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DC-SIGN is an antigen uptake receptor expressed on dendritic cells (DCs) with specificity for glycans present on a broad variety of pathogens and is capable of directing its cargo to MHC-I and MHC-II pathways for the induction of CD8+ and CD4+ T cell responses, respectively. Therefore, DC-SIGN is a very promising target for the delivery of antigen for anti-cancer vaccination. Although the endocytic route leading to MHC-II presentation is characterized to a large extent, the mechanisms controlling DC-SIGN targeted cross-presentation of exogenous peptides on MHC-I, are not completely resolved yet. In this paper, we used imaging flow cytometry and antigen-specific CD8+ T cells to investigate the intracellular fate of DC-SIGN and its cargo in human DCs. Our data demonstrates that immature DCs and toll-like receptor 4 (TLR4) stimulated DCs had similar internalization capacity and were both able to cross-present antigen targeted via DC-SIGN. Interestingly, simultaneous triggering of TLR4 and DC-SIGN on DCs resulted in the translocation of cargo to the cytosol, leading to proteasome-dependent processing and increased CD8+ T cell activation. Understanding the dynamics of DC-SIGN-mediated uptake and processing is essential for the design of optimal DC-SIGN-targeting vaccination strategies aimed at enhancing CD8+ T cell responses. [ABSTRACT FROM AUTHOR]

Highlights: [•] DC-SIGN is a multifunctional C-type lectin receptor on antigen-presenting cells. [•] Its functions include adhesion, migration, signaling, and antigen uptake/presentation. [•] All evidence supporting DC-SIGN functions is based on in vitro assays. [•] The absence of a functional DC-SIGN ortholog hampers the study of its physiological role. [Copyright &y& Elsevier]

Altered glycosylation in epithelial cancers may play an important role in tumour progression, as it may affect tumour cell migration and antigen presentation by antigen presenting cells. We specifically characterise the glycosylation patterns of two tumour antigens that are highly expressed in cancer tissue and often detected in their secreted form in serum: the epithelial mucin MUC1 and carcinoembryonic antigen (CEA, also called CEACAM5). We analysed 48 colorectal cancer patients, comparing normal colon and tumour epithelium within each patient. Lectin binding was studied by a standardised CEA/MUC1 capture ELISA, using several plant lectins, and the human C-type lectins MGL and DC-SIGN, and Galectin-3. Peanut agglutinin (PNA) bound to MUC1 from tumour tissue in particular, suggests increased expression of the Thomsen-Friedenreich antigen (TF-antigen) (Core 1, Galβ1-3GalNAc-Ser/Thr). Only small amounts of Tn-antigen (GalNAcα-Ser/Thr) expression was observed, but the human C-type lectin MGL showed increased binding to tumour-associated MUC1. Furthermore, sialylation was greatly enhanced. In sharp contrast, tumour-associated CEA (CEACAM5) contained high levels of the blood-group related carbohydrates, Lewis X and Lewis Y. This correlated strongly with the interaction of the human C-type lectin DC-SIGN to tumour-associated CEA, suggesting that CEA can be recognized and taken up by antigen presenting cells. In addition, increased mannose expression was observed and branched N-glycans were prominent, and this correlated well with human Galectin-3 binding. These data demonstrate that individual tumour antigens contain distinct glycan structures associated with cancer and, since glycans affect cellular interactions with its microenvironment, this may have consequences for progression of the disease. [ABSTRACT FROM AUTHOR]

Leptospirosis is a global zoonotic disease, caused by pathogenic Leptospira species including Leptospira interrogans, that causes public health and livestock problems. Pathogenesis, immune response and cellular receptors for Leptospira are not well understood. Interaction of dendritic cells (DCs) with L. interrogans serovar Autumnalis L-643 and BL-6 isolated from leptospirosis patients, and both virulent and avirulent serovar Pyrogenes 2317 strains isolated from animal were investigated. Carbohydrate analysis using lectins showed that all of these leptospires contained high mannose components as a common backbone and DC-SIGN was involved in leptospires' attachment. Interaction of the L. interrogans strains with DCs induced maturation, but had different effects on IL-10, IL-12p70 and tumor necrosis factor (TNF)-α production. Both virulent and avirulent Pyrogenes 2317 and Autumnalis BL-6 but not L-643 strains induced IL-12p70 and TNF-α production, but minimal IL-10 secretion. These data demonstrated that L. interrogans binds DC-SIGN and induces DCs maturation and cytokine production, which should provide new insights into cellular immune processes during leptospirosis. [ABSTRACT FROM AUTHOR]

