Monitoring tumor antigen specific T-cell responses in cancer patients and phase I clinical trials of peptide-based vaccination.

Numerous phase I and II clinical trials testing the safety and immunogenicity of various peptide vaccine formulations based on CTL-defined tumor antigens in cancer patients have been reported during the last 7 years. While specific T-cell responses can be detected in a variable fraction of immunized patients, an even smaller but significant fraction of these patients have objective tumor responses. Efficient therapeutic vaccination should aim at boosting naturally occurring antitumor T- and B-cell responses and at sustaining a large number of tumor antigen specific and fully functional effector T cells at tumor sites. Recent progress in our ability to quantitatively and qualitatively monitor tumor antigen specific CD8 T-cell responses will greatly help in making rapid progress in this field.

Detection of colorectal carcinoma by emission-computerized tomography after injection of 123I-labeled Fab or F(ab')2 fragments from monoclonal anti-carcinoembryonic antigen antibodies.

This clinical study was based on experimental results obtained in nude mice grafted with human colon carcinoma, showing that injected 131I-labeled F(ab')2 and Fab fragments from high affinity anti-carcinoembryonic antigen (CEA) monoclonal antibodies (MAb) gave markedly higher ratios of tumor to normal tissue localization than intact MAb. 31 patients with known colorectal carcinoma, including 10 primary tumors, 13 local tumor recurrences, and 21 metastatic involvements, were injected with 123I-labeled F(ab')2 (n = 14) or Fab (n = 17) fragments from MAb anti-CEA. The patients were examined by emission-computerized tomography (ECT) at 6, 24, and sometimes 48 h after injection using a rotating dual head scintillation camera. All 23 primary tumors and local recurrences except one were clearly visualized on at least two sections of different tomographic planes. Interestingly, nine of these patients had almost normal circulating CEA levels, and three of the visualized tumors weighed only 3-5 g. Among 19 known metastatic tumor involvements, 14 were correctly localized by ECT. Two additional liver and several bone metastases were discovered by immunoscintigraphy. Altogether, 86% of the tumor sites were detected, 82% with F(ab')2 and 89% with Fab fragments. The contrast of the tumor images obtained with Fab fragments suggests that this improved method of immunoscintigraphy has the potential to detect early tumor recurrences and thus to increase the survival of patients. The results of this retrospective study, however, should be confirmed in a prospective study before this method can be recommended for the routine diagnosis of cancer.

Immunogenicity of the carcinoembryonic antigen derived peptide 694 in HLA-A2 healthy donors and colorectal carcinoma patients.

Carcinoembryonic antigen (CEACAM5) is commonly overexpressed in human colon cancer. Several antigenic peptides recognized by cytolytic CD8+ T-cells have been identified and used in colon cancer phase-I vaccination clinical trials. The HLA-A*0201-binding CEA(694-702) peptide was recently isolated from acid eluted MHC-I associated peptides from a human colon tumor cell line. However, the immunogenicity of this peptide in humans remains unknown. We found that the peptide CEA(694-702) binds weakly to HLA-A*0201 molecules and is ineffective at inducing specific CD8+ T-cell responses in healthy donors. Immunogenic-altered peptide ligands with increased affinity for HLA-A*0201 were identified. Importantly, the elicited cytolytic T lymphocyte (CTL) lines and clones cross-reacted with the wild-type CEA(694-702) peptide. Tumor cells expressing CEA were recognized in a peptide and HLA-A*0201 restricted fashion, but high-CEA expression levels appear to be required for CTL recognition. Finally, CEA-specific T-cell precursors could be readily expanded by in vitro stimulation of peripheral blood mononuclear cell (PBMC) from colon cancer patients with altered CEA peptide. However, the CEA-specific CD8+ T-cell clones derived from cancer patients revealed low-functional avidity and impaired tumor-cell recognition. Together, using T-cells to demonstrate the processing and presentation of the peptide CEA694-702, we were able to corroborate its presentation by tumor cells. However, the low avidity of the specific CTLs generated from cancer patients as well as the high-antigen expression levels required for CTL recognition pose serious concerns for the use of CEA694-702 in cancer immunotherapy.

Vaccination with a recombinant protein encoding the tumor-specific antigen NY-ESO-1 elicits an A2/157-165-specific CTL repertoire structurally distinct and of reduced tumor reactivity than that elicited by spontaneous immune responses to NY-ESO-1-expressing Tumors.

