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.

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).

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.

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.

Unique interplay between sugar and lipid in determining the antigenic potency of bacterial antigens for NKT cells.

Invariant natural killer T (iNKT) cells are an evolutionary conserved T cell population characterized by features of both the innate and adaptive immune response. Studies have shown that iNKT cells are required for protective responses to Gram-positive pathogens such as Streptococcus pneumoniae, and that these cells recognize bacterial diacylglycerol antigens presented by CD1d, a non-classical antigen-presenting molecule. The combination of a lipid backbone containing an unusual fatty acid, vaccenic acid, as well as a glucose sugar that is weaker or not stimulatory when linked to other lipids, is required for iNKT cell stimulation by these antigens. Here we have carried out structural and biophysical studies that illuminate the reasons for the stringent requirement for this unique combination. The data indicate that vaccenic acid bound to the CD1d groove orients the protruding glucose sugar for TCR recognition, and it allows for an additional hydrogen bond of the glucose with CD1d when in complex with the TCR. Furthermore, TCR binding causes an induced fit in both the sugar and CD1d, and we have identified the CD1d amino acids important for iNKT TCR recognition and the stability of the ternary complex. The studies show also how hydrogen bonds formed by the glucose sugar can account for the distinct binding kinetics of the TCR for this CD1d-glycolipid complex. Therefore, our studies illuminate the mechanism of glycolipid recognition for antigens from important pathogens.

Long-term humoral and cellular immune responses elicited by a heterologous Plasmodium vivax apical membrane antigen 1 protein prime/adenovirus boost immunization protocol.

Apical membrane antigen 1 (AMA-1) is an invasion-related Plasmodium antigen that is expressed during both intracellular and extracellular asexual stages of the parasite's life cycle, making it an ideal target for induction of humoral and cellular immune responses that can protect against malaria. We show here that when it is administered as a recombinant protein (P) in Montanide ISA720 adjuvant, followed by a recombinant human type 5 adenovirus (Ad), intense and long-lasting Plasmodium vivax AMA-1-specific antibody responses (including both IgG1 and IgG2a), as well as proliferative memory T cell responses, can be detected in immunized mice. Memory T cells displayed both central (CD44(hi) CD62L(hi)) and effector (CD44(hi) CD62L(lo)) phenotypes, with the central memory phenotype prevailing (56% of AMA-1-specific proliferating cells). Considering the main traits of the memory immune responses induced against AMA-1, this particular sequence of immunogens (P followed by Ad), but no others (Ad/Ad, Ad/P, or P/P), displayed an optimal synergistic effect. These results give further support to the need for preclinical studies of P. vivax vaccine candidate AMA-1 administered in prime/boost protocols that include recombinant proteins and adenoviral vectors.

Adjuvants for enhancing the immunogenicity of whole tumor cell vaccines.

Whole tumor cell lysates can serve as excellent multivalent vaccines for priming tumor-specific CD8(+) and CD4(+) T cells. Whole cell vaccines can be prepared with hypochlorous acid oxidation, UVB-irradiation and repeat cycles of freeze and thaw. One major obstacle to successful immunotherapy is breaking self-tolerance to tumor antigens. Clinically approved adjuvants, including Montanide™ ISA-51 and 720, and keyhole-limpet proteins can be used to enhance tumor cell immunogenicity by stimulating both humoral and cellular anti-tumor responses. Other potential adjuvants, such as Toll-like receptor agonists (e.g., CpG, MPLA and PolyI:C), and cytokines (e.g., granulocyte-macrophage colony stimulating factor), have also been investigated.

A phase I study of vaccination with NY-ESO-1f peptide mixed with Picibanil OK-432 and Montanide ISA-51 in patients with cancers expressing the NY-ESO-1 antigen.

We conducted a phase I clinical trial of a cancer vaccine using a 20-mer NY-ESO-1f peptide (NY-ESO-1 91-110) that includes multiple epitopes recognized by antibodies, and CD4 and CD8 T cells. Ten patients were immunized with 600 µg of NY-ESO-1f peptide mixed with 0.2 KE Picibanil OK-432 and 1.25 ml Montanide ISA-51. Primary end points of the study were safety and immune response. Subcutaneous injection of the NY-ESO-1f peptide vaccine was well tolerated. Vaccine-related adverse events observed were fever (Grade 1), injection-site reaction (Grade 1 or 2) and induration (Grade 2). Vaccination with the NY-ESO-1f peptide resulted in an increase or induction of NY-ESO-1 antibody responses in nine of ten patients. The sera reacted with recombinant NY-ESO-1 whole protein as well as the NY-ESO-1f peptide. An increase in CD4 and CD8 T cell responses was observed in nine of ten patients. Vaccine-induced CD4 and CD8 T cells responded to NY-ESO-1 91-108 in all patients with various HLA types with a less frequent response to neighboring peptides. The findings indicate that the 20-mer NY-ESO-1f peptide includes multiple epitopes recognized by CD4 and CD8 T cells with distinct specificity. Of ten patients, two with lung cancer and one with esophageal cancer showed stable disease. Our study shows that the NY-ESO-1f peptide vaccine was well tolerated and elicited humoral, CD4 and CD8 T cell responses in immunized patients.