Abstract: Early during infection neutrophils are the most important immune cells that are involved in killing of pathogenic bacteria and regulation of innate immune responses at the site of infection. It has become clear that neutrophils also modulate adaptive immunity through interactions with dendritic cells (DCs) that are pivotal in the induction of T cell responses. Upon activation, neutrophils release TNF-α and induce maturation of DCs that enables these antigen-presenting cells to stimulate T cell proliferation and to induce T helper 1 polarization. DC maturation by neutrophils also requires cellular interactions that are mediated by binding of the DC-specific receptor DC-SIGN to Mac-1 on the neutrophil. Here, we demonstrate that also CEACAM1 is an important ligand for DC-SIGN on neutrophils. Binding of DC-SIGN to both CEACAM1 and Mac-1 is required to establish cellular interactions with neutrophils. DC-SIGN is a C-type lectin that has specificity for Lewisx, and we show that DC-SIGN mediates binding to CEACAM1 through Lewisx moieties that are specifically expressed on CEACAM1 derived from neutrophils. This indicates that glycosylation-driven binding of both Mac-1 and CEACAM1 to DC-SIGN is essential for interactions of neutrophils with DCs and enables neutrophils to modulate T cell responses through interactions with DCs. [Copyright &y& Elsevier]

Dendritic cells (DCs) are highly efficient antigen-presenting cells (APCs) that collect antigen in body tissues and transport them to draining lymph nodes. Antigenic peptides are loaded onto major histocompatibility complex (MHC) molecules for presentation to naïve T cells, resulting in the induction of cellular and humoral immune responses. DCs take up antigen through phagocytosis, pinocytosis, and endocytosis via different groups of receptor families, such as Fc receptors for antigen-antibody complexes, C-type lectin receptors (CLRs) for glycoproteins, and pattern recognition receptors, such as Toll-like receptors (TLRs), for microbial antigens. Uptake of antigen by CLRs leads to presentation of antigens on MHC class I and II molecules. DCs are well equipped to distinguish between self- and nonself antigens by the variable expression of cell-surface receptors such as CLRs and TLRs. In the steady state, DCs are not immunologically quiescent but use their antigen handling capacities to maintain peripheral tolerance. DCs are continuously sampling and presenting self- and harmless environmental proteins to silence immune activation. Uptake of self-components in the intestine and airways are good examples of sites where continuous presentation of self- and foreign antigens occurs without immune activation. In contrast, efficient antigen-specific immune activation occurs upon encounter of DCs with nonself-pathogens. Recognition of pathogens by DCs triggers specific receptors such as TLRs that result in DC maturation and subsequently immune activation. Here we discuss the concept that cross talk between TLRs and CLRs, differentially expressed by subsets of DCs, accounts for the different pathways to peripheral tolerance, such as deletion and suppression, and immune activation. [ABSTRACT FROM AUTHOR]

Dendritic cells (DC) are vital in the defense against pathogens. To sense pathogens DC express pathogen recognition receptors such as toll-like receptors (TLR) and C-type lectins that recognize different fragments of pathogens, and subsequently activate or present pathogen fragments to T cells. It is now becoming evident that some pathogens subvert DC functions to escape immune surveillance. HIV-1 targets the DC-specific C-type lectin DC-SIGN to hijack DC for viral dissemination. HIV-1 binding to DC-SIGN protects HIV-1 from antigen processing and facilitates its transport to lymphoid tissues, where DC-SIGN promotes HIV-1 infection of T cells. Recent studies demonstrate that DC-SIGN is a more universal pathogen receptor that also recognizes Ebola, cytomegalovirus and mycobacteria. Mycobacterium tuberculosis targets DC-SIGN by a mechanism that is distinct from that of HIV-1, leading to inhibition of the immunostimulatory function of DC and pathogen survival. Thus, a better understanding of DC-SIGN-pathogen interactions and their effects on DC function is necessary to combat infections. [ABSTRACT FROM AUTHOR]

