Uptake Of Antigen-presenting Cells Research Articles

Mannose modified targeted immersion vaccine delivery system improves protective immunity against Infectious spleen and kidney necrosis virus in mandarin fish (Siniperca chuatsi)

Vaccination is an effective method to prevent viral diseases. However, the biological barrier prevents the immersion vaccine from achieving the best effect without adding adjuvants and carriers. Researches on the targeted presentation technology of vaccines with nanocarriers are helpful to develop immersion vaccines for fish that can break through biological barriers and play an effective role in fish defense. In our study, functionally modified single-walled carbon nanotubes (SWCNTs) were used as carriers to construct a targeted immersion vaccine (SWCNTs-M−MCP) with mannose modified major capsid protein (MCP) to target antigen-presenting cells (APCs), against iridovirus diseases. After bath immunization, our results showed that SWCNTs-M−MCP induced the presentation process and uptake of APCs, triggering a powerful immune response. Moreover, the highest relative percent survival (RPS) was 81.3% in SWCNTs-M−MCP group, which was only 41.5% in SWCNTs-MCP group. Altogether, this study indicates that the SWCNTs-based targeted immersion vaccine induces strong immune response and provided an effective protection against iridovirus diseases.

Enhancing humoral and mucosal immune response of PED vaccine candidate by fusing S1 protein to nanoparticle multimerization

Porcine epidemic diarrhea virus (PEDV) is a highly infectious pathogen with a high mortality rate, which poses a serious threat to newborn piglets. A rapid, safe and effective vaccine is necessary for protecting pigs from PED infection. Nanoparticles have become molecular scaffolds for displaying soluble antigens due to their unique physical and chemical properties. Here, a vaccine candidate was based on the display of PEDV S1 protein on a mi3 nanoparticle platform using SpyTag/SpyCatcher technology. The size, zeta potential and microstructure of the S1-mi3 NPs were investigated, and their effects on the uptake of antigen-presenting cells (APCs) and maturation of dendritic cells (DCs) were analyzed. Mice were immunized via muscular and intranasal administrations, and the levels of humoral, cellular and mucosal immune responses were analyzed. As a result, S1 proteins were surface-displayed on NPs successfully, which self-assembled into nanoparticles composed of 60 subunits and showed superior safety and stability. In addition, mi3 NPs promoted antigen internalization and dendritic cell (DCs) maturation. In the mouse model, S1-mi3 NPs significantly increased the PEDV-specific antibody including serum IgG, secretory IgA (SIgA) and neutralizing antibodies (NAb). Furthermore, S1-mi3 NPs elicited more CD3+CD4+ and CD3+CD8+ T cell and cellular immune-related cytokines (IFN-γ and IL-4) compared to monomeric S1. In particular, it can induce an effective germinal center-specific (GC) B cell response, which is closely related to the production of neutralizing antibodies. Overall, S1-mi3 NPs are a promising subunit vaccine candidate against PEDV, and this self-assembly NPs also provide an attractive platform for improving vaccine efficacy against emerging pathogens.

Nanoparticle-based DNA vaccine protects against SARS-CoV-2 variants in female preclinical models

A safe and effective vaccine with long-term protection against SARS-CoV-2 variants of concern (VOCs) is a global health priority. Here, we develop lipid nanoparticles (LNPs) to provide safe and effective delivery of plasmid DNA (pDNA) and show protection against VOCs in female small animal models. Using a library of LNPs encapsulating unique barcoded DNA (b-DNA), we screen for b-DNA delivery after intramuscular administration. The top-performing LNPs are further tested for their capacity of pDNA uptake in antigen-presenting cells in vitro. The lead LNP is used to encapsulate pDNA encoding the HexaPro version of SARS-CoV-2 spike (LNP-HPS) and immunogenicity and protection is tested in vivo. LNP-HPS elicit a robust protective effect against SARS-CoV-2 Gamma (P.1), correlating with reduced lethality, decreased viral load in the lungs and reduced lung damage. LNP-HPS induce potent humoral and T cell responses against P.1, and generate high levels of neutralizing antibodies against P.1 and Omicron (B.1.1.529). Our findings indicate that the protective efficacy and immunogenicity elicited by LNP-HPS are comparable to those achieved by the approved COVID-19 vaccine from Biontech/Pfizer in animal models. Together, these findings suggest that LNP-HPS hold great promise as a vaccine candidate against VOCs.