Mice drinking goji berry juice (Lycium barbarum) are protected from UV radiation-induced skin damage via antioxidant pathways.

The goji berry, Lycium barbarum, has long been recognised in traditional Chinese medicine for various therapeutic properties based on its antioxidant and immune-modulating effects. This study describes the potential for orally consumed goji berry juice to alter the photodamage induced in the skin of mice by acute solar simulated UV (SSUV) irradiation. In Skh:hr-1 hairless mice, 5% goji berry juice significantly reduced the inflammatory oedema of the sunburn reaction. Dilutions of goji berry juice between 1% and 10% dose-dependently protected against SSUV-induced immunosuppression, and against suppression induced by the mediator, cis-urocanic acid, measured by the contact hypersensitivity reaction. The immune protection could not be ascribed to either the minor excipients in the goji juice, pear and apple juice, nor the vitamin C content, nor the preservative, and appeared to be a property of the goji berry itself. Antioxidant activity in the skin was demonstrated by the significant protection by 5% goji juice against lipid peroxidation induced by UVA radiation. Furthermore, two known inducible endogenous skin antioxidants, haem oxygenase-1 and metallothionein, were found to be involved in the photoimmune protection. The results suggest that consumption of this juice could provide additional photoprotection for susceptible humans.

Safety and immunogenicity study of NY-ESO-1b peptide and montanide ISA-51 vaccination of patients with epithelial ovarian cancer in high-risk first remission.

PURPOSE: The cancer-testis antigen NY-ESO-1 is expressed by >40% of advanced epithelial ovarian cancers and is a promising immunotherapeutic target. In this study, we describe the effects of vaccination with the HLA-A*0201-restricted NY-ESO-1b peptide on patients with epithelial ovarian cancer in high-risk first remission. EXPERIMENTAL DESIGN: After primary surgery and chemotherapy, high-risk epithelial ovarian cancer patients in first clinical remission received NY-ESO-1b peptide and Montanide every 3 weeks for five vaccinations. Tumor expression was evaluated by immunohistochemistry. Toxicity was monitored using National Cancer Institute Common Toxicity Criteria Scale Version 2. NY-ESO-1 specific humoral immunity (ELISA), T-cell immunity (tetramer and ELISPOT), and delayed-type hypersensitivity were assessed on weeks 0, 1, 4, 7, 10, 13, and 16. RESULTS: Treatment-related adverse events included grade 1 fatigue, anemia, pruritus, myalgias, and hyperthyroidism and grade 2 hypothyroidism. There were no grade 3/grade 4 adverse events. Three of four patients (75%) with NY-ESO-1-positive tumor showed T-cell immunity by tetramer (0.6-9.5%) and ELISPOT (range, 35-260 spots). Four of five patients (80%) with NY-ESO-1-negative tumor showed T-cell immunity by tetramer (1.0-12.1%) and/or ELISPOT (range, 35-400 spots). With a median follow-up of 11.3 months, six of nine patients (67%) have recurred, with a median progression-free survival of 13 months (95% confidence interval, 11.2 months-not reached). Three of nine patients remain in complete clinical remission at 25, 38, and 52 months. CONCLUSION: Vaccination of high-risk HLA-A*0201-positive epithelial ovarian cancer patients with NY-ESO-1b and Montanide has minimal toxicity and induces specific T-cell immunity in patients with both NY-ESO-1-positive and NY-ESO-1-negative tumors. Additional study is warranted.

Bovine milk fat enriched in conjugated linoleic and vaccenic acids attenuates allergic airway disease in mice.

BACKGROUND: It has been argued that a reduction in the Western diet of anti-inflammatory unsaturated lipids, such as n-3 polyunsaturated fatty acids, has contributed to the increase in the frequency and severity of allergic diseases. OBJECTIVE: We investigated whether feeding milk fat enriched in conjugated linoleic acid and vaccenic acids (VAs) ('enriched' milk fat), produced by supplementing the diet of pasture-fed cows with fish and sunflower oil, will prevent development of allergic airway responses. METHODS: C57BL/6 mice were fed a control diet containing soybean oil and diets supplemented with milk lipids. They were sensitized by intraperitoneal injection of ovalbumin (OVA) on days 14 and 28, and challenged intranasally with OVA on day 42. Bronchoalveolar lavage fluid, lung tissues and serum samples were collected 6 days after the intranasal challenge. RESULTS: Feeding of enriched milk fat led to marked suppression of airway inflammation as evidenced by reductions in eosinophilia and lymphocytosis in the airways, compared with feeding of normal milk fat and control diet. Enriched milk fat significantly reduced circulating allergen-specific IgE and IgG1 levels, together with reductions in bronchoalveolar lavage fluid of IL-5 and CCL11. Treatment significantly inhibited changes in the airway including airway epithelial cell hypertrophy, goblet cell metaplasia and mucus hypersecretion. The two major components of enriched milk fat, cis-9, trans-11 conjugated linoleic acid and VA, inhibited airway inflammation when fed together to mice, whereas alone they were not effective. CONCLUSION: Milk fat enriched in conjugated linoleic and VAs suppresses inflammation and changes to the airways in an animal model of allergic airway disease.

