Efficient targeting of NY-ESO-1 tumor antigen to human cDC1s by lymphotactin results in cross-presentation and antigen-specific T cell expansion.

BACKGROUND: Type 1 conventional dendritic cells (cDC1s) are characterized by their ability to induce potent CD8+ T cell responses. In efforts to generate novel vaccination strategies, notably against cancer, human cDC1s emerge as an ideal target to deliver antigens. cDC1s uniquely express XCR1, a seven transmembrane G protein-coupled receptor. Due to its restricted expression and endocytic nature, XCR1 represents an attractive receptor to mediate antigen-delivery to human cDC1s. METHODS: To explore tumor antigen delivery to human cDC1s, we used an engineered version of XCR1-binding lymphotactin (XCL1), XCL1(CC3). Site-specific sortase-mediated transpeptidation was performed to conjugate XCL1(CC3) to an analog of the HLA-A*02:01 epitope of the cancer testis antigen New York Esophageal Squamous Cell Carcinoma-1 (NY-ESO-1). While poor epitope solubility prevented isolation of stable XCL1-antigen conjugates, incorporation of a single polyethylene glycol (PEG) chain upstream of the epitope-containing peptide enabled generation of soluble XCL1(CC3)-antigen fusion constructs. Binding and chemotactic characteristics of the XCL1-antigen conjugate, as well as its ability to induce antigen-specific CD8+ T cell activation by cDC1s, was assessed. RESULTS: PEGylated XCL1(CC3)-antigen conjugates retained binding to XCR1, and induced cDC1 chemoattraction in vitro. The model epitope was efficiently cross-presented by human cDC1s to activate NY-ESO-1-specific CD8+ T cells. Importantly, vaccine activity was increased by targeting XCR1 at the surface of cDC1s. CONCLUSION: Our results present a novel strategy for the generation of targeted vaccines fused to insoluble antigens. Moreover, our data emphasize the potential of targeting XCR1 at the surface of primary human cDC1s to induce potent CD8+ T cell responses.

Cancer immunotherapy using artificial adjuvant vector cells to deliver NY-ESO-1 antigen to dendritic cells in situ.

NY-ESO-1 is a cancer/testis antigen expressed in various cancer types. However, the induction of NY-ESO-1-specific CTLs through vaccines is somewhat difficult. Thus, we developed a new type of artificial adjuvant vector cell (aAVC-NY-ESO-1) expressing a CD1d-NKT cell ligand complex and a tumor-associated antigen, NY-ESO-1. First, we determined the activation of invariant natural killer T (iNKT) and natural killer (NK) cell responses by aAVC-NY-ESO-1. We then showed that the NY-ESO-1-specific CTL response was successfully elicited through aAVC-NY-ESO-1 therapy. After injection of aAVC-NY-ESO-1, we found that dendritic cells (DCs) in situ expressed high levels of costimulatory molecules and produced interleukn-12 (IL-12), indicating that DCs undergo maturation in vivo. Furthermore, the NY-ESO-1 antigen from aAVC-NY-ESO-1 was delivered to the DCs in vivo, and it was presented on MHC class I molecules. The cross-presentation of the NY-ESO-1 antigen was absent in conventional DC-deficient mice, suggesting a host DC-mediated CTL response. Thus, this strategy helps generate sufficient CD8+ NY-ESO-1-specific CTLs along with iNKT and NK cell activation, resulting in a strong antitumor effect. Furthermore, we established a human DC-transferred NOD/Shi-scid/IL-2γcnull immunodeficient mouse model and showed that the NY-ESO-1 antigen from aAVC-NY-ESO-1 was cross-presented to antigen-specific CTLs through human DCs. Taken together, these data suggest that aAVC-NY-ESO-1 has potential for harnessing innate and adaptive immunity against NY-ESO-1-expressing malignancies.

Fibroblast Activation Protein Targeted Photodynamic Therapy Selectively Kills Activated Skin Fibroblasts from Systemic Sclerosis Patients and Prevents Tissue Contraction.