Supramolecular complex glycoconjugate vaccine generates self-enhancement effects for carbohydrate antigen delivery

Targeting delivery of tumor-associated carbohydrate antigen (TACA)-based vaccine to antigen-presenting cells (APCs) mediated by endogenous antibodies can improve the immunogenicity of TACA. However, an essential requirement of this approach is to generate high titers of endogenous antibodies in vivo through pre-immunization, which complicates the immunization procedure and may cause side effects. Herein, we report a new generation of APC-targeting TACA-based supramolecular complex vaccine, assembled by sialyl Thomsen-nouveau-bovine serum albumin-adamantine (sTn-BSA-Ada) and heptavalent rhamnose (Rha)-modified β-cyclodextrin (β-CD) via host–guest interaction. The complex vaccine retained anti-Rha antibodies recruiting capability and facilitated the APCs uptake of the vaccine via the interaction of the Fc-domain with the Fc receptors on APCs. We demonstrate that direct immunization of complex vaccine elicited anti-Rha and anti-sTn specific immune response synchronously, generating a novel self-enhancement effect that can improve the antigen delivery to APCs in high efficacy. The structure–activity relationship (SAR) study proved that complex vaccine 4 with polyethylene glycol 6 (PEG6) linker in host molecule provoked a robust and specific sTn immune response comparable to the pre-immunization approach. The antisera induced by complex vaccine, either through direct immunization or pre-immunization, exhibited equal potency of cytotoxicity against the sTn expression cancer cells. This study provides a general platform for TACA-based vaccines with self-enhancement effects without the need for pre-immunization.

Orf Virus-Based Vectors Preferentially Target Professional Antigen-Presenting Cells, Activate the STING Pathway and Induce Strong Antigen-Specific T Cell Responses.

BackgroundOrf virus (ORFV)-based vectors are attractive for vaccine development as they enable the induction of potent immune responses against specific transgenes. Nevertheless, the precise mechanisms of immune activation remain unknown. This study therefore aimed to characterize underlying mechanisms in human immune cells.MethodsPeripheral blood mononuclear cells were infected with attenuated ORFV strain D1701-VrV and analyzed for ORFV infection and activation markers. ORFV entry in susceptible cells was examined using established pharmacological inhibitors. Using the THP1-Dual™ reporter cell line, activation of nuclear factor-κB and interferon regulatory factor pathways were simultaneously evaluated. Infection with an ORFV recombinant encoding immunogenic peptides (PepTrio-ORFV) was used to assess the induction of antigen-specific CD8+ T cells.ResultsORFV was found to preferentially target professional antigen-presenting cells (APCs) in vitro, with ORFV uptake mediated primarily by macropinocytosis. ORFV-infected APCs exhibited an activated phenotype, required for subsequent lymphocyte activation. Reporter cells revealed that the stimulator of interferon genes pathway is a prerequisite for ORFV-mediated cellular activation. PepTrio-ORFV efficiently induced antigen-specific CD8+ T cell recall responses in a dose-dependent manner. Further, activation and expansion of naïve antigen-specific CD8+ T cells was observed in response.DiscussionOur findings confirm that ORFV induces a strong antigen-specific immune response dependent on APC uptake and activation. These data support the notion that ORFV D1701-VrV is a promising vector for vaccine development and the design of innovative immunotherapeutic applications.

Anticancer Vaccination with Immunogenic Micelles That Capture and Release Pristine CD8+ T-Cell Epitopes and Adjuvants.

The development of nanocarriers capable of codelivering antigens and immune-activating adjuvants is an emerging area of research and is relevant for cancer vaccines that target induction of antigen-specific CD8+ T-cell responses. Here, we report a system for delivery of short peptide antigens to dendritic cells for strong cellular immune responses, based on block copolymers chemically modified with a hydrophobic and self-immolative linker. After modification, micelles effectively and reversibly capture antigens and adjuvants via a covalent bond within several minutes in an aqueous solution. After uptake in antigen presenting cells, the polymer disulfide bond is cleaved by intracellular glutathione, leading to release of pristine antigens, along with the upregulated expression of costimulatory molecules. The induced antigen-specific CD8+ T cells have strong tumor cell killing efficacy in the murine B16OVA and human papilloma virus-E6/E7 subcutaneous and lung metastasis tumor models. In addition, delivery to lymph nodes can be imaged to visualize vaccine trafficking. Taken together, multifunctional self-immolative micelles represent a versatile class of a vaccine delivery system for the generation of a cellular immune response that warrants further exploration as a component of cancer immunotherapy.

