Sequentially Self-Assembled Nanoreactor Comprising Tannic Acid and Phenylboronic Acid-Conjugated Polymers Inducing Tumor-Selective Enzymatic Activity.

The construction of enzyme delivery systems, which can control enzymatic activity at a target site, is important for efficient enzyme-prodrug therapy/diagnosis. Herein we report a facile technique to construct a systemically applicable ß-galactosidase (ß-Gal)-loaded ternary complex comprising tannic acid (TA) and phenylboronic acid-conjugated polymers through sequential self-assembly in aqueous solution. At physiological conditions, the ternary complex exhibited a hydrodynamic diameter of ∼40 nm and protected the loaded ß-Gal from unfavorable degradation by proteinase. Upon cellular internalization, the ternary complex recovered ß-Gal activity by releasing the loaded ß-Gal. The intravenously injected ternary complex thereby delivered ß-Gal to the target tumor in a subcutaneous tumor model and exerted enhanced and selective enzymatic activity at the tumor site. Sequential self-assembly with TA and phenylboronic acid-conjugated polymers may offer a novel approach for enzyme-prodrug theragnosis.

A Single Injection of an Optimized Adeno-Associated Viral Vector into Cerebrospinal Fluid Corrects Neurological Disease in a Murine Model of GM1 Gangliosidosis.

GM1 gangliosidosis is a rare neurodegenerative lysosomal storage disease caused by loss-of-function mutations in the gene encoding beta-galactosidase (ß-gal). There are no approved treatments for GM1 gangliosidosis. Previous studies in animal models have demonstrated that adeno-associated viral (AAV) vector-mediated gene transfer to the brain can restore ß-gal expression and prevent the onset of neurological signs. We developed an optimized AAV vector expressing human ß-gal and evaluated the efficacy of a single intracerebroventricular injection of this vector into the cerebrospinal fluid (CSF) of a murine disease model. The AAV vector administration into the CSF increased ß-gal activity in the brain, reduced neuronal lysosomal storage lesions, prevented the onset of neurological signs and gait abnormalities, and increased survival. These findings demonstrate the potential therapeutic activity of this vector and support its subsequent development for the treatment of GM1 gangliosidosis.

Pharmacokinetics and pharmacodynamics of JR-051, a biosimilar of agalsidase beta, in healthy adults and patients with Fabry disease: Phase I and II/III clinical studies.

Fabry disease is a rare X-linked lysosomal disease, in which mutations in the gene encoding α-galactosidase A result in progressive cellular accumulation of globotriaosylceramide (GL-3) in various organs including the skin, kidney, and heart, often leading to life-threatening conditions. Enzyme replacement therapy is currently the standard therapy for the disease, to which two α-galactosidase A formulations have been approved: agalsidase α (Replagal®, Shire) and agalsidase ß (Fabrazyme®, Sanofi). We have recently developed a biosimilar of agalsidase ß, JR-051, and investigated its pharmacokinetics and pharmacodynamics to assess its bioequivalence to agalsidase ß. In a randomized phase I study, healthy adult male volunteers were treated with JR-051 or agalsidase ß and the pharmacokinetics of the drugs were compared. The ratio of geometric means (90% confidence interval [CI]) of the AUC0-24 and Cmax for JR-051 over agalsidase ß were 0.91 (0.8294, 1.0082) and 0.90 (0.7992, 1.0125), respectively. In a 52-week, single-arm, phase II/III study, patients with Fabry disease switched therapy from agalsidase ß to JR-051 to evaluate its pharmacodynamics. The mean (95% CI) plasma GL-3 concentrations at weeks 26 and 52 relative to pre-JR-051 administration were 1.03 (0.91, 1.15) and 0.96 (0.86, 1.06), respectively, which were within the pre-determined bioequivalence acceptance range (0.70, 1.43). The mean (95% CI) plasma globotriaosylsphingosine (lyso-GL-3) concentrations at weeks 26 and 52 relative to pre-JR-051 administration were 1.07 (0.92, 1.23) and 1.13 (1.03, 1.22), respectively. Estimated glomerular filtration rate and left ventricular mass index, as renal and cardiac function indicators, showed no notable changes from baseline throughout the study period, and no new safety concerns were identified. In conclusion, these studies demonstrated bioequivalence of JR-051 to agalsidase ß in terms of its pharmacokinetics and pharmacodynamics. JR-051 offers a potential new treatment option for patients with Fabry disease.