Immunotherapy of prostate cancer in a murine model using a novel GnRH based vaccine candidate.

Previous studies with gonadotrophin releasing hormone (GnRH/LHRH) vaccines have shown the usefulness of immunization against this hormone in prostate cancer. To this end, we have generated a completely synthetic peptide modified at position 6 and attached to the 830-844 tetanic toxoid (TT) helper T cell sequence. Through this work we have demonstrated that the GnRHm1-TT molecule was highly immunogenic when it is formulated as an oil-based emulsion adjuvated with Montanide ISA 51. That results correlated directly with testosterone reduction and tumor growth inhibition of the Dunning R3327-H androgen responsive prostate tumor model in rats. GnRHm1-TT, proved to be safe and useful for future clinical trials.

Vaccination with NY-ESO-1 protein and CpG in Montanide induces integrated antibody/Th1 responses and CD8 T cells through cross-priming.

The use of recombinant tumor antigen proteins is a realistic approach for the development of generic cancer vaccines, but the potential of this type of vaccines to induce specific CD8(+) T cell responses, through in vivo cross-priming, has remained unclear. In this article, we report that repeated vaccination of cancer patients with recombinant NY-ESO-1 protein, Montanide ISA-51, and CpG ODN 7909, a potent stimulator of B cells and T helper type 1 (Th1)-type immunity, resulted in the early induction of specific integrated CD4(+) Th cells and antibody responses in most vaccinated patients, followed by the development of later CD8(+) T cell responses in a fraction of them. The correlation between antibody and T cell responses, together with the ability of vaccine-induced antibodies to promote in vitro cross-presentation of NY-ESO-1 by dendritic cells to vaccine-induced CD8(+) T cells, indicated that elicitation of NY-ESO-1-specific CD8(+) T cell responses by cross-priming in vivo was associated with the induction of adequate levels of specific antibodies. Together, our data provide clear evidence of in vivo cross-priming of specific cytotoxic T lymphocytes by a recombinant tumor antigen vaccine, underline the importance of specific antibody induction for the cross-priming to occur, and support the use of this type of formulation for the further development of efficient cancer vaccines.

Modifications increasing the efficacy of recombinant vaccines; marked increase in antibody titers with moderately repetitive variants of a therapeutic allergy vaccine.

Development of vaccines targeting important self-molecules like tumor antigens, IgE, cytokines or other regulatory molecules, brings about challenges that are not met in classical vaccine development. Tolerance inducing mechanisms reduce the levels of therapeutic antibodies in the vaccinated subject, and anti-self antibody titers are frequently more than 50-fold lower than the anti-non-self response to the carrier. In order to overcome this limitation in efficacy we have explored various methods to enhance the immunogenicity of the vaccine antigen. Vaccination with a molecule containing two IgE Cepsilon3 domains and thereby a low level of repetitiveness markedly increased the efficacy. The anti-IgE antibody titers in the animals treated with the dimeric vaccine antigen were 4.5, 5 and 8 times higher than in the animals treated with the monomer, in three independent experiments. In addition, this increase in efficacy was not masked by the use of potent adjuvants. The effect persisted even in the presence of Freunds or Montanide ISA 51, two mineral oil based adjuvants. This in contrast to most Toll-like receptor (TLR) agonists, which appear to enhance the immune response only when administrated together with weak adjuvants. This clearly shows that the introduction of a moderately repetitive structure is enough to substantially increase the efficacy of a therapeutic vaccine.

Toll-like receptor agonists influence the magnitude and quality of memory T cell responses after prime-boost immunization in nonhuman primates.

There is a remarkable heterogeneity in the functional profile (quality) of T cell responses. Importantly, the magnitude and/or quality of a response required for protection may be different depending on the infection. Here, we assessed the capacity of different Toll like receptor (TLR)-binding compounds to influence T helper cell (Th)1 and CD8+ T cell responses when used as adjuvants in nonhuman primates (NHP) with HIV Gag as a model antigen. NHP were immunized with HIV Gag protein emulsified in Montanide ISA 51, an oil-based adjuvant, with or without a TLR7/8 agonist, a TLR8 agonist, or the TLR9 ligand cytosine phosphate guanosine oligodeoxynucleotides (CpG ODN), and boosted 12 wk later with a replication-defective adenovirus-expressing HIV-Gag (rAD-Gag). Animals vaccinated with HIV Gag protein/Montanide and CpG ODN or the TLR7/8 agonist had higher frequencies of Th1 responses after primary immunization compared to all other vaccine groups. Although the rAD-Gag boost did not elevate the frequency of Th1 memory cytokine responses, there was a striking increase in HIV Gag-specific CD8+ T cell responses after the boost in all animals that had received a primary immunization with any of the TLR adjuvants. Importantly, the presence and type of TLR adjuvant used during primary immunization conferred stability and dramatically influenced the magnitude and quality of the Th1 and CD8+ T cell responses after the rAD-Gag boost. These data provide insights for designing prime-boost immunization regimens to optimize Th1 and CD8+ T cell responses.