Preparation of mRNA Polyplexes with Post-conjugated Endosome-Disruptive Peptides.

Successful delivery of mRNA into the cytosol of professional antigen-presenting cells (APCs) poses one of the biggest challenges in developing effective mRNA vaccines to treat various cancers and viral infectious diseases. However, most polymeric mRNA delivery systems fail to transfect APCs. We have discovered that decoration of pH-sensitive endosome-disruptive GALA peptides on the surface of mRNA polyplexes leads to efficient targeting and transfection of APCs. GALA peptides not only enhance specific uptake in APCs through binding to sialic acid moieties, they also facilitate the endosomal escape of mRNA especially in dendritic cells (DCs). Here, we describe in detail the production of stabilized mRNA polyplexes post-conjugated with GALA peptides via copper-free click chemistry. Methods described here include the synthesis and purification of GALA peptides and its conjugation to mRNA polyplexes.

The Effects of Pre-Existing Antibodies on Live-Attenuated Viral Vaccines.

Live-attenuated vaccines (LAVs) have achieved remarkable successes in controlling virus spread, as well as for other applications such as cancer immunotherapy. However, with rapid increases in international travel, globalization, geographic spread of viral vectors, and widespread use of vaccines, there is an increasing need to consider how pre-exposure to viruses which share similar antigenic regions can impact vaccine efficacy. Pre-existing antibodies, derived from either from maternal–fetal transmission, or by previous infection or vaccination, have been demonstrated to interfere with vaccine immunogenicity of measles, adenovirus, and influenza LAVs. Immune interference of LAVs can be caused by the formation of virus–antibody complexes that neutralize virus infection in antigen-presenting cells, or by the cross-linking of the B-cell receptor with the inhibitory receptor, FcγRIIB. On the other hand, pre-existing antibodies can augment flaviviral LAV efficacy such as that of dengue and yellow fever virus, especially when pre-existing antibodies are present at sub-neutralizing levels. The increased vaccine immunogenicity can be facilitated by antibody-dependent enhancement of virus infection, enhancing virus uptake in antigen-presenting cells, and robust induction of innate immune responses that promote vaccine immunogenicity. This review examines the literature on this topic and examines the circumstances where pre-existing antibodies can inhibit or enhance LAV efficacy. A better knowledge of the underlying mechanisms involved could allow us to better manage immunization in seropositive individuals and even identify possibilities that could allow us to exploit pre-existing antibodies to boost vaccine-induced responses for improved vaccine efficacy.

β-glucan as a new tool in vaccine development.

Vaccination constitutes one of the major breakthroughs in human medicine. At the same time, development of more immunogenic vaccine alternatives to using aluminium-based adjuvants is one of the most important phases of vaccination development. Among different sources of carbohydrate polymers, including plants, microbes and synthetic sources tested, glucans were found to be the most promising vaccine adjuvant, as they alone stimulate various immune reactions including antibody production without any negative side effects. The use of glucan particles as a delivery system is a viable option based on the documented efficient antigen loading and receptor-targeted uptake in antigen-presenting cells. In addition to particles, soluble glucans can be used as novel hydrogels or as direct immunocyte-targeting delivery systems employing novel complexes with oligodeoxynucleotides. This review focuses on recent advances in glucan-based vaccine development from glucan-based conjugates to a glucan-based delivery and adjuvant platform.

Antigen-Inorganic Hybrid Flowers-Based Vaccines with Enhanced Room Temperature Stability and Effective Anticancer Immunity.

