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Cancer vaccines have emerged as a potent strategy to improve cancer immunity, with or without the combination of checkpoint blockade. In our investigation, liposomal formulations containing synthetic long peptides and α-Galactosylceramide, along with a DC-SIGN-targeting ligand, Lewis Y (LeY), were studied for their anti-tumor potential. The formulated liposomes boosted with anti-CD40 adjuvant demonstrated robust invariant natural killer (iNKT), CD4+, and CD8+ T-cell activation in vivo. The incorporation of LeY facilitated the targeting of antigen-presenting cells expressing DC-SIGN in vitro and in vivo. Surprisingly, mice vaccinated with LeY-modified liposomes exhibited comparable tumor reduction and survival rates to those treated with untargeted counterparts despite a decrease in antigen-specific CD8+ T-cell responses. These results suggest that impaired induction of antigen-specific CD8+ T-cells via DC-SIGN targeting does not compromise anti-tumor potential, hinting at alternative immune activation routes beyond CD8+ T-cell activation. [ABSTRACT FROM AUTHOR]

Cancer vaccination aims to activate immunity towards cancer cells and can be achieved by delivery of cancer antigens together with immune stimulatory adjuvants to antigen presenting cells (APC). APC maturation and antigen processing is a subsequent prerequisite for T cell priming and anti-tumor immunity. In order to specifically target APC, nanoparticles, such as liposomes, can be used for the delivery of antigen and adjuvant. We have previously shown that liposomal inclusion of the ganglioside GM3, an endogenous ligand for CD169, led to robust uptake by CD169-expressing APC and resulted in strong immune responses when supplemented with a soluble adjuvant. To minimize the adverse effects related to a soluble adjuvant, immune stimulatory molecules can be incorporated in liposomes to achieve targeted delivery of both antigen and adjuvant. In this study, we incorporated TLR4 (MPLA) or TLR7/8 (3M-052) ligands in combination with inflammasome stimuli, 1-palmitoyl-2-glutaryl-sn-glycero-3-phosphocholine (PGPC) or muramyl dipeptide (MDP), into GM3 liposomes. Incorporation of TLR and inflammasome ligands did not interfere with the uptake of GM3 liposomes by CD169-expressing cells. GM3 liposomes containing a TLR ligand efficiently matured human and mouse dendritic cells in vitro and in vivo , while inclusion of PGPC or MDP had minor effects on maturation. Immunization with MPLA-containing GM3 liposomes containing an immunogenic synthetic long peptide stimulated CD4+ and CD8+ T cell responses, but additional incorporation of either PGPC or MDP did not translate into stronger immune responses. In conclusion, our study indicates that TLRL-containing GM3 liposomes are effective vectors to induce DC maturation and T cell priming and thus provide guidance for further selection of liposomal components to optimally stimulate anti-cancer immune responses. [ABSTRACT FROM AUTHOR]

At the end of December 2019, the COVID-19 pandemic began in Wuhan of China. COVID-19 affects different people with a wide spectrum of clinical manifestations, ranging from asymptomatic with recovery without hospitalization up to a severe acute respiratory syndrome (SARS). The innate and adaptive immunity appears responsible for the defense against the virus and recovery from the disease. The innate immune system, as the first line of defense, is essential for the detection of virus and subsequent activation of acquired immunity. The innate immune response is carried out by sentinel cells such as monocytes/macrophages and dendritic cells and by receptors known as pattern recognition receptors (PRR). These receptors can recognize various components of the virus, which lead to intracellular signaling and subsequently the synthesis of various cytokines. These cytokines then recruit other immune cells, activate adaptive immune responses, and inhibit viral spreading. The most common receptors include Toll-like receptors, C-type lectin receptors, and RIG-I like receptors. This review describes the current knowledge about the interplay between innate immune responses and SARS-CoV-2 with a focus on the innate immune cells and the role of their receptors in viral RNA recognition, as well as their mechanisms for recognizing SARS-CoV-2. [ABSTRACT FROM AUTHOR]