Abstract: Dendritic cells are the most efficient professional antigen-presenting cells in pathogen recognition and play a pivotal role in the control of the immune response. Pathogen recognition is ensured by the expression of a vast variety of pattern-recognition receptors. Amongst them are C-type lectins, a large family of receptors characterized by a domain that – in many cases – mediates calcium-dependent glycan binding. C-type lectins facilitate antigen uptake for efficient processing and presentation and, in some cases, also trigger signaling to modulate T cell responses. These properties make C-type lectin receptors ideal candidates for the targeting of antigens to dendritic cells for vaccination. DC-SIGN is a paradigmatic example of C-type lectin receptors on dendritic cells that facilitate vaccination strategies. DC-SIGN is highly expressed on immature conventional dendritic cells, particularly at the mucosa and the dermis, where DCs first encounter pathogens, but also can easily be accessed for vaccination. Upon ligand binding, DC-SIGN rapidly internalizes and directs its cargo into the endo-lysosomal pathway, which results in MHC-II presentation. But antigens targeted to DC-SIGN are also presented efficiently to CD8+ T cells, suggesting there is an additional endocytic route that leads to cross-presentation. Simultaneous triggering of DC-SIGN and TLRs results in the modulation of cytokine responses and facilitates cross-presentation to enhance CD4+ and CD8+ T cell responses. Because the glycan specificity of DC-SIGN has been characterized in detail, glycans can be used for the targeting of antigens to DCs in a DC-SIGN-dependent manner. Glycans represent a great advantage over monoclonal antibodies, they diminish the risk of side effects, are very small, and their production can rely entirely in organic chemistry approaches. Here, we discuss the capacity of glycan-based vaccines to enhance antigen-specific CD4+ and CD8+ T cell responses in human skin and mouse model systems. [Copyright &y& Elsevier]

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]

Summary: Dendritic cells (DCs) are essential in regulating adaptive immunity. DC-SIGN (DC-specific ICAM-grabbing nonintegrin) is a C-type lectin receptor that is expressed mainly by DCs. Accumulating evidence supports that certain pathogens target DC-SIGN to escape host immunity. To investigate a possible role of DC-SIGN in Burkholderia pseudomallei infection, we initially screened its DC-SIGN binding activity by an ELISA method utilizing a DC-SIGN-Fc chimeric protein and found that all of the B. pseudomallei strains tested failed to bind DC-SIGN. However, one strain, the LPS mutant SRM117, which lacks the type II O-polysaccharide expression, actually bound DC-SIGN, in contrast to its wild-type counterpart 1026b (P < 0.001). We also found that, although the LPS mutant could readily activate monocyte-derived human DCs, it induced lower levels of IL-12p70 and IL-10 production than its wild-type counterpart (P < 0.01). By contrast, the wild-type and the LPS mutants were indistinguishable from one another in terms of TH1/TH2 differentiation. Altogether, these data suggest that, unlike other certain host pathogen interactions, activation of DCs by B. pseudomallei is not dependent on DC-SIGN. We also found evidence that the LPS mutant that binds DC-SIGN has a suppressive effect on DC cytokine production. [Copyright &y& Elsevier]

Dendritic cell (DC)–targeting vaccines show great promise in increasing antitumor immunity. Glycan-engineered vaccines facilitate both DC targeting and increased uptake by DCs for processing and presentation to CD4+ and CD8+ T cells to induce tumor-specific T-cell responses. However, the complexity of various DC subsets in skin tissues, expressing different glycan-binding receptors that can mediate vaccine uptake or drainage of vaccines via lymphatics directly to the lymph node–resident DCs, complicates the success of vaccines. Moreover, the influx of inflammatory immune cells to the site of vaccination, such as monocytes that differentiate to DCs and coexpress glycan-binding receptors, may contribute to the strength of DC-targeting glycovaccines for future clinical use. [ABSTRACT FROM AUTHOR]