The Combination Vaccine Adjuvant System Alum/c-di-AMP Results in Quantitative and Qualitative Enhanced Immune Responses Post Immunization.

The development of new effective vaccines strongly depends on adjuvants and formulations able to stimulate not only strong humoral responses against a certain pathogen but also effector as well as memory CD4+ and CD8+ T cells (Dubensky et al., 2013). However, the majority of vaccines licensed for human use or currently under clinical investigation fail to stimulate efficient cellular responses. For example, vaccines against hepatitis B virus (HBV), human papillomavirus (HPV), diphtheria, tetanus and influenza are usually administered by intramuscular (i.m.) injection and contain aluminum salts (alum) as adjuvant. Alum has been shown to stimulate Th2 immune cells resulting in increased production of antigen-specific antibodies but to be incapable of stimulating robust Th1 or cytotoxic responses. To overcome such limitations recent research has focused on the development of adjuvant combinations (e.g., MF59, AS03 or AS04) to not only further strengthen antigen-specific immune responses but to also allow their modulation. We have shown previously that bis-(3',5')-cyclic dimeric adenosine monophosphate (c-di-AMP) constitutes a promising adjuvant candidate stimulating both effective Th1/Th2 and cytotoxic immune responses when included in mucosal or parenteral vaccine formulations. In the present work we demonstrate that c-di-AMP can be also combined with other adjuvants like alum resulting in increases in not only humoral responses but more striking also in cellular immune responses. This leads to improved vaccine efficacy against intracellular pathogens.

Enzyme delivery using the 30Kc19 protein and human serum albumin nanoparticles.

Nanoparticles have been widely used for delivering various chemical and biomolecular drugs, such as anti-cancer drugs and therapeutic proteins. Among nanoparticles, protein nanoparticles have advantages of non-cytotoxicity and biodegradability. In this study, a recombinant 30Kc19 protein was applied to human serum albumin (HSA) nanoparticles to enhance cellular uptake and stability of a nanoparticle cargo enzyme. The 30Kc19 protein, which originates from silkworm, has cell-penetrating and enzyme-stabilizing abilities. Therefore, 30Kc19-HSA nanoparticles were expected to enhance cellular uptake and stability of an enzyme loaded on the nanoparticles. Here, nanoparticles loaded with ß-galactosidase were prepared using the desolvation method. The 30Kc19-HSA nanoparticles were uniformly spherical in shape, dispersed evenly in phosphate buffered saline and cell culture media, and released ß-galactosidase in a sustained manner. The 30Kc19-HSA nanoparticles had negligible toxicity to animal cells and exhibited enhanced cellular uptake and intracellular stability of ß-galactosidase in HeLa and HEK293 cells when compared with those of HSA nanoparticles. These results suggest that 30Kc19-HSA protein nanoparticles could be used as a versatile tool for drug delivery to various cells.

Molecular logistics using cytocleavable polyrotaxanes for the reactivation of enzymes delivered in living cells.

The intracellular delivery of enzymes is an essential methodology to extend their therapeutic application. Herein, we have developed dissociable supermolecule-enzyme polyelectrolyte complexes based on reduction-cleavable cationic polyrotaxanes (PRXs) for the reactivation of delivered enzymes. These PRXs are characterized by their supramolecular frameworks of a polymeric chain threading into cyclic molecules, which can form polyelectrolyte complexes with anionic enzymes while retaining their three dimensional structure, although their enzymatic activity is reduced. Upon the addition of a reductant, the PRXs dissociate into their constituent molecules and release the enzymes, resulting in a complete recovery of enzymatic activity. Under the intracellular environment, the PRX-based enzyme complexes showed the highest intracellular enzymatic activity and efficient activation of anticancer prodrugs to induce cytotoxic effects in comparison with the non-dissociable complexes and the commercial cell-penetrating peptide-based reagents. Thus, the intracellularly dissociable supermolecules are an attractive system for delivering therapeutic enzymes into living cells.