Systemic sclerosis (SSc) is a rare, severe, auto-immune disease characterized by inflammation, vasculopathy and fibrosis. Activated (myo)fibroblasts are crucial drivers of this fibrosis. By exploiting their expression of fibroblast activation protein (FAP) to perform targeted photodynamic therapy (tPDT), we can locoregionally deplete these pathogenic cells. In this study, we explored the use of FAP-tPDT in primary skin fibroblasts from SSc patients, both in 2D and 3D cultures. Method: The FAP targeting antibody 28H1 was conjugated with the photosensitizer IRDye700DX. Primary skin fibroblasts were obtained from lesional skin biopsies of SSc patients via spontaneous outgrowth and subsequently cultured on plastic or collagen type I. For 2D FAP-tPDT, cells were incubated in buffer with or without the antibody-photosensitizer construct, washed after 4 h and exposed to λ = 689 nm light. Cell viability was measured using CellTiter Glo®®. For 3D FAP-tPDT, cells were seeded in collagen plugs and underwent the same treatment procedure. Contraction of the plugs was followed over time to determine myofibroblast activity. Results: FAP-tPDT resulted in antibody-dose dependent cytotoxicity in primary skin fibroblasts upon light exposure. Cells not exposed to light or incubated with an irrelevant antibody-photosensitizer construct did not show this response. FAP-tPDT fully prevented contraction of collagen plugs seeded with primary SSc fibroblasts. Even incubation with a very low dose of antibody (0.4 nM) inhibited contraction in 2 out of 3 donors. Conclusions: Here we have shown, for the first time, the potential of FAP-tPDT for the treatment of fibrosis in SSc skin.

High-resolution epitope mapping by AllerScan reveals relationships between IgE and IgG repertoires during peanut oral immunotherapy.

Peanut allergy can result in life-threatening reactions and is a major public health concern. Oral immunotherapy (OIT) induces desensitization to food allergens through administration of increasing amounts of allergen. To dissect peanut-specific immunoglobulin E (IgE) and IgG responses in subjects undergoing OIT, we have developed AllerScan, a method that leverages phage-display and next-generation sequencing to identify the epitope targets of peanut-specific antibodies. We observe a striking diversification and boosting of the peanut-specific IgG repertoire after OIT and a reduction in pre-existing IgE levels against individual epitopes. High-resolution epitope mapping reveals shared recognition of public epitopes in Ara h 1, 2, 3, and 7. In individual subjects, OIT-induced IgG specificities overlap extensively with IgE and exhibit strikingly similar antibody footprints, suggesting related clonal lineages or convergent evolution of peanut-specific IgE and IgG B cells. Individual differences in epitope recognition identified via AllerScan could inform safer and more effective personalized immunotherapy.

Neoadjuvant In Situ Immunomodulation Enhances Systemic Antitumor Immunity against Highly Metastatic Tumors.

Neoadjuvant immunotherapy, given before surgical resection, is a promising approach to develop systemic antitumor immunity for the treatment of high-risk resectable disease. Here, using syngeneic and orthotopic mouse models of triple-negative breast cancer, we have tested the hypothesis that generation of tumor-specific T-cell responses by induction and activation of tumor-residing Batf3-dependent conventional type 1 dendritic cells (cDC1) before resection improves control of distant metastatic disease and survival. Mice bearing highly metastatic orthotopic tumors were treated with a combinatorial in situ immunomodulation (ISIM) regimen comprised of intratumoral administration of Flt3L, local radiotherapy, and in situ TLR3/CD40 stimulations, followed by surgical resection. Neoadjuvant ISIM (neo-ISIM) generated tumor-specific CD8+ T cells that infiltrated into distant nonirradiated metastatic sites, which delayed the progression of lung metastases and improved survival after the resection of primary tumors. The efficacy of neo-ISIM was dependent on de novo adaptive T-cell immunity elicited by Batf3-dependent dendritic cells and was enhanced by increasing dose and fractionation of radiotherapy, and early surgical resection after the completion of neo-ISIM. Importantly, neo-ISIM synergized with programmed cell death protein-1 ligand-1 (PD-L1) blockade to improve control of distant metastases and prolong survival, while removal of tumor-draining lymph nodes abrogated the antimetastatic efficacy of neo-ISIM. Our findings illustrate the therapeutic potential of neoadjuvant multimodal intralesional therapy for the treatment of resectable tumors with high risk of relapse. SIGNIFICANCE: Neoadjuvant induction and activation of cDC1s in primary tumors enhances systemic antitumor immunity, suppresses metastatic progression, improves survival, and synergizes with anti-PD-L1 therapy.