Abstract: Cancer immunotherapy requires potent tumor-specific CD8+ and CD4+ T-cell responses, initiated by dendritic cells (DCs). Tumor antigens can be specifically targeted to DCs in vivo by exploiting their expression of C-type lectin receptors (CLR), which bind carbohydrate structures on antigens, resulting in internalization and antigen presentation to T-cells. We explored the potential of glycan-modified liposomes to target antigens to DCs to boost murine and human T-cell responses. Since DC-SIGN is a CLR expressed on DCs, liposomes were modified with DC-SIGN-binding glycans Lewis (Le)B or LeX. Glycan modification of liposomes resulted in increased binding and internalization by BMDCs expressing human DC-SIGN. In the presence of LPS, this led to 100-fold more efficient presentation of the encapsulated antigens to CD4+ and CD8+ T-cells compared to unmodified liposomes or soluble antigen. Similarly, incubation of human moDC with melanoma antigen MART-1-encapsulated liposomes coated with LeX in the presence of LPS led to enhanced antigen-presentation to MART-1-specific CD8+ T-cell clones. Moreover, this formulation drove primary CD8+ T-cells to differentiate into high numbers of tetramer-specific, IFN-γ-producing effector T-cells. Together, our data demonstrate the potency of a glycoliposome-based vaccine targeting DC-SIGN for CD4+ and CD8+ effector T-cell activation. This approach may offer improved options for treatment of cancer patients and opens the way to in situ DC-targeted vaccination. [Copyright &y& Elsevier]

Abstract: Dendritic cells (DCs) are key antigen presenting cells that have the unique ability to present antigens on MHC molecules, which can lead to either priming or suppression of T cell mediated immune responses. C-type lectin receptors expressed by DCs are involved in antigen uptake and presentation through recognition of carbohydrate structures on antigens. Here we have explored the feasibility of modification of liposomes with glycans for targeting purposes to boost immune responses. The potential of targeting glycoliposomal constructs to the C-type lectin DC-SIGN on DCs was studied using either PEGylated or non-PEGylated liposomes. Our data demonstrate that formulation of the glycoliposomes as PEGylated negatively affected their potential to target to DCs. [Copyright &y& Elsevier]

Abstract: Dendritic cells have gained much interest in the field of anti-cancer vaccine development because of their central function in immune regulation. One of the receptors that facilitate DC-specific targeting of antigens is the DC-specific C-type lectin DC-SIGN. Although DC-SIGN is specifically expressed on human DCs, its murine homologue is not present on any murine DC subsets, which makes in vivo evaluation of potential DC-SIGN targeting vaccines very difficult. Here we describe the use of DC-SIGN transgenic mice, as a good model system to evaluate DC-SIGN targeting vaccines. We demonstrate that glycan modification of OVA with DC-SIGN targeting glycans, targets antigen specifically to bone marrow (BM)** derived DCs and splenic DCs. Glycan modification of OVA with Lewis X or Lewis B oligosaccharides, that target DC-SIGN transgenic DCs, resulted in efficient 10-fold induction of OT-II compared to unmodified OVA. Interestingly, glycan modified OVA proteins were significantly cross-presented to OT-I T cells by wild type DC, 10-fold more than native OVA, and the expression of DC-SIGN further enhanced this cross-presentation. Targeting of glycosylated OVA was neither accompanied with any DC maturation, nor the production of inflammatory or anti-inflammatory cytokines. Thus, we conclude that glycan modification of antigens and targeting to DC-SIGN enhance both CD4 and CD8 T cell responses. Furthermore, our data demonstrate that DC-SIGN transgenic mice are valuable tool for optimisation and efficiency testing of DC vaccination strategies that are designed to target in particular the human DC-SIGN receptor. [Copyright &y& Elsevier]