Protein transfection of mouse lung.

Increasing protein expression enables researchers to better understand the functional role of that protein in regulating key biological processes(1). In the lung, this has been achieved typically through genetic approaches that utilize transgenic mice(2,3) or viral or non-viral vectors that elevate protein levels via increased gene expression(4). Transgenic mice are costly and time-consuming to generate and the random insertion of a transgene or chronic gene expression can alter normal lung development and thus limit the utility of the model(5). While conditional transgenics avert problems associated with chronic gene expression(6), the reverse tetracycline-controlled transactivator (rtTA) mice, which are used to generate conditional expression, develop spontaneous air space enlargement(7). As with transgenics, the use of viral and non-viral vectors is expensive(8) and can provoke dose-dependent inflammatory responses that confound results(9) and hinder expression(10). Moreover, the efficacy of repeated doses are limited by enhanced immune responses to the vector(11,12). Researchers are developing adeno-associated viral (AAV) vectors that provoke less inflammation and have longer expression within the lung(13). Using ß-galactosidase, we present a method for rapidly and effectively increasing protein expression within the lung using a direct protein transfection technique. This protocol mixes a fixed amount of purified protein with 20 µl of a lipid-based transfection reagent (Pro-Ject, Pierce Bio) to allow penetration into the lung tissue itself. The liposomal protein mixture is then injected into the lungs of the mice via the trachea using a microsprayer (Penn Century, Philadelphia, PA). The microsprayer generates a fine plume of liquid aerosol throughout the lungs. Using the technique we have demonstrated uniform deposition of the injected protein throughout the airways and the alveoli of mice(14). The lipid transfection technique allows the use of a small amount of protein to achieve effect. This limits the inflammatory response that otherwise would be provoked by high protein administration. Indeed, using this technique we published that we were able to significantly increase PP2A activity in the lung without affecting lung lavage cellularity(15). Lung lavage cellularity taken 24 hr after challenge was comparable to controls (27 ± 4 control vs. 31 ± 5 albumin transfected; N=6 per group). Moreover, it increases protein levels without inducing lung developmental changes or architectural changes that can occur in transgenic models. However, the need for repeated administrations may make this technique less favorable for studies examining the effects of long-term increases in protein expression. This would be particularly true for proteins with short half-lives.

Optimization of water-in-oil-in-water microencapsulated ß-galactosidase by response surface methodology.

This study was carried out to determine the optimum conditions for water-in-oil-in-water (W/O/W) microencapsulated lactase (ß-galactosidase) in order to prevent the intolerance of lactose in milk. The core material was lactase and the coating materials were medium-chain triglyceride for W/O phase, and whey protein isolate (WPI), maltodextrin, gum arabic, and its mixtures for W/O/W phase. Polyglycerol polyricinoleate (PGPR) was used as a primary emulsifier, and polyoxyethylene sorbitan monolaurate (PSML) was selected as a secondary emulsifier based on emulsion stability index. To determine the most efficient conditions for the W/O/W-lactase microencapsulation, the ratio of core to coating materials and amounts of emulsifiers were investigated by response surface methodology. The optimum ratio of core to coating materials in W/O, amount of PGPR, ratio of core to coating material in W/O/W, and amount of PSML were found to be 0.5-9.5, 0.75% (w/v), 1.7-8.3, and 0.25% (w/v), respectively. The average size of the microcapsules was about 10 µm under optimum conditions. Microcapsules of 30% (w/v) WPI as a secondary coating material could evenly distribute the pocket of lactase. Based on the data obtained from this study, lactase microcapsules could effectively be produced by the method of W/O/W double emulsion.

Brain-targeted delivery of protein using chitosan- and RVG peptide-conjugated, pluronic-based nano-carrier.