In a recent vaccination trial assessing the immunogenicity of an NY-ESO-1 (ESO) recombinant protein administered with Montanide and CpG, we have obtained evidence that this vaccine induces specific cytolytic T lymphocytes (CTL) in half of the patients. Most vaccine-induced CTLs were directed against epitopes located in the central part of the protein, between amino acids 81 and 110. This immunodominant region, however, is distinct from another ESO CTL region, 157-165, that is a frequent target of spontaneous CTL responses in A2+ patients bearing ESO tumors. In this study, we have investigated the CTL responses to ESO 157-165 in A2+ patients vaccinated with the recombinant protein. Our data indicate that after vaccination with the protein, CTL responses to ESO 157-165 are induced in some, but not all, A2+ patients. ESO 157-165-specific CTLs induced by vaccination with the ESO protein were functionally heterogeneous in terms of tumor recognition and often displayed decreased tumor reactivity as compared with ESO 157-165-specific CTLs isolated from patients with spontaneous immune responses to ESO. Remarkably, protein-induced CTLs used T-cell receptors similar to those previously isolated from patients vaccinated with synthetic ESO peptides (Vbeta4.1) and distinct from those used by highly tumor-reactive CTLs isolated from patients with spontaneous immune responses (Vbeta1.1, Vbeta8.1, and Vbeta13.1). Together, these results demonstrate that vaccination with the ESO protein elicits a repertoire of ESO 157-165-specific CTLs bearing T-cell receptors that are structurally distinct from those of CTLs found in spontaneous immune responses to the antigen and that are heterogeneous in terms of tumor reactivity, being often poorly tumor reactive.

Multi-well fungal co-culture for de novo metabolite-induction in time-series studies based on untargeted metabolomics.

The induction of fungal metabolites by fungal co-cultures grown on solid media was explored using multi-well co-cultures in 2 cm diameter Petri dishes. Fungi were grown in 12-well plates to easily and rapidly obtain the large number of replicates necessary for employing metabolomic approaches. Fungal culture using such a format accelerated the production of metabolites by several weeks compared with using the large-format 9 cm Petri dishes. This strategy was applied to a co-culture of a Fusarium and an Aspergillus strain. The metabolite composition of the cultures was assessed using ultra-high pressure liquid chromatography coupled to electrospray ionisation and time-of-flight mass spectrometry, followed by automated data mining. The de novo production of metabolites was dramatically increased by nutriment reduction. A time-series study of the induction of the fungal metabolites of interest over nine days revealed that they exhibited various induction patterns. The concentrations of most of the de novo induced metabolites increased over time. However, interesting patterns were observed, such as with the presence of some compounds only at certain time points. This result indicates the complexity and dynamic nature of fungal metabolism. The large-scale production of the compounds of interest was verified by co-culture in 15 cm Petri dishes; most of the induced metabolites of interest (16/18) were found to be produced as effectively as on a small scale, although not in the same time frames. Large-scale production is a practical solution for the future production, identification and biological evaluation of these metabolites.

Comparison of three antigenic extracts of Eurotium amstelodami in serological diagnosis of farmer's lung disease.

In France and Finland, farmer's lung disease (FLD), a hypersensitivity pneumonitis common in agricultural areas, is mainly caused by Eurotium species. The presence of antibodies in patients' serum is an important criterion for diagnosis. Our study aimed to improve the serological diagnosis of FLD by using common fungal particles that pollute the farm environment as antigens. Fungal particles of the Eurotium species were observed in handled hay. A strain of Eurotium amstelodami was grown in vitro using selected culture media; and antigen extracts from sexual (ascospores), asexual (conidia), and vegetative (hyphae) forms were made. Antigens were tested by enzyme-linked immunosorbent assay (ELISA), which was used to test for immunoglobulin G antibodies from the sera of 17 FLD patients, 40 healthy exposed farmers, and 20 nonexposed controls. The antigens were compared by receiver operating characteristic analysis, and a threshold was then established. The ascospores contained in asci enclosed within cleistothecia were present in 38% of the hay blades observed; conidial heads of aspergillus were less prevalent. The same protocol was followed to make the three antigen extracts. A comparison of the results for FLD patients and exposed controls showed the area under the curve to be 0.850 for the ascospore antigen, 0.731 for the conidia, and 0.690 for the hyphae. The cutoffs that we determined, with the standard deviation for measures being taken into account, showed 67% for sensitivity and 92% for specificity with the ascospore antigen. In conclusion, the serological diagnosis of FLD by ELISA was improved by the adjunction of ascospore antigen.