A protocol for qualitative and quantitative measurement of endosomal processing using hot spot analysis.

A detailed quantification of antigen processing by endosomal compartments provides important information on the pattern of protein fragmentation. Here, we describe a protocol that combines gradient purified endosomes, incubated with antigens, followed by hot spot analysis of MS/MS-sequenced peptides. The analysis identifies differences in endosomal antigen processing by dendritic cells under diverse experimental conditions. For complete details on the use and execution of this protocol, please refer to Clement et al. (2021).

Deep immune profiling reveals targetable mechanisms of immune evasion in immune checkpoint inhibitor-refractory glioblastoma.

BACKGROUND: Glioblastoma (GBM) is refractory to immune checkpoint inhibitor (ICI) therapy. We sought to determine to what extent this immune evasion is due to intrinsic properties of the tumor cells versus the specialized immune context of the brain, and if it can be reversed. METHODS: We used CyTOF mass cytometry to compare the tumor immune microenvironments (TIME) of human tumors that are generally ICI-refractory (GBM and sarcoma) or ICI-responsive (renal cell carcinoma), as well as mouse models of GBM that are ICI-responsive (GL261) or ICI-refractory (SB28). We further compared SB28 tumors grown intracerebrally versus subcutaneously to determine how tumor site affects TIME and responsiveness to dual CTLA-4/PD-1 blockade. Informed by these data, we explored rational immunotherapeutic combinations. RESULTS: ICI-sensitivity in human and mouse tumors was associated with increased T cells and dendritic cells (DCs), and fewer myeloid cells, in particular PD-L1+ tumor-associated macrophages. The SB28 mouse model of GBM responded to ICI when grown subcutaneously but not intracerebrally, providing a system to explore mechanisms underlying ICI resistance in GBM. The response to ICI in the subcutaneous SB28 model required CD4 T cells and NK cells, but not CD8 T cells. Recombinant FLT3L expanded DCs, improved antigen-specific T cell priming, and prolonged survival of mice with intracerebral SB28 tumors, but at the cost of increased Tregs. Targeting PD-L1 also prolonged survival, especially when combined with stereotactic radiation. CONCLUSIONS: Our data suggest that a major obstacle for effective immunotherapy of GBM is poor antigen presentation in the brain, rather than intrinsic immunosuppressive properties of GBM tumor cells. Deep immune profiling identified DCs and PD-L1+ tumor-associated macrophages as promising targetable cell populations, which was confirmed using therapeutic interventions in vivo.

Orphan GPR116 mediates the insulin sensitizing effects of the hepatokine FNDC4 in adipose tissue.

The proper functional interaction between different tissues represents a key component in systemic metabolic control. Indeed, disruption of endocrine inter-tissue communication is a hallmark of severe metabolic dysfunction in obesity and diabetes. Here, we show that the FNDC4-GPR116, liver-white adipose tissue endocrine axis controls glucose homeostasis. We found that the liver primarily controlled the circulating levels of soluble FNDC4 (sFNDC4) and lowering of the hepatokine FNDC4 led to prediabetes in mice. Further, we identified the orphan adhesion GPCR GPR116 as a receptor of sFNDC4 in the white adipose tissue. Upon direct and high affinity binding of sFNDC4 to GPR116, sFNDC4 promoted insulin signaling and insulin-mediated glucose uptake in white adipocytes. Indeed, supplementation with FcsFNDC4 in prediabetic mice improved glucose tolerance and inflammatory markers in a white-adipocyte selective and GPR116-dependent manner. Of note, the sFNDC4-GPR116, liver-adipose tissue axis was dampened in (pre) diabetic human patients. Thus our findings will now allow for harnessing this endocrine circuit for alternative therapeutic strategies in obesity-related pre-diabetes.

Evaluation of multi-epitope recombinant protein as a candidate for a contraceptive vaccine.