Monocytes are antigen-presenting cells (APCs) that play diverse roles in promoting or regulating inflammatory responses, but their role in T cell stimulation is not well defined. In inflammatory conditions, monocytes frequently show increased expression of CD169/Siglec-1, a type-I interferon (IFN-I)-regulated protein. However, little is known about the phenotype and function of these CD169+ monocytes. Here, we have investigated the phenotype of human CD169+ monocytes in different diseases, their capacity to activate CD8+ T cells, and the potential for a targeted-vaccination approach. Using spectral flow cytometry, we detected CD169 expression by CD14+ CD16- classical and CD14+ CD16+ intermediate monocytes and unbiased analysis showed that they were distinct from dendritic cells, including the recently described CD14-expressing DC3. CD169+ monocytes expressed higher levels of co-stimulatory and HLA molecules, suggesting an increased activation state. IFNα treatment highly upregulated CD169 expression on CD14+ monocytes and boosted their capacity to cross-present antigen to CD8+ T cells. Furthermore, we observed CD169+ monocytes in virally-infected patients, including in the blood and bronchoalveolar lavage fluid of COVID-19 patients, as well as in the blood of patients with different types of cancers. Finally, we evaluated two CD169-targeting nanovaccine platforms, antibody-based and liposome-based, and we showed that CD169+ monocytes efficiently presented tumor-associated peptides gp100 and WT1 to antigen-specific CD8+ T cells. In conclusion, our data indicate that CD169+ monocytes are activated monocytes with enhanced CD8+ T cell stimulatory capacity and that they emerge as an interesting target in nanovaccine strategies, because of their presence in health and different diseases. [ABSTRACT FROM AUTHOR]

Changes in the glycosylation profile of cancer cells have been strongly associated with cancer progression. To increase our insights into the role of glycosylation in human pancreatic ductal adenocarcinoma (PDAC), we performed a study on O -glycans and glycosphingolipid (GSL) glycans of the PDAC cell lines Pa-Tu-8988T (PaTu-T) and Pa-Tu-8988S (PaTu-S). These cell lines are derived from the same patient, but show an almost opposite phenotype, morphology and capacity to metastasize, and may thus provide an attractive model to study the role of glycosylation in progression of PDAC. Gene-array analysis revealed that 24% of the glycosylation-related genes showed a ≥ 1.5-fold difference in expression level between the two cell lines. Subsequent validation of the data by porous graphitized carbon nano-liquid chromatography coupled to a tandem ion trap mass spectrometry and flow cytometry established major differences in O -glycans and GSL-glycans between the cell lines, including lower levels of T and sialylated Tn (sTn) antigens, neoexpression of globosides (Gb3 and Gb4), and higher levels of gangliosides in the mesenchymal-like PaTu-T cells compared to the epithelial-like PaTu-S. In addition, PaTu-S cells demonstrated a significantly higher binding of the immune-lectins macrophage galactose-type lectin and galectin-4 compared to PaTu-T. In summary, our data provide a comprehensive and differential glycan profile of two PDAC cell lines with disparate phenotypes and metastatic behavior. This will allow approaches to modulate and monitor the glycosylation of these PDAC cell lines, which opens up avenues to study the biology and metastatic behavior of PDAC. [ABSTRACT FROM AUTHOR]

