified in any patient throughout the observation period. Three of the six patients had stable disease, and a tumor contraction effect was observed in one patient. CONCLUSIONS: LY6K was expressed in 85 % of observed gastric cancers. Vaccination with LY6K-177 peptide/Montanide ISA 51 appeared to be tolerated by advanced gastric cancer patients, and moreover anticancer efficacy was suggested. This trial was registered with ClinicalTrial.gov (no. NCT00845611).

Peptide vaccination in Montanide adjuvant induces and GM-CSF increases CXCR3 and cutaneous lymphocyte antigen expression by tumor antigen-specific CD8 T cells.

T cell infiltration of melanoma is associated with enhanced clinical efficacy and is a desirable endpoint of immunotherapeutic vaccination. Infiltration is regulated, in part, by chemokine receptors and selectin ligands on the surface of tumor-specific lymphocytes. Therefore, we investigated the expression of two homing molecules--CXCR3 and CLA - on vaccine-induced CD8 T cells, in the context of a clinical trial of a melanoma-specific peptide vaccine. Both CXCR3 and CLA have been associated with T cell infiltration of melanoma. We demonstrate that a single subcutaneous/intradermal administration of peptide vaccine in Montanide adjuvant induces tumor-specific CD8 T cells that are predominantly positive for CXCR3, with a subpopulation of CXCR3(+)CLA(+) cells. Addition of GM-CSF significantly enhances CXCR3 expression and increases the proportion of CLA-expressing cells. Concurrent with CXCR3 and CLA expression, vaccine-induced CD8 cells express high levels of Tbet, IFN-γ, and IL-12Rß1. Collectively, these studies demonstrate that peptide vaccination in adjuvant induces CD8 T cells with a phenotype that may support infiltration of melanoma.

A multi-peptide, dual-adjuvant telomerase vaccine (GX301) is highly immunogenic in patients with prostate and renal cancer.

BACKGROUND: Anti-tumor vaccination is a new frontier in cancer treatment applicable to immunogenic neoplasms such as prostate and renal cancers. GX301 is a vaccine constituted by four telomerase peptides and two adjuvants, Montanide ISA-51 and Imiquimod. OBJECTIVE: The aim of this study was to analyze safety and tolerability of GX301 in an open-label, phase I/II trial. Immunological and clinical responses were also evaluated as secondary endpoints. EXPERIMENTAL DESIGN: GX301 was administered by intradermally injecting 500 µg of each peptide (dissolved in Montanide ISA-51) in the skin of the abdomen. Imiquimod was applied as a cream at the injection sites. The protocol included 8 administrations at days 1, 3, 5, 7, 14, 21, 35, 63. Eligible patients were affected with stage IV prostate or renal cancer resistant to conventional treatments. Patients were clinically and immunologically monitored up to 6 months from the first immunization. RESULTS: No grade 3-4 adverse events were observed. Evidence of vaccine-specific immunological responses was detected in 100 % of patients. Disease stabilization occurred in 4 patients. Prolonged progression-free survival and overall survival were observed in patients showing a full pattern of vaccine-specific immunological responses. CONCLUSION: GX301 demonstrated to be safe and highly immunogenic. Further studies are needed to determine its clinical efficacy.

A unifying mechanism for cancer cell death through ion channel activation by HAMLET.

Ion channels and ion fluxes control many aspects of tissue homeostasis. During oncogenic transformation, critical ion channel functions may be perturbed but conserved tumor specific ion fluxes remain to be defined. Here we used the tumoricidal protein-lipid complex HAMLET as a probe to identify ion fluxes involved in tumor cell death. We show that HAMLET activates a non-selective cation current, which reached a magnitude of 2.74±0.88 nA within 1.43±0.13 min from HAMLET application. Rapid ion fluxes were essential for HAMLET-induced carcinoma cell death as inhibitors (amiloride, BaCl2), preventing the changes in free cellular Na(+) and K(+) concentrations also prevented essential steps accompanying carcinoma cell death, including changes in morphology, uptake, global transcription, and MAP kinase activation. Through global transcriptional analysis and phosphorylation arrays, a strong ion flux dependent p38 MAPK response was detected and inhibition of p38 signaling delayed HAMLET-induced death. Healthy, differentiated cells were resistant to HAMLET challenge, which was accompanied by innate immunity rather than p38-activation. The results suggest, for the first time, a unifying mechanism for the initiation of HAMLET's broad and rapid lethal effect on tumor cells. These findings are particularly significant in view of HAMLET's documented therapeutic efficacy in human studies and animal models. The results also suggest that HAMLET offers a two-tiered therapeutic approach, killing cancer cells while stimulating an innate immune response in surrounding healthy tissues.