Brain-targeted delivery of drug or imaging agent is hard to achieve efficiently due to the infiltrative nature of the blood-brain barrier (BBB). Moreover, delivery of therapeutic proteins to brain tissue is further limited by the size and physic-chemical properties of proteins. In this work, we developed a chitosan-conjugated Pluronic-based nano-carrier with a specific target peptide for the brain (rabies virus glycoprotein; RVG29) and applied for the protein delivery to the brain. The in-vivo brain accumulation of the nano-carrier in mice followed i.v injection was optically monitored with Cy5.5-conjugation to the nano-carrier, and the result showed that the Pluronic-based nano-carrier conjugated with both chitosan and the peptide was very efficient for the accumulation in brain tissue and was remarkably better than the nano-carrier conjugated with the peptide only. ß-galactosidase, a model protein, was also delivered and accumulated efficiently in the brain by loading in the nano-carrier, analyzed by the bio-distribution of ß-galactosidase. The delivered protein in the brain also maintained its bioactivity. Therefore, RVG29- and chitosan-conjugated Pluronic-based nano-carrier could be potentially useful for the diagnosis and therapy of brain diseases.

Magnetically-enabled and MR-monitored selective brain tumor protein delivery in rats via magnetic nanocarriers.

The delivery of bioactive proteins to tumors is associated with many difficulties that have impeded clinical translation of these promising therapeutics. Herein we present an approach, including (1) use of magnetically-responsive and MRI-visible nanoparticles as drug carriers, (2) topography-optimized intra-arterial magnetic targeting, (3) MRI-guided subject alignment within the magnetic field, and (4) surface modification of the protein drug with membrane-permeable polyethyleneimine (PEI), to prevail over the obstacles in protein delivery. Applying these methodologies, we demonstrated the delivery of a significant quantity of ß-galactosidase selectively into brain tumors of glioma-bearing rats, while limiting the exposure of normal brain regions. Clinical viability of the technologies utilized, and the ability to deliver proteins at high nanomolar-range tumor concentrations, sufficient to completely eradicate a tumor lesion with existing picomolar-potency protein toxins, renders the prospect of enabling protein-based cancer therapy extremely promising.

Bis-(3',5')-cyclic dimeric adenosine monophosphate: strong Th1/Th2/Th17 promoting mucosal adjuvant.

New effective adjuvants are required to improve the performance of subunit vaccines. Here, we showed that bis-(3',5')-cyclic dimeric adenosine monophosphate (c-di-AMP), a second messenger molecule in bacteria and archaea, exerts strong adjuvant activities when delivered by mucosal route. In vitro studies showed that c-di-AMP was able to both stimulate pre-activated murine macrophages and promote the activation and maturation of dendritic cells of murine and human origin. Co-administration of c-di-AMP with ß-galactosidase (ß-Gal) by intranasal route to BALB/c mice resulted in the elicitation of significantly higher serum antigen-specific IgG titres than in controls. The induction of local immune responses was shown by the production of antigen-specific secretory IgA in different mucosal territories. In addition, strong cellular immune responses were observed against both the ß-Gal protein and a peptide encompassing its MHC class I-restricted epitope. The ratio of ß-Gal-specific antibodies and the secreted cytokine profiles by in vitro re-stimulated splenocytes suggested that a balanced Th1/Th2/Th17 response pattern is promoted by c-di-AMP. When C57BL/6 mice were immunized with OVA and c-di-AMP, vigorous in vivo CTL responses were also observed. These results indicated that c-di-AMP exhibits a high potential as adjuvant for the development of mucosal vaccines, in particular when cellular immunity is needed.

Functionalized linear poly(amidoamine)s are efficient vectors for intracellular protein delivery.