Glioblastoma is the most prevalent and aggressive primary brain tumour for which total tumour lysate-pulsed dendritic cell vaccination is currently under clinical evaluation. Glioblastoma extracellular vesicles (EVs) may represent an enriched cell-free source of tumour-associated (neo-) antigens to pulse dendritic cells (DCs) for the initiation of an anti-tumour immune response. Capture and uptake of EVs by DCs could occur in a receptor-mediated and presumably glycan-dependent way, yet the glycan composition of glioblastoma EVs is unknown. Here, we set out to characterize the glycocalyx composition of glioblastoma EVs by lectin-binding ELISA and comprehensive immunogold transmission electron microscopy (immuno-TEM). The surface glycan profile of human glioblastoma cell line-derived EVs (50–200 nm) was dominated by α-2,3- and α-2,6 linked sialic acid-capped complex N-glycans and bi-antennary N-glycans. Since sialic acids can trigger immune inhibitory sialic acid–binding Ig-like lectin (Siglec) receptors, we screened for Siglec ligands on the EVs. Glioblastoma EVs showed significant binding to Siglec-9, which is highly expressed on DCs. Surprisingly, however, glioblastoma EVs lack glycans that could bind Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin (DC-SIGN, CD209), a receptor that mediates uptake and induction of CD4+ and CD8+ T cell activation. Therefore, we explored whether modification of the EV glycan surface could reduce immune inhibitory Siglec binding, while enhancing EV internalization by DCs in a DC-SIGN dependent manner. Desialylation with a pan-sialic acid hydrolase led to reduction of sialic acid expression on EVs. Moreover, insertion of a high-affinity ligand (LewisY) for DC-SIGN resulted in a four-fold increase of uptake by monocyte-derived DCs. In conclusion, we show that the glycocalyx composition of EVs is a key factor of efficient DC targeting and that modification of the EV glycocalyx potentiates EVs as anti-cancer vaccine. [ABSTRACT FROM AUTHOR]

CD169+ macrophages are part of the innate immune system and capture pathogens that enter secondary lymphoid organs such as the spleen and the lymph nodes. Their strategic location in the marginal zone of the spleen and the subcapsular sinus in the lymph node enables them to capture antigens from the blood and the lymph respectively. Interestingly, these specific CD169+ macrophages do not destroy the antigens they obtain, but instead, transfer it to B cells and dendritic cells (DCs) which facilitates the induction of strong adaptive immune responses. This latter characteristic of the CD169+ macrophages can be exploited by specifically targeting tumor antigens to CD169+ macrophages for the induction of specific T cell immunity. In the current study we target protein and peptide antigen as antibody-antigen conjugates to CD169+ macrophages. We monitored the primary, memory, and recall T cell responses and evaluated the anti-tumor immune responses after immunization. In conclusion, both protein and peptide targeting to CD169 resulted in strong primary, memory, and recall T cell responses and protective immunity against melanoma, which indicates that both forms of antigen can be further explored as anti-cancer vaccination strategy. [ABSTRACT FROM AUTHOR]

The efficacy of vaccination studies aimed at targeting antigens to human DC-SIGN (hDC-SIGN) have been notoriously difficult to study in vivo, as eight dendritic cell-specific intercellular adhesion molecule-3 grabbing non-integrin (DC-SIGN) homologs have been described in mice. CD209a/SIGNR5 has been coined as the mouse DC-SIGN (mDCSIGN) ortholog, based on its expression and location in the genome. Nonetheless, which properties of hDC-SIGN are covered by mDC-SIGN is poorly investigated. One of the most important functions of DC-SIGN is the induction of adaptive immunity. As such, the aim of this study is to determine the capability of mDC-SIGN to induce adaptive immune responses. Here, we show that mDC-SIGN is expressed on GM-CSF cultured bone marrow-derived dendritic cells (BMDCs) and macrophages. However, mDC-SIGN is an internalizing receptor which, unlike hDC-SIGN, quickly resurfaces after internalization. Binding of OVA-coupled anti-mDC-SIGN antibody by BMDCs leads to quick internalization, processing, and presentation to antigen-specific CD8+ and CD4+ T cells, which can be boosted using the TLR4 ligand, monophosphoryl lipid A. In the homeostatic condition, mDC-SIGN is mostly expressed on myeloid cells in the skin and spleen. A subcutaneous injection of fluorescent anti-mDC-SIGN reveals specific targeting to mDC-SIGN+ skin dendritic cells (DCs) and monocyte-derived DCs in situ. A subcutaneous vaccination strategy containing OVA-coupled anti-mDC-SIGN antibody generated antigen-specific polyfunctional CD8+ T cell and CD4+ T cell responses and a strong isotype-switched OVA-specific antibody response in vivo. We conclude that mDC-SIGN shows partly overlapping similarities to hDC-SIGN and that targeting mDC-SIGN provides a valuable approach to investigate the immunological function of DC-SIGN in vivo. [ABSTRACT FROM AUTHOR]