Effect of Montanide and poly-ICLC adjuvant on human self/tumor antigen-specific CD4+ T cells in phase I overlapping long peptide vaccine trial.

Vaccination of patients with ovarian cancer with overlapping long peptides (OLP) from cancer-testis antigen NY-ESO-1 and poly-ICLC in Montanide-ISA-51 (Montanide) was found to consistently induce integrated immune responses (antibody, CD4(+), and CD8(+) T cells). Using detailed methods, we investigated the respective effects of poly-ICLC and Montanide adjuvant on pre- and postvaccine NY-ESO-1-specific CD4(+) T cells, because of their central function for induction and maintenance of both antibody and CD8(+) T cells. Polyclonal NY-ESO-1-specific CD4(+) T-cell lines were generated from 12 patients using CD154-based selection of precursors before and after vaccination with (i) OLP alone, (ii) OLP in Montanide, or (iii) OLP and poly-ICLC in Montanide. Kinetics, quantification, fine specificity, avidity, and cytokine-producing pattern were analyzed in depth and compared between vaccine cohorts. Vaccination with OLP alone did not elicit CD4(+) T-cell responses; it suppressed high-avidity CD4(+) T-cell precursors that recognized naturally processed NY-ESO-1 protein before vaccination. Emulsification of OLP in Montanide was required for the expansion of high-avidity NY-ESO-1-specific CD4(+) T-cell precursors. Poly-ICLC significantly enhanced CD4(+) Th1 responses while suppressing the induction of interleukin (IL)-4-producing Th2 and IL-9-producing Th9 cells. In summary, Montanide and poly-ICLC had distinct and cooperative effects for the induction of NY-ESO-1-specific Th1 cells and integrated immune responses by OLP vaccination. These results support the use of admixing poly-ICLC in Montanide adjuvant to rapidly induce antitumor type I immune responses by OLP from self/tumor antigens in human cancer vaccines.

Effects on immunogenicity by formulations of emulsion-based adjuvants for malaria vaccines.

New malaria vaccines are urgently needed to improve vaccine protective efficacy. PfCelTOS is a recombinant malaria vaccine antigen that has shown protective efficacy in a small-animal challenge model when combined with a water-in-oil emulsion adjuvant (Montanide ISA 720). In this report, we show that PfCelTOS vaccines containing GLA-SE (a stable oil-in-water emulsion combined with a Toll-like receptor 4 [TLR4] agonist) elicit strong Th1-type immune responses in BALB/c mice. These responses include higher antigen-specific IgG2a antibody titers and more gamma interferon (IFN-γ) production than those seen with a PfCelTOS vaccine containing Montanide ISA 720. Furthermore, reducing the emulsion dose from 2% to 1% or 0.5% (vol/vol) squalene in GLA-SE did not compromise immunogenicity. Emulsion dose titration in the absence of formulated GLA caused some reduction in humoral and cellular immune responses compared to those with the 2% squalene emulsion dose.

Phase I trial of Wilms' Tumor 1 (WT1) peptide vaccine with GM-CSF or CpG in patients with solid malignancy.

BACKGROUND: The aim of this study was to investigate the safety and efficacy of combinatorial use of granulocyte-macrophage colony-stimulating factor (GM-CSF) and CpG oligodeoxynucleotides (CpG-ODN) as immunoenhancement adjuvants in Wilms' Tumor 1 (WT1) vaccine therapy for patients with solid malignancy. PATIENTS AND METHODS: The patients were placed into treatment groups as follows: WT1 peptide alone, WT1 peptide with GM-CSF (100 µg) and WT1 peptide with CpG-ODN (100 µg). HLA-A *2402 or *0201/*0206-restricted, WT1 peptide emulsified with Montanide ISA51 was injected intradermally every week for eight weeks. Toxicities were evaluated according to the National Cancer Institute Common Terminology Criteria for Adverse Events ver. 3.0. Tumor size, which was measured by computed tomography, was determined every four weeks. The responses were analyzed according to Response Evaluation Criteria in Solid Tumors. RESULTS: The protocol was well tolerated; only local erythema occurred at the WT1 vaccine injection site. The disease control rate of the groups treated with WT1 peptide alone (n=10), with combinatorial use of GM-CSF (n=8) and with combinatorial use of CpG-ODN (n=10), in the initial two months was 20%, 25% and 60%, respectively. CONCLUSION: Addition of GM-CSF or CpG-ODN to the WT1 peptide vaccine for patients with solid malignancy was safe and improved the effectiveness of clinical response.