Abstract: Immunization using human papilloma virus (HPV)-L1 virus-like particles (VLPs) induces a robust and effective immune response, which has recently resulted in the implementation of the HPV-L1 VLP vaccination in health programs. However, during infection, HPV can escape immune surveillance leading to latency and disease. Dendritic cells (DCs) induce effective immune responses after vaccination, but might also induce immune modulation during infection. The interaction of HPV-L1 VLPs with mucosal DCs determines the immune response. However, little is known about the receptors on mucosal DC subsets involved in HPV-L1 VLP binding. Therefore, we set out to investigate the interaction of HPV-L1 VLPs with the different mucosal DC subsets; the subepithelial DCs and Langerhans cells (LCs). We observed strong binding of HPV-L1 VLPs to both DCs and LCs. We did not observe an involvement for C-type lectins such as dendritic cell-specific ICAM-3 grabbing non-integrin (DC-SIGN) and langerin. The HPV-L1 VLP binding to DCs was mediated through heparan sulfates, since it was abrogated by heparinase-II treatment. The heparan sulfate proteoglycan syndecan-3 binds VLPs and is expressed on both DCs and LCs. Binding of VLPs to DCs, but not to LCs, strongly correlated with the levels of heparan sulfates and syndecan-3, suggesting that syndecan-3 is the main receptor for HPV-L1 VLPs on DCs. VLP interaction with DCs resulted in the up-regulation of co-stimulatory molecules and the production of the cytokines IL-6, IL-8, IL-10 and IL-12p40. Our results support an important role for syndecan-3 as a HPV receptor on DCs, which could be important for both vaccine development and understanding HPV pathogenesis. [Copyright &y& Elsevier]

Abstract: The thymus is the principal organ for development of T-cells. Thymocyte precursors from bone marrow-derived progenitor cells enter the thymus where they differentiate involving several differentiation stages into mature T-cells that can leave the thymus to the periphery. Migration of thymocytes through the thymus and their development are tightly controlled by the interaction of thymocytes with components of the thymic microenvironments. Several studies have demonstrated the pivotal importance of glycosylation in cell–cell interactions or interactions of cells with extracellular matrix components (ECM) in various physiologic processes in the body. The knowledge on glycosylation of thymic microenvironments is however limited although the presence of C-type lectin receptors such as DC-SIGN, mannose receptor and DEC-205, which are specifically recognizing distinct carbohydrate moieties emphasize the importance of glycosylation in the thymus. In order to outline the distribution of glycoconjugates in microenvironments of the human thymus we studied the glycosylation of the human thymic microarchitecture by using plant lectins in situ. Eleven plant lectin–biotin conjugates with distinct specificity were used and analyzed by fluorescence microscopy. Mannose glycoconjugates, specifically detected by the lectins GNA and NPA, were abundant in the cortex but not in the medulla. Dendritic cells present in the thymic cortex were specifically co-stained with the galactose-specific lectins DSA and PNA. Several lectins bound to the thymic vasculature. The α2-fucose-specific lectin UEA stained thymic blood vessels in the interlobular space and medulla and capillaries in the cortex. In addition to UEA, thymic blood vessels and capillaries also reacted with the lectins DSA, PNA and the α-GalNac-specific lectin HPA. In contrast, lymph vessels present in the interlobular space do not interact with UEA, DSA and PNA, but only with HPA, revealing a disparate glycosylation pattern of lymph and blood vessels that may be important to determine the direction of thymocytes entering or leaving the thymus. In conclusion, the restricted expression patterns of carbohydrates defined microenvironments in the human thymus highlight the importance of glycosylation in various steps of T-cell development. [Copyright &y& Elsevier]