Tuberculosis remains a major global health problem and efforts to develop a more effective vaccine have been unsuccessful so far. Targeting antigens (Ags) to dendritic cells (DCs) in vivo has emerged as a new promising vaccine strategy. In this approach, Ags are delivered directly to DCs via antibodies that bind to endocytic cell-surface receptors. Here, we explored DC-specific-ICAM3-grabbing-nonintegrin (DC-SIGN) targeting as a potential vaccine against tuberculosis. For this, we made use of the hSIGN mouse model that expresses human DC-SIGN under the control of the murine CD11c promoter. We show that in vitro and in vivo delivery of anti-DC-SIGN antibodies conjugated to Ag85B and peptide 25 of Ag85B in combination with anti-CD40, the fungal cell wall component zymosan, and the cholera toxin-derived fusion protein CTA1-DD induces strong Ag-specific CD4+ T-cell responses. Improved anti-mycobacterial immunity was accompanied by increased frequencies of Ag-specific IFN-γ+ IL-2+ TNF-α+ polyfunctional CD4+ T cells in vaccinated mice compared with controls. Taken together, in this study we provide the proof of concept that the human DC-SIGN receptor can be efficiently exploited for vaccine purposes to promote immunity against mycobacterial infections. [ABSTRACT FROM AUTHOR]

Ag delivery to specific APCs is an attractive approach in developing strategies for vaccination. CD169+ macrophages in the marginal zone of the spleen represent a suitable target for delivery of Ag because of their strategic location, which is optimal for the capture of blood-borne Ag and their close proximity to B cells and T cells in the white pulp. Here we show that Ag targeting to CD169+ macrophages in mice resulted in strong, isotype-switched, high-affinity Ab production and the preferential induction and long-term persistence of Ag-specific GC B cells and follicular Th cells. In agreement with these observations, CD169+ macrophages retained intact Ag, induced cognate activation of B cells, and increased expression of costimulatory molecules upon activation. In addition, macrophages were required for the production of cytokines that promote B-cell responses. Our results identify CD169+ macrophages as promoters of high-affinity humoral immune responses and emphasize the value of CD169 as target for Ag delivery to improve vaccine responses. [ABSTRACT FROM AUTHOR]

Toll-like receptor (TLR) ligands are attractive candidate adjuvants for therapeutic cancer vaccines, since TLR signaling stimulates and tunes both humoral and cellular immune responses induced by dendritic cells (DCs). Given that human skin contains a dense network of DCs, which are easily accessible via (intra-)dermal delivery of vaccines, skin is actively explored as an antitumor vaccination site. Here we used a human skin explant model to explore the potential of TLR ligands as adjuvants for DC activation in their complex microenvironment. We show that topical application of Aldara skin cream, 5% of which comprises the TLR7 agonist imiquimod, significantly enhanced DC migration as compared with that resulting from intradermal injection of the TLR7/8 ligand R848 or the soluble form of imiquimod. Moreover, Aldara-treated DCs showed highest levels of the costimulatory molecules CD86, CD83, CD40, and CD70. Topical Aldara induced the highest production of pro-inflammatory cytokines in skin biopsies. When combined with intradermal peptide vaccination, Aldara-stimulated DCs showed enhanced crosspresentation of the melanoma antigen MART-1, which resulted in increased priming and activation of MART-1-specific CD8+ T cells. These results point to advantageous effects of combining the topical application of Aldara with antitumor peptide vaccination. [ABSTRACT FROM AUTHOR]

It is becoming increasingly clear that glycosylation plays important role in intercellular communication within the immune system. Glycosylation-dependent interactions are crucial for the innate and adaptive immune system and regulate immune cell trafficking, synapse formation, activation, and survival. These functions take place by the cis or trans interaction of lectins with glycans. Classical immunological and biochemical methods have been used for the study of lectin function; however, the investigation of their counterparts, glycans, requires very specialized methodologies that have been extensively developed in the past decade within the Glycobiology scientific community. This Mini-Review intends to summarize the available technology for the study of glycan biosynthesis, its regulation and characterization for their application to the study of glycans in Immunology. [ABSTRACT FROM AUTHOR]