Contraceptive vaccine (CV) is a valuable, non-invasive, and alternative method for purposeful contraception. Sperm antigens are useful targets for producing CVs due to their specialized expression in sperm. In this study, a recombinant protein containing three main sperm epitopes (IZUMO1, SACA3, and PH-20) was designed and evaluated as CV to control fertility in male mice. The chimeric recombinant protein was expressed and purified in E. coli. Male mice were immunized by 100 µg purified protein and sera were collected to assess IgG antibodies. Evaluating the reproductive performance, immunized male mice mated with normal-fertile female mice and mating rate and the number of newborns was studied. Immunized mice were sacrificed and necropsy and histopathology studies were conducted. The results revealed that the designed chimeric protein stimulated the immune system of the mice effectively. The level of IgG antibody was significantly higher in vaccinated mouse rather than control mouse. Eighty percent of the vaccinated mice became infertile and in the remaining ones, the number of children decreased to 4-6 offspring instead of 10-12 in normal mice. Histopathological studies showed that no organs including heart, brain, lung, liver, kidney and intestine were damaged. However, Normal spermatogenesis has been disrupted and necrotic spermatogonia cells were reported in Seminiferous tubules. We concluded that the designed chimeric protein containing IZUMO1, SACA3, and PH-20 epitopes can stimulate the immune system and cause male contraception without any side effects.

Controlled Delivery of Slit3 Proteins from Alginate Microbeads Inhibits In Vitro Angiogenesis.

BACKGROUND: The Slit-Robo pathway is a key regulator of angiogenesis and cellular function in experimental models. Slit3 proteins exhibit both proangiogenic and antiangiogenic properties, but the exact mechanism remains unclear. It is theorized that Slit3 may be a potential treatment for vascular diseases and cancer. METHODS: Slit3 labeled with I-125 was encapsulated in microbeads composed of low-viscosity alginate of high-glucuronic acid content, first coated with poly-L-ornithine for various durations and finally with low-viscosity high mannuronic acid. Gamma counter was used to measure microbead encapsulation efficiency and Slit3 release. Markers of angiogenesis were assessed with Boyden chamber, scratch wound, and Matrigel tube formation assays using human umbilical vein and mouse endothelial cells. RESULTS: On incubation of Slit3-loaded microbeads, there was an initial burst phase release of Slit3 for the first 24 h followed by sustained release for 6 to 12 d. Microbead composition determined encapsulation efficiency and rate of release; Slit3 encapsulation was most efficient in microbeads with lower low-viscosity alginate of high-glucuronic acid content concentrations (1.5%) and no poly-L-ornithine coating. Compared with controls (media alone), Slit3 microbeads significantly inhibited in vitro cellular migration, endothelial cell migration for wound closure at 24 and 48 h and endothelial tube formation (P < 0.001, respectively). CONCLUSIONS: Slit3 can be effectively encapsulated and delivered via a controlled release pattern using alginate microbeads. Microbead encapsulation reduces in vitro endothelial tube formation and inhibits cellular migration to impair angiogenesis. Thus, Slit3 microparticles may be explored as a therapeutic option to mitigate tumor proliferation.

Respiratory FimA-Specific Secretory IgA Antibodies Upregulated by DC-Targeting Nasal Double DNA Adjuvant Are Essential for Elimination of Porphyromonas gingivalis.

Our previous studies showed that a combination of a DNA plasmid encoding Flt3 ligand (pFL) and CpG oligodeoxynucleotides 1826 (CpG ODN) (FL/CpG) as a nasal adjuvant provoked antigen-specific immune responses. In this study, we investigated the efficacy of a nasal vaccine consisting of FimA as the structural subunit of Porphyromonas gingivalis (P. gingivalis) fimbriae and FL/CpG for the induction of FimA-specific antibody (Ab) responses and their protective roles against nasal and lung infection by P. gingivalis, a keystone pathogen in the etiology of periodontal disease. C57BL/6 mice were nasally immunized with recombinant FimA (rFimA) plus FL/CpG three times at weekly intervals. As a control, mice were given nasal rFimA alone. Nasal washes (NWs) and bronchoalveolar lavage fluid (BALF) of mice given nasal rFimA plus FL/CpG resulted in increased levels of rFimA-specific secretory IgA (SIgA) and IgG Ab responses when compared with those in controls. Significantly increased numbers of CD8- or CD11b-expressing mature-type dendritic cells (DCs) were detected in the respiratory inductive and effector tissues of mice given rFimA plus FL/CpG. Additionally, significantly upregulated Th1/Th2-type cytokine responses by rFimA-stimulated CD4+ T cells were noted in the respiratory effector tissues. When mice were challenged with live P. gingivalis via the nasal route, mice immunized nasally with rFimA plus FL/CpG inhibited P. gingivalis colonization in the nasal cavities and lungs. In contrast, controls failed to show protection. Of interest, when IgA-deficient mice given nasal rFimA plus FL/CpG were challenged with nasal P. gingivalis, the inhibition of bacterial colonization in the respiratory tracts was not seen. Taken together, these results show that nasal FL/CpG effectively enhanced DCs and provided balanced Th1- and Th2-type cytokine response-mediated rFimA-specific IgA protective immunity in the respiratory tract against P. gingivalis. A nasal administration with rFimA and FL/CpG could be a candidate for potent mucosal vaccines for the elimination of inhaled P. gingivalis in periodontal patients.