Lentivector immunization induces tumor antigen-specific B and T cell responses in vivo.

Expression of the cancer/germ-line antigen NY-ESO-1 by tumors elicits spontaneous humoral and cellular immune responses in some cancer patients. Development of vaccines capable of stimulating such comprehensive immune responses is desirable. We have produced recombinant lentivectors directing the intracellular synthesis of NY-ESO-1 (rLV/ESO) and have analyzed the in vivo immune response elicited by this vector. Single injection of rLV/ESO into HLA-A2-transgenic mice elicited long-lasting B and T cell responses against NY-ESO-1. CD8+ T cells against the HLA-A2-restricted peptide NY-ESO-1(157-165) were readily detectable ex vivo and showed restricted TCR Vbeta usage. Moreover, rLV/ESO elicited a far greater anti-NY-ESO-1(157-165) CD8+ T cell response than peptide- or protein-based vaccines. Anti-NY-ESO-1 antibodies were rapidly induced after immunization and their detection preceded that of the antigen-specific CD8+ T cells. The rLV/ESO also induced CD4+ T cells. These cells played an essential role as their depletion completely abrogated B cell and CD8+ T cell responses against NY-ESO-1. The induced CD4+ T cells were primarily directed against a single NY-ESO-1 epitope spanning amino acids 81-100. Altogether, our study shows that rLV/ESO induces potent and comprehensive immune responses in vivo.

Impact of targeted antifungal prophylaxis in heart transplant recipients at high risk for early invasive fungal infection.

BACKGROUND: Invasive fungal infection (IFI) is associated with high mortality after heart transplantation (HTx). After two undiagnosed fatal cases of early disseminated fungal infections in our heart transplant program, a retrospective analysis was conducted to identify risk factors for the development of IFI and implement a new antifungal prophylaxis policy. METHODS: Clinical characteristics of HTx recipients hospitalized in our center (2004-2010) were recorded (Period 1), and risk factors associated with IFI were investigated using Cox regression analysis. From October 2010 to October 2012 (Period 2), targeted caspofungin prophylaxis was administered to all recipients at high risk for IFI, based on the results of the Period 1 analysis. RESULTS: During Period 1, 10% (6/59) of the patients developed IFI at a median onset of 9 days after transplantation. By multivariate analysis, the use of posttransplant extracorporeal membrane oxygenation (ECMO) was the strongest predictor for fungal infection (OR, 29.93; 95% CI, 1.51-592.57, P=0.03), whereas renal replacement therapy (RRT) and Aspergillus colonization were significant predictors only by univariate analysis. During Period 2, only 4% (1/26) of the patients developed IFI. In patients at high risk for IFI, antifungal prophylaxis was administered to 17% (4/23) in Period 1 versus 100% (13/13) in Period 2 (P<0.01). By survival analysis, antifungal prophylaxis was associated with a reduction in 90-day IFI incidence (HR, 0.14; 95% CI, 0.03-0.84, P=0.03) and 30-day mortality (HR, 0.25; 95% CI, 0.09-0.8, P=0.02). CONCLUSION: Extracorporeal membrane oxygenation was identified an important risk factor for IFI after HTx, and its use may require targeted administration of antifungal prophylaxis in the immediate posttransplant period.

Milbemycins: more than efflux inhibitors for fungal pathogens.

Existing antifungal agents are still confronted to activities limited to specific fungal species and to the development of resistance. Several improvements are possible either by tackling and overcoming resistance or exacerbating the activity of existing antifungal agents. In Candida glabrata, azole resistance is almost exclusively mediated by ABC transporters (including C. glabrata CDR1 [CgCDR1] and CgCDR2) via gain-of-function mutations in the transcriptional activator CgPDR1 or by mitochondrial dysfunctions. We also observed that azole resistance was correlating with increasing virulence and fitness of C. glabrata in animal models of infection. This observation motivated the re-exploitation of ABC transporter inhibitors as a possible therapeutic intervention to decrease not only the development of azole resistance but also to interfere with the virulence of C. glabrata. Milbemycins are known ABC transporter inhibitors, and here we used commercially available milbemycin A3/A4 oxim derivatives to verify this effect. As expected, the derivatives were inhibiting C. glabrata efflux with the highest activity for A3 oxim below 1 μg/ml. More surprising was that oxim derivatives had intrinsic fungicidal activity above 3.2 μg/ml, thus highlighting effects additional to the efflux inhibition. Similar values were obtained with C. albicans. Our data show that the fungicidal activity could be related to reactive oxygen species formation in these species. Transcriptional analysis performed both in C. glabrata and C. albicans exposed to A3 oxim highlighted a core of commonly regulated genes involved in stress responses, including genes involved in oxidoreductive processes, protein ubiquitination, and vesicle trafficking, as well as mitogen-activated protein kinases. However, the transcript profiles contained also species-specific signatures. Following these observations, experimental treatments of invasive infections were performed in mice treated with the commercial A3/A4 oxim preparation alone or in combination with fluconazole. Tissue burden analysis revealed that oxims on their own were able to decrease fungal burdens in both Candida species. In azole-resistant isolates, oxims acted synergistically in vivo with fluconazole to reduce fungal burden to levels of azole-susceptible isolates. In conclusion, we show here the potential of milbemycins not only as drug efflux inhibitors but also as effective fungal growth inhibitors in C. glabrata and C. albicans.