Vaccination is one of the oldest yet still most effective methods to prevent infectious diseases. However, eradication of intracellular pathogens and treatment of certain diseases like cancer requiring efficient cytotoxic immune responses remain a medical challenge. In mice, a successful approach to induce strong cytotoxic CD8+ T-cell (CTL) reactions is to target antigens to DCs using specific antibodies against surface receptors in combination with adjuvants. A major drawback for translating this strategy into one for the clinic is the lack of analogous targets in human DCs. DC- SIGN ( DC-specific- ICAM3-grabbing-nonintegrin/ CD209) is a C-type lectin receptor with potent endocytic capacity and a highly restricted expression on human immature DCs. Therefore, DC- SIGN represents an ideal candidate for DC targeting. Using transgenic mice that express human DC- SIGN under the control of the murine CD11c promoter (h SIGN mice), we explored the efficacy of anti- DC- SIGN antibodies to target antigens to DCs and induce protective immune responses in vivo. We show that anti- DC- SIGN antibodies conjugated to OVA induced strong and persistent antigen-specific CD4+ and CD8+ T-cell responses, which efficiently protected from infection with OVA-expressing Listeria monocytogenes. Thus, we propose DC targeting via DC- SIGN as a promising strategy for novel vaccination protocols against intracellular pathogens. [ABSTRACT FROM AUTHOR]

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The article focuses on a study related to the modification of an antigen with tumour-associated glycan glycan N-acetylgalactosamine (GalNAc). The study reveals that the modification targets antigen to the macrophage galactose-type lectin 2 (MGL2) on bone marrow derived dendritic cells (DCs) and splenic DCs. The study reflects on the expression of MGL on dendritic cells by analysing DCs cultured from bone marrow of C57BL/6 mice. Several graphs related to the same are also presented.

Wounds in the mouth heal faster and with less scarification and inflammation than those in the skin. Saliva is thought to be essential for the superior oral wound healing, but the involved mechanism is still unclear. We have previously discovered that a human-specific peptide, histatin, might be implicated in the wound-healing properties of saliva. Here we report that histatin enhances reepithelialization in a human full-skin wound model closely resembling normal skin. The peptide does not stimulate proliferation but induces cell spreading and migration, two key initiating steps in reepithelialization. Activation of cells by histafin requires a C-protein-coupled receptor that activates the ERK1/2 pathway. Using a stepwise-truncation method, we determined the minimal domain (SHREFPFYGDYGS) of the 38-mer-parent peptide that is required for activity. Strikingly, N- to C-terminal cyclization of histatin-I potentiates the molar activity ∼1000-fold, indicating that the recognition of histatin by its cognate receptor requires a specific spatial conformation of the peptide. Our results emphasize the importance of histatin in human saliva for tissue protection and recovery and establish the experimental basis for the development of synthetic histatins as novel skin wound-healing agents. [ABSTRACT FROM AUTHOR]

Wounds in the oral cavity heal much faster than skin lesions. Among other factors, saliva is generally assumed to be of relevance to this feature. Rodent saliva contains large mounts of growth factors such as epidermal growth factor (EGF) and nerve growth factor (NGF). In humans, however, the identity of the involved compounds has remained elusive, especially since EGF and NGF concentrations are ~100,000 times lower than those in rodent saliva. Using an in vitro model for wound closure, we examined the properties of human saliva and the fractions that were obtained from saliva by high-performance liquid chromotography (HPLC) separation. We identified histatin 1 (Hst1) and histatin 2 (Hst2) as major wound-closing factors in human saliva. In contrast, the D-enantiomer of Hst2 did not induce wound closure, indicating stereospecific activation. Furthermore, histatins were actively internalized by epithelial cells and specifically used the extracellular signal-regulated kinases &frac;12 (ERK&frac;12) pathway, thereby enhancing epithelial migration. This study demonstrates that members of the histatin family, which up to now were implicated in the antifungal weaponry of saliva, exert a novel function that likely is relevant for oral wound healing. [ABSTRACT FROM AUTHOR]