Abstract: In schistosomiasis, a parasitic disease caused by helminths, the parasite eggs induce a T helper 2 cell (TH2) response in the host. Here, the specific role of human monocyte-derived dendritic cells (DCs) in initiation and polarization of the egg-specific T cell responses was examined. We demonstrate that immature DCs (iDCs) pulsed with schistosome soluble egg antigens (SEA) do not show an increase in expression of co-stimulatory molecules or cytokines, indicating that no conventional maturation was induced. The ability of SEA to affect the Toll-like receptor (TLR) induced maturation of iDCs was examined by copulsing the DCs with SEA and TLR-ligands. SEA suppressed both the maturation of iDCs induced by poly-I:C and LPS, as indicated by a decrease in co-stimulatory molecule expression and production of IL-12, IL-6 and TNF-α. In addition, SEA suppressed TH1 responses induced by the poly-I:C-pulsed DCs, and skewed the LPS-induced mixed response towards a TH2 response. Immature DCs rapidly internalized SEA through the C-type lectins DC-SIGN, MGL and the mannose receptor and the antigens were targeted to MHC class II-positive lysosomal compartments. The internalization of SEA by multiple C-type lectins may be important to regulate the response of the iDCs to TLR-induced signals. [Copyright &y& Elsevier]

Abstract: Protein–carbohydrate interactions play crucial roles in numerous biological processes. To study these interactions, we developed a simple and fast procedure for the biotinylation of carbohydrates based on reductive amination. The method allows complete and stable biotinylation of small quantities of oligosaccharides and includes a rapid and simple procedure to remove excess labeling reagent. After biotinylation, the structural and biological integrity of the glycans was intact as determined by HPLC, mass spectrometry, and a plant lectin assay. By using the human C-type lectin DC–SIGN (dendritic cell-specific ICAM-3-grabbing nonintegrin), we demonstrate that the biotinylated glycans can be used in a glycan array to determine binding specificities of lectins. Moreover, we show that fluorescent beads coated with selected biotinylated glycans bind to DC–SIGN-expressing dendritic cells in vitro. Finally, by using biotinylated high-mannose N-glycans, we could visualize DC–SIGN-expressing cells in lymph node tissue. The availability of easy biotinylation methods for oligosaccharides such as those described here greatly facilitates the functional analysis of lectins. In addition, the biotinylated glycans will be great tools for investigating functional lectin receptors in situ. [Copyright &y& Elsevier]

Abstract: The Gram-positive bacterium Streptococcus pneumoniae is the leading causative pathogen in community-acquired pneumonia. The ever-increasing frequency of antibiotic-resistant S. pneumoniae strains severely hampers effective treatments. Thus, a better understanding of the mechanisms involved in the pathogenesis of pneumococcal disease is needed; in particular, of the initial interactions that take place between the host and the bacterium. Recognition of pathogens by dendritic cells is one of the most crucial steps in the induction of an immune response. For efficient pathogen recognition, dendritic cells express various kinds of receptors, including the DC-specific C-type lectin DC-SIGN. Pathogens such as Mycobacterium tuberculosis and HIV target DC-SIGN to escape immunity. Here the in vitro binding of DC-SIGN with S. pneumoniae was investigated. DC-SIGN specifically interacts with S. pneumoniae serotype 3 and 14 in contrast to other serotypes such as 19F. While the data described here suggest that DC-SIGN interacts with S. pneumoniae serotype 14 through a ligand expressed by the capsular polysaccharide, the binding to S. pneumoniae serotype 3 appears to depend on an as yet unidentified ligand. Despite the binding capacity of the capsular polysaccharide of S. pneumoniae 14 to DC-SIGN, no immunomodulatory effects on the dendritic cells were observed. The immunological consequences of the serotype-specific capacity to interact with DC-SIGN should be further explored and might result in new insights in the development of new and more potent vaccines. [Copyright &y& Elsevier]