Neurokinin 3 Receptor Antagonism Ameliorates Key Metabolic Features in a Hyperandrogenic PCOS Mouse Model.

Polycystic ovary syndrome (PCOS) is a prevalent endocrine condition characterized by a range of endocrine, reproductive, and metabolic abnormalities. At present, management of women with PCOS is suboptimal as treatment is only symptomatic. Clinical and experimental advances in our understanding of PCOS etiology support a pivotal role for androgen neuroendocrine actions in PCOS pathogenesis. Hyperandrogenism is a key PCOS trait and androgen actions play a role in regulating the kisspeptin-/neurokinin B-/dynorphin (KNDy) system. This study aimed to investigate if targeted antagonism of neurokinin B signaling through the neurokinin 3 receptor (NK3R) would reverse PCOS traits in a dihydrotestosterone (DHT)-induced mouse model of PCOS. After 3 months, DHT exposure induced key reproductive PCOS traits of cycle irregularity and ovulatory dysfunction, and PCOS-like metabolic traits including increased body weight; white and brown fat pad weights; fasting serum triglyceride and glucose levels, and blood glucose incremental area under the curve. Treatment with a NK3R antagonist (MLE4901) did not impact the observed reproductive defects. In contrast, following NK3R antagonist treatment, PCOS-like females displayed decreased total body weight, adiposity, and adipocyte hypertrophy, but increased respiratory exchange ratio, suggesting NK3R antagonism altered the metabolic status of the PCOS-like females. NK3R antagonism did not improve circulating serum triglyceride or fasted glucose levels. Collectively, these findings demonstrate that NK3R antagonism may be beneficial in the treatment of adverse metabolic features associated with PCOS and support neuroendocrine targeting in the development of novel therapeutic strategies for PCOS.

Protein Delivery by Peptide-Based Stealth Liposomes: A Biomolecular Insight into Enzyme Replacement Therapy.

Infantile neural ceroid lipofuscinosis (INCL) is a lysosomal storage disorder characterized by mutations in the CLN1 gene that leads to lack of the lysosomal enzyme palmitoyl-protein thioesterase-1 (PPT1), which causes the progressive death of cortical neurons. Enzyme replacement therapy (ERT) is one of the most promising treatments, but its translation toward a clinical use is hampered by the need to deliver the enzyme to the central nervous system and a more detailed understanding of its capability to restore physiologic conditions at the biochemical and protein level, beyond the simple regulation of enzymatic activity. Targeted nanoparticles can promote protein delivery to the central nervous system and affect biological pathways inside cells. Here, we describe an innovative peptide-based stealth nanoparticle that inhibits serum protein adsorption exploiting transferrin-driven internalization to convey the PPT1 enzyme to transferrin receptor-mediated pathways (endocytosis in this work, or transcytosis, in perspective, in vivo). These enzyme-loaded nanoparticles were able to restore stable levels of enzymatic activity in CLN1 patient's fibroblasts, comparable with the free enzyme, demonstrating that delivery after encapsulation in the nanocarrier does not alter uptake or intracellular trafficking. We also investigate, for the first time, dysregulated pathways of proteome and palmitoylome and their alteration upon enzyme delivery. Our nanoparticles were able of halving palmitoylated protein levels restoring conditions similar to the normal cells. From proteomic analysis, we also highlighted the reduction of the different groups of proteins after treatments with the free or encapsulated enzyme. In conclusion, our system is able to deliver the enzyme to a model of CLN1 disease restoring normal conditions in cells. Investigation of molecular details of pathologic state and enzyme-based correction reveals dysregulated pathways with unprecedented details for CLN1. Finally, we unveil for the first time the dysregulation landscape of palmitoylome and proteome in primary patient-derived fibroblasts and their modifications in response to enzyme administration. These findings will provide a guideline for the validation of future therapeutic strategies based on enzyme replacement therapy or acting at different metabolic levels.