Despite promising progress in cancer vaccination, therapeutic effectiveness is often insufficient. Cancer vaccine effectiveness could be enhanced by targeting vaccine antigens to antigen-presenting cells, thereby increasing T-cell activation. CD169-expressing splenic macrophages efficiently capture particulate antigens from the blood and transfer these antigens to dendritic cells for the activation of CD8+ T cells. In this study, we incorporated a physiological ligand for CD169, the ganglioside GM3, into liposomes to enhance liposome uptake by CD169+ macrophages. We assessed how variation in the amount of GM3, surface-attached PEG and liposomal size affected the binding to, and uptake by, CD169+ macrophages in vitro and in vivo. As a proof of concept, we prepared GM3-targeted liposomes containing a long synthetic ovalbumin peptide and tested the capacity of these liposomes to induce CD8+ and CD4+ T-cell responses compared to control liposomes or soluble peptide. The data indicate that the delivery of liposomes to splenic CD169+ macrophages can be optimized by the selection of liposomal constituents and liposomal size. Moreover, optimized GM3-mediated liposomal targeting to CD169+ macrophages induces potent immune responses and therefore presents as an interesting delivery strategy for cancer vaccination. [ABSTRACT FROM AUTHOR]

In vivo dendritic-cell targeting constitutes a promising strategy for anticancer vaccination. Here, we discuss the usage of multivalent DC-SIGN-targeting glycan platforms that allow for the efficient routing of antigens to the endo-lysosomal pathway as well as to a yet uncharacterized cross-presentation mechanism inducing CD4+ and CD8+ T-cell responses. [ABSTRACT FROM AUTHOR]

Background: Multiple sclerosis (MS) involves a misdirected immune attack against myelin in the brain and spinal cord, leading to profound neuroinflammation and neurodegeneration. While the mechanisms of disease pathogenesis have been widely studied, the suppression mechanisms that lead to the resolution of the autoimmune response are still poorly understood. Here, we investigated the role of the C-type lectin receptor macrophage galactose-type lectin (MGL), usually expressed on tolerogenic antigen-presenting cells (APCs), as a negative regulator of autoimmune-driven neuroinflammation.Methods: We used in silico, immunohistochemical, immunofluorescence, quantitative real-time polymerase chain reaction (qRT-PCR) and flow cytometry analysis to explore the expression and functionality of MGL in human macrophages and microglia, as well as in MS post-mortem tissue. In vitro, we studied the capacity of MGL to mediate apoptosis of experimental autoimmune encephalomyelitis (EAE)-derived T cells and mouse CD4+ T cells. Finally, we evaluated in vivo and ex vivo the immunomodulatory potential of MGL in EAE.Results: MGL plays a critical role in the resolution phase of EAE as MGL1-deficient (Clec10a-/-) mice showed a similar day of onset but experienced a higher clinical score to that of WT littermates. We demonstrate that the mouse ortholog MGL1 induces apoptosis of autoreactive T cells and diminishes the expression of pro-inflammatory cytokines and inflammatory autoantibodies. Moreover, we show that MGL1 but not MGL2 induces apoptosis of activated mouse CD4+ T cells in vitro. In human settings, we show that MGL expression is increased in active MS lesions and on alternatively activated microglia and macrophages which, in turn, induces the secretion of the immunoregulatory cytokine IL-10, underscoring the clinical relevance of this lectin.Conclusions: Our results show a new role of MGL-expressing APCs as an anti-inflammatory mechanism in autoimmune neuroinflammation by dampening pathogenic T and B cell responses, uncovering a novel clue for neuroprotective therapeutic strategies with relevance for in MS clinical applications. [ABSTRACT FROM AUTHOR]