A natural structural variant of the mouse TCR beta-chain displays intrinsic receptor function and antigen specificity.

The Cbeta0 alternate cassette exon is located between the Jbeta1 and Cbeta1 genes in the mouse TCR beta-locus. In T cells with a VDJbeta1 rearrangement, the Cbeta0 exon may be included in TCRbeta transcripts (herein called TCRbeta-Cbeta0 transcripts), potentially inserting an additional 24 aa between the V and C domains of the TCR beta-chain. These TCRbeta splice isoforms may be differentially regulated after Ag activation, because we detected TCRbeta-Cbeta0 transcripts in a high proportion (>60%) of immature and mature T cells having VDJbeta1 rearrangements but found a substantially reduced frequency (<35%) of TCRbeta-Cbeta0 expression among CD8 T cells selected by Ag in vivo. To study the potential activity of the TCRbeta-Cbeta0 splice variant, we cloned full-length TCR cDNAs by single-cell RT-PCR into retroviral expression vectors. We found that the TCRbeta-Cbeta0 splice isoform can function during an early stage of T cell development normally dependent on TCR beta-chain expression. We also demonstrate that T hybridoma-derived cells expressing a TCRbeta-Cbeta0 isoform together with the clonally associated TCR alpha-chain recognize the same cognate peptide-MHC ligand as the corresponding normal alphabetaTCR. This maintenance of receptor function and specificity upon insertion of the Cbeta0 peptide cassette signifies a remarkable adaptability for the TCR beta-chain, and our findings open the possibility that this splice isoform may function in vivo.

Monophyly of beta-tubulin and H+-ATPase gene variants in Glomus intraradices: consequences for molecular evolutionary studies of AM fungal genes.

Arbuscular mycorrhizal fungi (AMF) are an ecologically important group of fungi. Previous studies showed the presence of divergent copies of beta-tubulin and V-type vacuolar H+-ATPase genes in AMF genomes and suggested horizontal gene transfer from host plants or mycoparasites to AMF. We sequenced these genes from DNA isolated from an in vitro cultured isolate of Glomus intraradices that was free of any obvious contaminants. We found two highly variable beta-tubulin sequences and variable H+-ATPase sequences. Despite this high variation, comparison of the sequences with those in gene banks supported a glomeromycotan origin of G. intraradices beta-tubulin and H+-ATPase sequences. Thus, our results are in sharp contrast with the previously reported polyphyletic origin of those genes. We present evidence that some highly divergent sequences of beta-tubulin and H+-ATPase deposited in the databases are likely to be contaminants. We therefore reject the prediction of horizontal transfer to AMF genomes. High differences in GC content between glomeromycotan sequences and sequences grouping in other lineages are shown and we suggest they can be used as an indicator to detect such contaminants. H+-ATPase phylogeny gave unexpected results and failed to resolve fungi as a natural group. beta-Tubulin phylogeny supported Glomeromeromycota as sister group of the Chytridiomycota. Contrasts between our results and trees previously generated using rDNA sequences are discussed.