Mitochondrial Fusion Promoter Alleviates Brain Damage in Rats with Cardiac Ischemia/Reperfusion Injury.

BACKGROUND: Cardiac ischemia/reperfusion (I/R) injury induces brain damage through increased blood-brain barrier (BBB) breakdown, microglial hyperactivity, pro-inflammatory cytokines, amyloid-ß deposition, loss of dendritic spines, brain mitochondrial dysfunction, and imbalanced mitochondrial dynamics. Previous studies demonstrated that mitochondrial fusion promoter reduced cardiac damage from cardiac I/R injury; however, following cardiac I/R injury, the roles of mitochondrial dynamics on the brain have not been investigated. OBJECTIVE: To investigate the effects of pharmacological modulation using mitochondrial fusion promoter (M1) in the brain of rats following cardiac I/R injury. METHODS: Twenty-four male Wistar rats were separated into two groups; 1) sham-operation (n = 8) and 2) cardiac I/R injury (n = 16). Rats in the cardiac I/R injury group were randomly received either normal saline solution as a vehicle or a mitochondrial fusion promoter (M1, 2 mg/kg) intravenously. Both treatments were given to the rats 15 minutes before cardiac I/R injury. At the end of the reperfusion protocol, the brain was rapidly removed to investigate brain mitochondrial function, mitochondrial dynamics proteins, microglial activity, and Alzheimer's disease (AD) related proteins. RESULTS: Cardiac I/R injury induced brain mitochondrial dynamics imbalance as indicated by reduced mitochondrial fusion proteins expression without alteration in mitochondrial fission, brain mitochondrial dysfunction, BBB breakdown, increased macrophage infiltration, apoptosis, and AD-related proteins. Pretreatment with M1 effectively increased the expression of mitofusin 2, a mitochondrial outer membrane fusion protein, reduced brain mitochondrial dysfunction, BBB breakdown, macrophage infiltration, apoptosis, and AD-related proteins in rats following cardiac I/R injury. CONCLUSION: This mitochondrial fusion promoter significantly protected rats with cardiac I/R injury against brain damage.

Tat-Cannabinoid Receptor Interacting Protein Reduces Ischemia-Induced Neuronal Damage and Its Possible Relationship with 14-3-3η.

Cannabinoid receptor-interacting protein 1a (CRIP1a) binds to the C-terminal domain of cannabinoid 1 receptor (CB1R) and regulates CB1R activities. In this study, we made Tat-CRIP1a fusion proteins to enhance CRIP1a penetration into neurons and brain and to evaluate the function of CRIP1a in neuroprotection following oxidative stress in HT22 hippocampal cells and transient forebrain ischemia in gerbils. Purified exogenous Tat-CRIP1a was penetrated into HT22 cells in a time and concentration-dependent manner and prevented H2O2-induced reactive oxygen species formation, DNA fragmentation, and cell damage. Tat-CRIP1a fusion protein also ameliorated the reduction of 14-3-3η expression by H2O2 treatment in HT22 cells. Ischemia-reperfusion damage caused motor hyperactivity in the open field test of gerbils; however, the treatment of Tat-CRIP1a significantly reduced hyperactivity 1 day after ischemia. Four days after ischemia, the administration of Tat-CRIP1a restored the loss of pyramidal neurons and decreased reactive astrocytosis and microgliosis induced by ischemic damage in the hippocampal cornu Ammonis (CA)1 region. Ischemic damage decreased 14-3-3η expression in all hippocampal sub-regions 4 days after ischemia; however, the treatment of Tat-CRIP1 ameliorated the reduction of 14-3-3η expression. These results suggest that Tat-CRIP1a attenuates neuronal damage and hyperactivity induced by ischemic damage, and it restores normal expression levels of 14-3-3η protein in the hippocampus.