An effective intracellular protein delivery system was developed based on functionalized linear poly(amidoamine)s (PAAs) that form self-assembled cationic nanocomplexes with oppositely charged proteins. Three differently functionalized PAAs were synthesized, two of these having repetitive disulfide bonds in the main chain, by Michael-type polyaddition of 4-amino-1-butanol (ABOL) to cystamine bisacrylamide (CBA), histamine (HIS) to CBA, and ABOL to bis(acryloyl)piperazine (BAP). These water-soluble PAAs efficiently condense ß-galactosidase by self-assembly into nanoscaled and positively-charged complexes. Stable under neutral extracellular conditions, the disulfide-containing nanocomplexes rapidly destabilized in a reductive intracellular environment. Cell-internalization and cytotoxicity experiments showed that the PAA-based nanocomplexes were essentially non-toxic. ß-Galactosidase was successfully internalized into cells, with up to 94% of the cells showing ß-galactosidase activity, whereas the enzyme alone was not taken up by the cells. The results indicate that these poly(amidoamine)s have excellent properties as highly potent and non-toxic intracellular protein carriers, which should create opportunities for novel applications in protein delivery.

Targeted delivery of large fusion protein into hippocampal neurons by systemic administration.

Targeted delivery of proteins into the hippocampus has not yet been achieved. Here, we show that systemic administration of the ß-galactosidase, fused to a 43-amino-acid peptide derived from rabies virus glycoprotein (RVG), results in targeted delivery of the fusion protein into the hippocampal neurons. This approach may enable the development of protein therapy for neurodegenerative diseases, such as Alzheimer's disease. This result may open new possibilities in using neurotropic virus glycoprotein-derived peptides for targeted delivery of therapeutic molecules into local brain regions.

Adenovirus-mediated peroxisome proliferator activated receptor gamma overexpression prevents nutritional fibrotic steatohepatitis in mice.

OBJECTIVE: The pathogenesis of non-alcoholic steatohepatitis is still unclear. We have demonstrated previously that peroxisome proliferator activated receptor gamma (PPARγ) ligand protects against inflammation and fibrogenesis in experimental non-alcoholic steatohepatitis. We aim to elucidate the effect and the mechanism of PPARγ itself on nutritional fibrotic steatohepatitis in mice. METHODS: C57BL/6J mice were fed with methionine-choline deficient (MCD) diet for 8 weeks to induce fibrotic steatohepatitis. Mice fed the MCD diet were treated with adenovirus carrying PPARγ (Ad-PPARγ), Ad-PPARγ plus PPARγ agonist rosiglitazone, or PPARγ antagonist 2-chloro-5-nitrobenzaniliden (GW9662), respectively. The effects of up-regulation of PPARγ in the presence or absence of its agonist/or antagonist were assessed by comparing the severity of hepatic injury, activation of hepatic stellate cells and the expression of adiponectin, heme oxygenase-1, and fibrogenic related genes. RESULTS: Mice fed with MCD diet for 8 weeks showed severe hepatic injury including hepatic steatosis, inflammatory infiltration, and fibrosis. Administration of Ad-PPARγ significantly lowered serum alanine aminotransferase level and ameliorated hepatic steatosis, necroinflammation, and fibrosis. These effects were associated with enhanced expression of PPARγ, up-regulated expression of adiponectin and heme oxygenase-1, and down-regulated expression of tumor necrosis factor alpha, interleukin-6, α-smooth muscle actin, transforming growth factor beta 1, matrix metallopeptidase-2, and -9. Administration of GW9662 promoted the severity of liver histology. CONCLUSIONS: The present study provided evidences for the protective role of overexpressing PPARγ in ameliorating hepatic fibrosing steatohepatitis in mice. Modulation of PPARγ expression might serve as a therapeutic approach for fibrotic steatohepatitis.

A carrier for non-covalent delivery of functional beta-galactosidase and antibodies against amyloid plaques and IgM to the brain.