The role of fungi in the precipitation of calcite : relationships between fungal filaments, nanofibres, and needle fibre calcite

RésuméLes champignons sont impliqués dans les cycles biogéochimiques de différentes manières. En particulier, ils sont reconnus en tant qu'acteurs clés dans la dégradation de la matière organique, comme fournisseurs d'éléments nutritifs via l'altération des minéraux mais aussi comme grands producteurs d'acide oxalique et de complexes oxalo-métalliques. Toutefois, peu de choses sont connues quant à leur contribution à la genèse d'autres types de minéraux, tel que le carbonate de calcium (CaCO3). Le CaCO3 est un minéral ubiquiste dans de nombreux écosystèmes et il joue un rôle essentiel dans les cycles biogéochimiques du carbone (C) et du calcium (Ca). Le CaCO3 peut être d'origine physico-chimique ou biogénique et de nombreux organismes sont connus pour contrôler ou induire sa biominéralisation. Les champignons ont souvent été soupçonnés d'être impliqué dans ce processus, cependant il existe très peu d'informations pour étayer cette hypothèse.Cette thèse a eu pour but l'étude de cet aspect négligé de l'impact des champignons dans les cycles biogéochimiques, par l'exploration de leur implication potentielle dans la formation d'un type particulier de CaCO3 secondaires observés dans les sols et dans les grottes des environnements calcaires. Dans les grottes, ces dépôts sont appelés moonmilk, alors que dans les sols on les appelle calcite en aiguilles. Cependant ces deux descriptions correspondent en fait au même assemblage microscopique de deux habitus particulier de la calcite: la calcite en aiguilles (au sens strict du terme cette fois-ci) et les nanofibres. Ces deux éléments sont des habitus aciculaires de la calcite, mais présentent des dimensions différentes. Leur origine, physico-chimique ou biologique, est l'objet de débats intenses depuis plusieurs années déjà.L'observation d'échantillons environnementaux avec des techniques de microscopie (microscopie électronique et micromorphologie), ainsi que de la microanalyse EDX, ont démontré plusieurs relations intéressantes entre la calcite en aiguilles, les nanofibres et des éléments organiques. Premièrement, il est montré que les nanofibres peuvent être organiques ou minérales. Deuxièmement, la calcite en aiguilles et les nanofibres présentent de fortes analogies avec des structures hyphales, ce qui permet de confirmer l'hypothèse de leur origine fongique. En outre, des expériences en laboratoire ont confirmé l'origine fongique des nanofibres, par des digestions enzymatiques d'hyphes fongiques. En effet, des structures à base de nanofibres, similaires à celles observées dans des échantillons naturels, ont pu être produites par cette approche. Finalement, des enrichissements en calcium ont été mesurés dans les parois des hyphes et dans des inclusions intrahyphales provenant d'échantillons naturels de rhizomorphes. Ces résultats suggèrent une implication de la séquestration de calcium dans la formation de la calcite en aiguilles et/ou des nanofibres.Plusieurs aspects restent à élucider, en particulier la compréhension des processus physiologiques impliqués dans la nucléation de calcite dans les hyphes fongiques. Cependant, les résultats obtenus dans cette thèse ont permis de confirmer l'implication des champignons dans la formation de la calcite en aiguilles et des nanofibres. Ces découvertes sont d'une grande importance dans les cycles biogéochimiques puisqu'ils apportent de nouveaux éléments dans le cycle couplé C-Ca. Classiquement, les champignons sont considérés comme étant impliqués principalement dans la minéralisation de la matière organique et dans l'altération minérale. Cette étude démontre que les champignons doivent aussi être pris en compte en tant qu'agents majeurs de la genèse de minéraux, en particulier de CaCO3. Ceci représente une toute nouvelle perspective en géomycologie quant à la participation des champignons au cycle biologique du C. En effet, la présence de ces précipitations de CaCO3 secondaires représente un court-circuit dans le cycle biologique du C puisque du C inorganique du sol se retrouve piégé dans de la calcite plutôt que d'être retourné dans l'atmosphère.AbstractFungi are known to be involved in biogeochemical cycles in numerous ways. In particular, they are recognized as key players in organic matter recycling, as nutrient suppliers via mineral weathering, as well as large producers of oxalic acid and metal-oxalate. However, little is known about their contribution to the genesis of other types of minerals such as calcium carbonate (CaCO3). Yet, CaC03 are ubiquitous minerals in many ecosystems and play an essential role in the biogeochemical cycles of both carbon (C) and calcium (Ca). CaC03 may be physicochemical or biogenic in origin and numerous organisms have been recognized to control or induce calcite biomineralization. While fungi have often been suspected to be involved in this process, only scarce information support this hypothesis.This Ph.D. thesis aims at investigating this disregarded aspect of fungal impact on biogeochemical cycles by exploring their possible implication in the formation of a particular type of secondary CaC03 deposit ubiquitously observed in soils and caves from calcareous environments. In caves, these deposits are known as moonmilk, whereas in soils, they are known as Needle Fibre Calcite (NFC - sensu lato). However, they both correspond to the same microscopic assemblage of two distinct and unusual habits of calcite: NFC {sensu stricto) and nanofibres. Both features are acicular habits of calcite displaying different dimensions. Whether these habits are physicochemical or biogenic in origin has been under discussion for a long time.Observations of natural samples using microscopic techniques (electron microscopy and micromorphology) and EDX microanalyses have demonstrated several interesting relationships between NFC, nanofibres, and organic features. First, it has shown that nanofibres can be either organic or minera! in nature. Second, both nanofibres and NFC display strong structural analogies with fungal hyphal features, supporting their fungal origin. Furthermore, laboratory experiments have confirmed the fungal origin of nanofibres through an enzymatic digestion of fungal hyphae. Indeed, structures made of nanofibres with similar features as those observed in natural samples have been produced. Finally, calcium enrichments have been measured in both cell walls and intrahyphal inclusions of hyphae from rhizomorphs sampled in the natural environment. These results point out an involvement of calcium sequestration in nanofibres and/or NFC genesis.Several aspects need further investigation, in particular the understanding of the physiological processes involved in hyphal calcite nucleation. However, the results obtained during this study have allowed the confirmation of the implication of fungi in the formation of both NFC and nanofibres. These findings are of great importance regarding global biogeochemical cycles as they bring new insights into the coupled C and Ca cycles. Conventionally, fungi are considered to be involved in organic matter mineralization and mineral weathering. In this study, we demonstrate that they must also be considered as major agents in mineral genesis, in particular CaC03. This is a completely new perspective in geomycology regarding the role of fungi in the short-term (or biological) C cycle. Indeed, the presence of these secondary CaC03 precipitations represents a bypass in the short- term carbon cycle, as soil inorganic C is not readily returned to the atmosphere.