Multi-functional nanocomplex codelivery of Trp2 and R837 to activate melanoma-specific immunity.

Antigen-adjuvant combination could induce a protective and long-lasting anti-tumor immune response. However, exploiting system which could co-deliver melanoma antigen peptide Trp2 (Tyrosinase-related protein 2) and Toll-like-receptor-7 (TLR7) agonists imiquimod (R837) both are poor aqueous solubility is still challenging. Our new nanocomplex was explored for specific delivery of Trp2 and R837 into antigen-presenting cells (APCs). R837 was loaded into mannosylated-ß-cyclodextrin (Man-CD) to target dendritic cells (DCs) by binding mannose receptors (MR) on DCs. A fusion peptide (WT) was constructed by incorporating the amino acid region of TAT (cell-penetrating peptide) into Trp2 to improve the TAT-mediated intracellular efficiency. Negatively charged sodium alginate (SA), a biocompatible polymer, which can induce adjuvant responses by affecting the functions of DCs, was complexed with Man-CD/R837 and WT via physical adsorption. The optimized nanocomplex promoted the cellular uptake and showed remarkable efficacy to enhance the secreting of Th1-cytokines. This multi-functional nanocomplex system may allow effective targeting-codelivery of multi-hydrophobic drugs and be a promising subunit vaccine candidate as a potential prevention action of tumor.

Cancer Vaccines: Adjuvant Potency, Importance of Age, Lifestyle, and Treatments.

Although the discovery and characterization of multiple tumor antigens have sparked the development of many antigen/derived cancer vaccines, many are poorly immunogenic and thus, lack clinical efficacy. Adjuvants are therefore incorporated into vaccine formulations to trigger strong and long-lasting immune responses. Adjuvants have generally been classified into two categories: those that 'depot' antigens (e.g. mineral salts such as aluminum hydroxide, emulsions, liposomes) and those that act as immunostimulants (Toll Like Receptor agonists, saponins, cytokines). In addition, several novel technologies using vector-based delivery of antigens have been used. Unfortunately, the immune system declines with age, a phenomenon known as immunosenescence, and this is characterized by functional changes in both innate and adaptive cellular immunity systems as well as in lymph node architecture. While many of the immune functions decline over time, others paradoxically increase. Indeed, aging is known to be associated with a low level of chronic inflammation-inflamm-aging. Given that the median age of cancer diagnosis is 66 years and that immunotherapeutic interventions such as cancer vaccines are currently given in combination with or after other forms of treatments which themselves have immune-modulating potential such as surgery, chemotherapy and radiotherapy, the choice of adjuvants requires careful consideration in order to achieve the maximum immune response in a compromised environment. In addition, more clinical trials need to be performed to carefully assess how less conventional form of immune adjuvants, such as exercise, diet and psychological care which have all be shown to influence immune responses can be incorporated to improve the efficacy of cancer vaccines. In this review, adjuvants will be discussed with respect to the above-mentioned important elements.

Immunization with the basic membrane protein (BMP) family ABC transporter elicits protection against Enterococcus faecium in a murine infection model.

Enterococcus faecium is evolving as a multi-resistant pathogen causing infections with high morbidity and mortality. A protective vaccine against E. faecium is lacking up till now. ATP-binding cassette (ABC) transporter proteins have important functions in bacteria to maintain survival and homeostasis. In the present study, we evaluated the basic membrane protein (BMP) family ABC transporter substrate-binding protein, designated herein as BMP, as a potential vaccine candidate against E. faecium. Recombinant BMP of E. faecium was expressed in Escherichia coli, and purified by metal affinity chromatography. Swiss albino mice were immunized with the recombinant BMP combined with Bacillus Calmette-Guérin (BCG) and/or alum as adjuvants. Mice immunized with BMP combined with alternating BCG and alum developed BMP-specific IgG and were protected against E. faecium challenge as evidenced from organ bioburden and histopathological examination. Furthermore, serum from immunized mice showed enhanced opsonophagocytic activity and protected mice against E. faecium challenge by passive immunization. Bioinformatic analysis revealed appreciable degrees of homology between E. faecium BMP and proteins from other pathogens which suggests BMP could be a useful vaccine against multiple pathogens. To our knowledge, this is the first report of in-vivo evaluation of BMP as a potential vaccine candidate against E. faecium.