BACKGROUND: Therapeutic intervention of numerous brain-associated disorders currently remains unrealized due to serious limitations imposed by the blood-brain-barrier (BBB). The BBB generally allows transport of small molecules, typically <600 daltons with high octanol/water partition coefficients, but denies passage to most larger molecules. However, some receptors present on the BBB allow passage of cognate proteins to the brain. Utilizing such receptor-ligand systems, several investigators have developed methods for delivering proteins to the brain, a critical requirement of which involves covalent linking of the target protein to a carrier entity. Such covalent modifications involve extensive preparative and post-preparative chemistry that poses daunting limitations in the context of delivery to any organ. Here, we report creation of a 36-amino acid peptide transporter, which can transport a protein to the brain after routine intravenous injection of the transporter-protein mixture. No covalent linkage of the protein with the transporter is necessary. APPROACH: A peptide transporter comprising sixteen lysine residues and 20 amino acids corresponding to the LDLR-binding domain of apolipoprotein E (ApoE) was synthesized. Transport of beta-galactosidase, IgG, IgM, and antibodies against amyloid plques to the brain upon iv injection of the protein-transporter mixture was evaluated through staining for enzyme activity or micro single photon emission tomography (micro-SPECT) or immunostaining. Effect of the transporter on the integrity of the BBB was also investigated. PRINCIPAL FINDINGS: The transporter enabled delivery to the mouse brain of functional beta-galactosidase, human IgG and IgM, and two antibodies that labeled brain-associated amyloid beta plaques in a mouse model of Alzheimer's disease. SIGNIFICANCE: The results suggest the transporter is able to transport most or all proteins to the brain without the need for chemically linking the transporter to a protein. Thus, the approach offers an avenue for rapid clinical evaluation of numerous candidate drugs against neurological diseases including cancer. (299 words).

Actividad de la ß-galactosidasa como marcador de senescencia en cultivos primarios del epitelio superficial del ovario / ß-Galactosidase activity as a marker of senescence in primary cultures of the ovarian surface epithelium

La actividad de la ß-galactosidasa refleja la tasa de envejecimiento celular in vitro. Mediante quimioluminiscencia se cuantificó dicha actividad a pH 6 en células epiteliales ováricas provenientes de 28 donantes sin antecedentes de cáncer. Las células fueron cultivadas en forma seriada hasta alcanzar el estado de detención permanente de crecimiento. Durante la fase de crecimiento exponencial, todos los cultivos mostraron un patrón semejante de crecimiento y una baja actividad ß-galactosidasa. Sin embargo, el inicio de la disminución de la capacidad replicativa que caracteriza el final de dicha fase, así como el inicio de la fase estacionaria o senescente presentaron un aumento significativo en la actividad enzimática. Nuestros resultados mostraron que la actividad ß-galactosidasa puede ser considerada como marcador de senescencia replicativa del epitelio superficial del ovario a pH 6.

ß-galactosidase activity reflects the rate of cellular aging in vitro. Such activity was quantified at pH 6 in ovarian epithelial cells from 28 donors without a history of cancer, by the chemoluminiscent method. The cells were serially cultured until they achieved the state of permanent growth arrest. During the exponential growth phase, all cultures showed a similar pattern of growth and low ß-galactosidase activity. However, both in the onset of decrease replicative activity, as well as in the onset of the stationary phase, there was a significant rise in the enzyme activity. Our results showed that ß-galactosidase activity can be considered as a replicative senescence marker of the ovarian surface epithelium at pH 6.

Establishment of protein delivery systems targeting podocytes.

BACKGROUND: Podocytes are uniquely structured cells that are critical to the kidney filtration barrier. Their anatomic location on the outer side of the glomerular capillaries expose podocytes to large quantities of both plasma and urinary components and thus are reachable for drug delivery. Recent years have made clear that interference with podocyte-specific disease pathways can modulate glomerular function and influence severity and progression of glomerular disease. METHODOLOGY/PRINCIPAL FINDINGS: Here, we describe studies that show efficient transport of proteins into the mammalian cells mouse 3T3 fibroblasts and podocytes, utilizing an approach termed profection. We are using synthetic lipid structures that allow the safe packing of proteins or antibodies resulting in the subsequent delivery of protein into the cell. The uptake of lipid coated protein is facilitated by the intrinsic characteristic of cells such as podocytes to engulf particles that are physiologically retained in the extracellular matrix. Profection of the restriction enzyme MunI in 3T3 mouse fibroblasts caused an increase in DNA degradation. Moreover, purified proteins such as beta-galactosidase and the large GTPase dynamin could be profected into podocytes using two different profection reagents with the success rate of 95-100%. The delivered beta-galactosidase enzyme was properly folded and able to cleave its substrate X-gal in podocytes. Diseased podocytes are also potential recipients of protein cargo as we also delivered fluorophore labeled IgG into puromycin treated podocytes. We are currently optimizing our protocol for in vivo profection. CONCLUSIONS: Protein transfer is developing as an exciting tool to study and target highly differentiated cells such as podocytes.