Abstract: The unsurpassed capacity of dendritic cells (DC) to prime naive T cells is thought to depend on the formation of an immunological synapse. DC-SIGN, a C-type lectin exclusively expressed at the cell surface of DC, functions as an adhesion receptor facilitating T cell binding and priming through recognition of glycosylated ICAM-3 on naive T cells. Yet, DC-SIGN also mediates binding to pathogens such as HIV by recognizing glycosylated gp120. The scope of the present study was to investigate whether DC-SIGN upon recognition of its cellular ligand and pathogenic ligand affects DC synapse formation and activation/mobilization of other adhesion receptors such as LFA-1 to the cell contact site. Using a DC-SIGN deletion mutant, we show that DC-SIGN is a constitutively active receptor that mediates ligand binding independent of signaling through the cytoplasmic domain. Surprisingly, initial binding of gp120 to DC-SIGN did not result in increased adhesion levels of LFA-1 to its ligand ICAM-1 in both immature DC and Raji-DC-SIGN cells. However, ligand binding to DC-SIGN induced recruitment of LFA-1 to the adhesion site. Moreover, we could demonstrate that activation of LFA-1 results in DC-SIGN-LFA-1 co-clustering in the cell membrane. This triggers binding of ligands to LFA-1 that are shared with DC-SIGN, such as ICAM-3, but not of ligands that are not shared with DC-SIGN, such as ICAM-1. Thus, we propose that upon ligand binding DC-SIGN recruits LFA-1 to the contact site, resulting in the formation of DC-SIGN-LFA-1 co-clusters, in which the initial DC-SIGN-mediated interactions with ligand are transient and eventually shift to more stable LFA-1-dependent interactions. [Copyright &y& Elsevier]

The interaction of DC-SIGN with gp120 provides an attractive target for intervention of HIV-1 transmission. Here, we have investigated the potency of gp120 antibodies to inhibit the DC-SIGN–gp120 interaction. We demonstrate that although the V3 loop is not essential for DC-SIGN binding, antibodies against the V3 loop partially inhibit DC-SIGN binding, suggesting that these antibodies sterically hinder DC-SIGN binding to gp120. Polyclonal antibodies raised against non-glycosylated gp120 inhibited both low and high avidity DC-SIGN–gp120 interactions in contrast to polyclonal antibodies raised against glycosylated gp120. Thus, glycans present on gp120 may prevent the generation of antibodies that block the DC-SIGN–gp120 interactions. Moreover, the polyclonal antibodies against non-glycosylated gp120 efficiently inhibited HIV-1 capture by both DC-SIGN transfectants and immature dendritic cells. Therefore, non-glycosylated gp120 may be an attractive immunogen to elicit gp120 antibodies that block the binding to DC-SIGN. Furthermore, we demonstrate that DC-SIGN binding to gp120 enhanced CD4 binding, suggesting that DC-SIGN induces conformational changes in gp120, which may provide new targets for neutralizing antibodies. [Copyright &y& Elsevier]

Injection of antigenic peptides has been widely used as a vaccine strategy to boost T cell immunity. However, the poor immunogenicity of single peptides can potentially be strengthened through modification of the tertiary structure and the selection of the accompanying adjuvant. Here, we generated antigenic peptides into non-linear trimers by solid phase peptide synthesis, thereby enhancing antigen presentation by dendritic cells to CD8+ T cells in vitro and in vivo. CD8+ T cells from mice vaccinated with trimers showed an KLRG1+ effector phenotype and were able to recognize and kill antigen-expressing tumor cells ex vivo. Importantly, trimers outperformed synthetic long peptide in terms of T cell response even when equal number of epitopes were used for immunization. To improve the synthesis of trimers containing difficult peptide sequences, we developed a novel small molecule that functions as conjugation platform for synthetic long peptides. This platform , termed Antigen MAtriX (AMAX) improved yield, purity and solubility of trimers over conventional solid phase synthesis strategies. AMAX outperformed synthetic long peptides in terms of both CD8+ and CD4+ T cell responses and allowed functionalization with DC-SIGN-binding carbohydrates for in vivo dendritic cell targeting strategies, boosting T cell responses even further. Moreover, we show that agonistic CD40 antibody combined with MF59 (AddaVax) emulsion synergistically improves the antigen-specific T cell response of the AMAX in vivo. Also, tumor-associated antigens and neo-antigens could be incorporated in AMAX for tumor-specific CD8+ T cell responses. Importantly, immunization with a mix of neoantigen AMAX could reduce tumor growth in a pre-clinical syngeneic mouse model. Hence, we provide pre-clinical support for the induction of effector CD8+ T cells through the adaptable AMAX platform as easy implementable peptidic vaccination strategy against any antigen of choice, including neoantigens for anti-tumor immunity. Image 1 [ABSTRACT FROM AUTHOR]