Dectin-1 is essential for reverse transcytosis of glycosylated SIgA-antigen complexes by intestinal M cells.

Intestinal microfold (M) cells possess a high transcytosis capacity and are able to transport a broad range of materials including particulate antigens, soluble macromolecules, and pathogens from the intestinal lumen to inductive sites of the mucosal immune system. M cells are also the primary pathway for delivery of secretory IgA (SIgA) to the gut-associated lymphoid tissue. However, although the consequences of SIgA uptake by M cells are now well known and described, the mechanisms whereby SIgA is selectively bound and taken up remain poorly understood. Here we first demonstrate that both the Cα1 region and glycosylation, more particularly sialic acid residues, are involved in M cell-mediated reverse transcytosis. Second, we found that SIgA is taken up by M cells via the Dectin-1 receptor, with the possible involvement of Siglec-5 acting as a co-receptor. Third, we establish that transcytosed SIgA is taken up by mucosal CX3CR1⁺ dendritic cells (DCs) via the DC-SIGN receptor. Fourth, we show that mucosal and systemic antibody responses against the HIV p24-SIgA complexes administered orally is strictly dependent on the expression of Dectin-1. Having deciphered the mechanisms leading to specific targeting of SIgA-based Ag complexes paves the way to the use of such a vehicle for mucosal vaccination against various infectious diseases.

Gas-filled microbubble-mediated delivery of antigen and the induction of immune responses.

The use of well characterized recombinant or purified protein antigens (Ag) for vaccination is of interest for safety reasons and in the case where inactivated pathogens are not available (cancer, allergy). However it requires the addition of adjuvants such as Ag carrier or immune stimulators to potentiate their immunogenicity. In this study, we demonstrated that gas-filled microbubbles (MB) can serve as an efficient Ag delivery system to promote phagocytosis of the model Ag ovalbumin (OVA) without the need of ultrasound application. Once internalized by DC, OVA was processed and presented to both CD4 and CD8 T cells in vitro; such observations were coupled with the capacity of MB to activate DC. In vivo administration of MB-associated OVA in naïve wild-type Balb/c mice resulted in the induction of OVA-specific antibody and T cell responses. Detailed characterization of the generated immune response demonstrated the production of both IgG1 and IgG2a serum antibodies, as well as the secretion of IFN-γ and IL-10 by splenocytes. Interestingly, similar results were obtained with human DC in regards of Ag delivery and cell activation. Therefore, the data presented here settle the proof of principle for the further evaluation of MB-based immunomodulation studies.