Particle-based antigen carriers as adjuvants play an important role in vaccine development. Herein, an antigen-inorganic hybrid flower-like particle is developed as a novel vaccine carrier. Model antigen ovalbumin (OVA)-copper (II) sulfate hybrid vaccines (OVA-Cu-HVs) are mildly and facilely constructed through a biomimetic mineralization process. OVA-Cu-HVs facilitate cellular uptake in antigen-presenting cells and the internalization of OVA-Cu-HVs involves macropinocytosis-mediated endocytosis. OVA-Cu-HVs can release OVA in a pH-responsive behavior and promote cytosolic release of antigen to enhance antigen cross-presentation. Immunization with OVA-Cu-HVs promotes the maturation of dendritic cells in draining lymph nodes, induces robust antigen-specific T lymphocyte response, and inhibits tumor growth in vivo. In addition, OVA-Cu-HVs are efficacious after being stored for 4 weeks at room temperature and are expected to simplify vaccine storage and lower the cost of cold storage for transportation. Looking forward, OVA-Cu-HVs may hold strong potential to be as an effective vaccine delivery platform, which will facilitate the application of organic-inorganic hybrid flowers in biomedical areas.

Neisserial PorB immune enhancing activity and use as a vaccine adjuvant

ABSTRACTOur laboratory has focused on Porin B (PorB), an outer membrane protein from Neisseria meningitidis and TLR2 ligand-based adjuvant, to characterize specific molecular and cellular pathways involved in improved immune responses induced by vaccine adjuvants. PorB’s ability to form micellar nanoparticular multi-molecular organized structures and its interaction with Toll-like receptor 2/1 complexes likely accounts for its potent adjuvant activity. Downstream from this stimulation, we have observed enhanced antigen uptake in antigen presenting cells (APC), greater antigen deposition in secondary lymphoid organs, and promotion of germinal center reactions. In mice, antigen-specific IgGs were increased after PorB adjuvanted vaccination using the model antigen ovalbumin (OVA). Likewise, this formulation resulted in more IL-4 and IFN-γ positive T cells. Mice that received PorB adjuvanted vaccinations benefitted from lower bacterial burdens when challenged with recombinant Listeria monocytogenes expressing OVA. Mouse models lacking MyD88 signaling in various APC types helped identify macrophages as an essential cell type for the adjuvant activity of PorB. We believe the work presented here provides examples of the mechanistic studies required to understand how vaccine adjuvants are contributing to the establishment of protective immunity.

Critical Size Limit of Biodegradable Nanoparticles for Enhanced Lymph Node Trafficking and Paracortex Penetration.

Lymph node (LN) targeting through interstitial drainage of nanoparticles (NPs) is an attractive strategy to stimulate a potent immune response, as LNs are the primary site for lymphocyte priming by antigen presenting cells (APCs) and triggering of an adaptive immune response. NP size has been shown to influence the efficiency of LN-targeting and retention after subcutaneous injection. For clinical translation, biodegradable NPs are preferred as carrier for vaccine delivery. However, the selective "size gate" for effective LN-drainage, particularly the kinetics of LN trafficking, is less well defined. This is partly due to the challenge in generating size-controlled NPs from biodegradable polymers in the sub-100-nm range. Here, we report the preparation of three sets of poly(lactic-co-glycolic)-b-poly(ethylene-glycol) (PLGA-b-PEG) NPs with number average diameters of 20-, 40-, and 100-nm and narrow size distributions using flash nanoprecipitation. Using NPs labeled with a near-infrared dye, we showed that 20-nm NPs drain rapidly across proximal and distal LNs following subcutaneous inoculation in mice and are retained in LNs more effectively than NPs with a number average diameter of 40-nm. The drainage of 100-nm NPs was negligible. Furthermore, the 20-nm NPs showed the highest degree of penetration around the paracortex region and had enhanced access to dendritic cells in the LNs. Together, these data confirmed that small, size-controlled PLGA-b-PEG NPs at the lower threshold of about 30-nm are most effective for LN trafficking, retention, and APC uptake after s.c. administration. This report could inform the design of LN-targeted NP carrier for the delivery of therapeutic or prophylactic vaccines.