The lack of tumor-reactive T cells is one reason why immune checkpoint inhibitor therapies still fail in a significant proportion of melanoma patients. A vaccination that induces melanoma-specific T cells could potentially enhance the efficacy of immune checkpoint inhibitors. Here, we describe a vaccination strategy in which melanoma antigens are targeted to mouse and human CD169 and thereby induce strong melanoma antigen-specific T cell responses. CD169 is a sialic acid receptor expressed on a subset of mouse splenic macrophages that captures antigen from the blood and transfers it to dendritic cells (DCs). In human and mouse spleen, we detected CD169+ cells at an equivalent location using immunofluorescence microscopy. Immunization with melanoma antigens conjugated to antibodies (Abs) specific for mouse CD169 efficiently induced gp100 and Trp2-specific T cell responses in mice. In HLA-A2.1 transgenic mice targeting of the human MART-1 peptide to CD169 induced strong MART-1-specific HLA-A2.1-restricted T cell responses. Human gp100 peptide conjugated to Abs specific for human CD169 bound to CD169-expressing monocyte-derived DCs (MoDCs) and resulted in activation of gp100-specific T cells. Together, these data indicate that Ab-mediated antigen targeting to CD169 is a potential strategy for the induction of melanoma-specific T cell responses in mice and in humans. [ABSTRACT FROM AUTHOR]

Bovine lactoferrin harbors 2 antimicrobial sequences (LFcin and LFampin), situated in close proximity in the N1-domain. To mimic their semi parallel configuration we have synthesized a chimeric peptide (LFchimera) in which these sequences are linked in a head-to-head fashion to the α- and ζ-amino group, respectively, of a single lysine. In line with previously described bactericidal effects, this peptide was also a stronger candidacidal agent than the antimicrobial peptides LFcin17-30 and LFampin265-284, or a combination of these 2. Conditions that strongly reduced the candidacidal activities of LFcin17-30 and LFampin265-284, such as high ionic strength and energy depletion, had little influence on the activity of LFchimera. Freeze-fracture electron microscopy showed that LFchimera severely affected the membrane morphology, resulting in disintegration of the membrane bilayer and in an efflux of small and high molecular weight molecules such as ATP and proteins. The differential effects displayed by the chimeric peptide and a mixture of its constituent peptides clearly demonstrate the synergistic effect of linking these peptides in a fashion that allows a similar spatial arrangement as in the parent protein, suggesting that in bovine lactoferrrin the corresponding fragments act in concert in its candidacidal activity. Key words: Candida albicans, yeast, pseudohyphae, lactoferricin, lactoferrampin, LFchimera. La lactoferrine bovine comprend deux sequences antimicrobiennes (LFcine et LFampine) situees a proximite du domaine N1. Afin de mimer leur configuration semi parallele, nous avons synthetise un peptide chimere (LF chimere) dans lequel ces sequences sont respectivement liees tete contre tete aux groupes amino a et e d'une lysine unique. Conformement aux effets bactericides deja decrits, ce peptide etait aussi efficace envers Candida que les deux peptides antimicrobiens LFcine17-30 et LFampine265-284 ou la combinaison de ceux-ci. Les conditions qui reduisent fortement l'activite des peptides LFcine17-30 et LFampine265-284 contre le Candida, comme une force ionique elevee ou une carence d'energie, avaient peu d' effets sur l' activite de la LF chimere. La microscopie electronique par cryofracture a montre que la LF chimere affectait serieusement la morphologie de la membrane, ce qui resultait en une desintegration de la bicouche membranaire et en un efflux de molecules de poids moleculaire faible ou eleve comme l'ATP et les proteines. Les effets differentiels exerces par le peptide chimere et le melange des peptides qui le constitue demontrent clairement l' effet synergique de la liaison de ces peptides d'une facon qui permette un arrangement spatial similaire a celui de la proteine d'origine, suggerant que dans la lactoferrine bovine, les fragments correspondants agissent de concert dans son activite contre le Candida. Mots-cles: Candida albicans, levure, pseudo-hyphe, lactoferrine, lactoferrampine, LF chimere., Introduction The opportunistic pathogen Candida albicans is one of the most frequently isolated pathogens that invade mucosal surfaces in humans (Kim and Sudbery 2011). It lives as a commensal organism [...]