Rapamycin-Loaded Biomimetic Nanoparticles Reverse Vascular Inflammation.

RATIONALE: Through localized delivery of rapamycin via a biomimetic drug delivery system, it is possible to reduce vascular inflammation and thus the progression of vascular disease. OBJECTIVE: Use biomimetic nanoparticles to deliver rapamycin to the vessel wall to reduce inflammation in an in vivo model of atherosclerosis after a short dosing schedule. METHODS AND RESULTS: Biomimetic nanoparticles (leukosomes) were synthesized using membrane proteins purified from activated J774 macrophages. Rapamycin-loaded nanoparticles were characterized using dynamic light scattering and were found to have a diameter of 108±2.3 nm, a surface charge of -15.4±14.4 mV, and a polydispersity index of 0.11 +/ 0.2. For in vivo studies, ApoE-/- mice were fed a high-fat diet for 12 weeks. Mice were injected with either PBS, free rapamycin (5 mg/kg), or rapamycin-loaded leukosomes (Leuko-Rapa; 5 mg/kg) once daily for 7 days. In mice treated with Leuko-Rapa, flow cytometry of disaggregated aortic tissue revealed fewer proliferating macrophages in the aorta (15.6±9.79 %) compared with untreated mice (30.2±13.34 %) and rapamycin alone (26.8±9.87 %). Decreased macrophage proliferation correlated with decreased levels of MCP (monocyte chemoattractant protein)-1 and IL (interleukin)-b1 in mice treated with Leuko-Rapa. Furthermore, Leuko-Rapa-treated mice also displayed significantly decreased MMP (matrix metalloproteinases) activity in the aorta (mean difference 2554±363.9, P=9.95122×10-6). No significant changes in metabolic or inflammation markers observed in liver metabolic assays. Histological analysis showed improvements in lung morphology, with no alterations in heart, spleen, lung, or liver in Leuko-Rapa-treated mice. CONCLUSIONS: We showed that our biomimetic nanoparticles showed a decrease in proliferating macrophage population that was accompanied by the reduction of key proinflammatory cytokines and changes in plaque morphology. This proof-of-concept showed that our platform was capable of suppressing macrophage proliferation within the aorta after a short dosing schedule (7 days) and with a favorable toxicity profile. This treatment could be a promising intervention for the acute stabilization of late-stage plaques.

Combined Vaccination with NY-ESO-1 Protein, Poly-ICLC, and Montanide Improves Humoral and Cellular Immune Responses in Patients with High-Risk Melanoma.

Given its ability to induce both humoral and cellular immune responses, NY-ESO-1 has been considered a suitable antigen for a cancer vaccine. Despite promising results from early-phase clinical studies in patients with melanoma, NY-ESO-1 vaccine immunotherapy has not been widely investigated in larger trials; consequently, many questions remain as to the optimal vaccine formulation, predictive biomarkers, and sequencing and timing of vaccines in melanoma treatment. We conducted an adjuvant phase I/II clinical trial in high-risk resected melanoma to optimize the delivery of poly-ICLC, a TLR-3/MDA-5 agonist, as a component of vaccine formulation. A phase I dose-escalation part was undertaken to identify the MTD of poly-ICLC administered in combination with NY-ESO-1 and montanide. This was followed by a randomized phase II part investigating the MTD of poly-ICLC with NY-ESO-1 with or without montanide. The vaccine regimens were generally well tolerated, with no treatment-related grade 3/4 adverse events. Both regimens induced integrated NY-ESO-1-specific CD4+ T-cell and humoral responses. CD8+ T-cell responses were mainly detected in patients receiving montanide. T-cell avidity toward NY-ESO-1 peptides was higher in patients vaccinated with montanide. In conclusion, NY-ESO-1 protein in combination with poly-ICLC is safe, well tolerated, and capable of inducing integrated antibody and CD4+ T-cell responses in most patients. Combination with montanide enhances antigen-specific T-cell avidity and CD8+ T-cell cross-priming in a fraction of patients, indicating that montanide contributes to the induction of specific CD8+ T-cell responses to NY-ESO-1.