Quality of the transgene-specific CD8+ T cell response induced by adenoviral vector immunization is critically influenced by virus dose and route of vaccination.

Adenoviral vectors have been widely used for experimental gene therapy and vaccination, yet there is a surprising lack of knowledge connecting the route and dose of adenovirus administration to the induced transgene-specific immune response. We have recently demonstrated polyfunctional CD8(+) T cells and protective memory responses using adenoviral vectors, which seem to contrast with recent reports suggesting that an exhausted CD8(+) T cell phenotype is induced by inoculation with adenoviral vectors. Accordingly, we investigated the route and dose interrelationship for transgene-specific CD8(+) T cells using adenoviral vectors encoding beta-galactosidase applied either s.c. or i.v. Irrespective of the route of inoculation, most of the adenoviral inoculum was found to disseminate systemically as the dose was raised beyond 10(9) particles. The number of transgene-specific CD8(+) T cells correlated positively with dissemination, whereas the functional capacity of the generated T cells correlated inversely with vector dissemination. A comparison of the immune response to s.c. or i.v. administration at moderate doses revealed that inoculation by both routes induced a transient peak of IFN-gamma-producing CD8(+) T cells 2 to 3 wk postinfection, but following i.v. administration, these cells were only detected in the liver. Two to four months after systemic, but not peripheral, immunization, dysfunctional transgene-specific CD8(+) T cells impaired in both cytokine production and important in vivo effector functions, accumulated in the spleen. These findings indicate that the localization of the adenoviral inoculum and not the total Ag load determines the quality of the CD8(+) T cell response induced with adenoviral vaccines.

Intracellular delivery of a membrane-impermeable enzyme in active form using functionalized gold nanoparticles.

Gold nanoparticles were coated with a short peptide to promote intracellular delivery of membrane-impermeable proteins. Through microscopy and enzyme assays, we demonstrated the particles were able to transport functional enzymes into a variety of cell lines. Significantly, the transported proteins were able to escape from endosomes. Moreover, these particles showed no apparent cytotoxicity.

The member of the cyclic di-nucleotide family bis-(3', 5')-cyclic dimeric inosine monophosphate exerts potent activity as mucosal adjuvant.

Here we demonstrated that bis-(3',5')-cyclic dimeric inosine monophosphate (c-di-IMP) exhibits potent adjuvant properties. BALB/c or C57BL/6 mice were immunized with the model antigens beta-galactosidase (beta-Gal) or Ovalbumin (OVA) alone or co-administered with c-di-IMP by the intranasal route. Animals receiving c-di-IMP showed significantly higher anti-beta-Gal or OVA immunoglobulin G titres (IgG) in sera than those vaccinated with beta-Gal or OVA alone. Furthermore, strong local immune responses were also detectable in different mucosal territories, as shown by the high levels of beta-Gal-specific secretory IgA (sIgA). The analysis of the antigen-specific IgG isotypes in sera, together with the profiles of the cytokines and chemokines secreted by lymphocytes from vaccinated animals showed that the use of c-di-IMP resulted in stimulation of a mixed T(H)1/T(H)2/T(H)17 response. Mucosal immunization of C57BL/6 mice with OVA using c-di-IMP as adjuvant also led to the stimulation of strong in vivo CTL responses (i.e., 60% of antigen-specific lysis) [corrected].Our results demonstrated that the novel compound c-di-IMP exhibits strong adjuvant properties when co-administered with an antigen by the mucosal route, thereby representing a promising candidate adjuvant for the development of mucosal vaccination strategies.