Genetically engineered cell factories produce glycoengineered vaccines that target antigen-presenting cells and reduce antigen-specific T-cell reactivity

Genetically engineered cell factories produce glycoengineered vaccines that target antigen-presenting cells and reduce antigen-specific T-cell reactivity

Lymph node targeting strategies to improve vaccination efficacy

With the rapid development of nanotechnology as well as growing interest in immunotherapy, a great number of vaccine delivery vehicles have been explored in order to elicit potent adaptive immune responses against various infections or tumors. Recent studies have shown that targeting vaccine to antigen-presenting cells (APCs) within lymph nodes is an effective strategy for improving antigen-specific adaptive immune response. However, the characteristics of vaccine vehicles, such as size, surface charge and the degree of PEGylation, affect lymph node transfer and subsequent APC uptake, leading to different levels of immune responses. Thus, a comprehensive review of barriers, difficulties and corresponding strategies in lymph node targeting is important. In this review, we summarize the critical factors that affect lymph node delivery and survey recently reported successful vaccine carriers, which may be helpful for the rational design of vaccine vehicles.

Display of DNA on Nanoparticles for Targeting Antigen Presenting Cells.

Efficient delivery of antigens is of paramount concern in immunotherapies. We aimed to target antigen presenting cells (APCs) by conjugating CpG oligonucleotides to an E2 protein nanoparticle surface (CpG-PEG-E2). Compared to E2 alone, we observed ~4-fold increase of in vitro APC uptake of both CpG-PEG-E2 and E2 conjugated to non-CpG DNA. Furthermore, compared to E2-alone or E2 functionalized solely with polyethylene glycol (PEG), the CpG-PEG-E2 showed enhanced lymph node retention up to at least 48 hr and 2-fold increase in APC uptake in vivo, parameters which are advantageous for vaccine success. This suggests that enhanced APC uptake of nanoparticles mediated by oligonucleotide display may help overcome delivery barriers in vaccine development.

Poly-L-lysine-coated nanoparticles are ineffective in inducing mucosal immunity against group a streptococcus

Background: Group A Streptococcus (GAS) can cause a range of maladies, from simple throat infections to lethal complication, such as rheumatic heart disease. The M-protein, a bacterial cell surface protein, is the major virulence factor of GAS. Several attempts have been made over the past few decades to develop vaccines against GAS that employed peptides derived from the M-protein. One suchapproach used lipopeptides or lipid core peptide (LCP) systems that incorporated a B cell epitope derived from the conserved region of the M-protein.Methods: In the present study, we prepared different biodegradable polymer [dextran, poly-(lacticcoglycolic- acid) (PLGA), and poly-L-lysine] nanoparticles (NPs)-based delivery systems for a lipopeptide vaccine candidate (LCP-1). The NPs were characterised by their size, charge, morphology, antigen-presenting cells (APCs) uptake and subsequent APCs maturations efficacy, followed by in vivo nasal immunisation in mice.Results: All produced NPs ranged in size from 100-205 nm, and their charge varied depending upon the nature of polymer. A high APCs uptake efficacy for dextran and poly-L-lysine NPs were observed, compared to PLGA NPs. Despite the high uptake by APCs, dextran and poly-L-lysine NPs failed to improve APCs maturation that resulted in low antibody titres. In contrast, while LCP-1 encapsulated into PLGA showed low APCs uptake,it induced significant maturation of DCs and higher antibody titres compared to other NPs.Conclusions: Positively-charged poly-L-lysine NPs were non-immunogenic, while negatively charged PLGA NPs induced similar responses to antigens adjuvanted with cholera toxin B (CTB).

Construction and immunogenicity of a new Fc-based subunit vaccine candidate against Mycobacterium tuberculosis.

As an ancient disease, tuberculosis (TB) is a major global health threat. Therefore, there is an urgent need for an effective and safe anti-TB vaccine. In the current study, a delivery system of Fc domain of mouse IgG2a and early secreted antigenic target protein 6 (ESAT-6) was evaluated for the selective uptake of antigens by antigen-presenting cells (APCs). Thus, it was based on the immunogenicity of a fusion protein. The study was initiated by the transfer of recombinant expression vectors of pPICZαA-ESAT-6:Fcγ2a and pPICZαA-ESAT-6: His into Pichia pastoris (P. pastoris). Recombinant proteins were assessed for immunogenicity following the immunoblotting analysis. High levels of IFN-γ and IL-12 were produced to induce Th1-type cellular responses through vaccination with both recombinant proteins [ESAT-6:Fcγ2a (EF) and ESAT-6:His (EH)]. The Fc-tagged recombinant protein induced more effective Th1-type cellular responses with a low increment in IL-4 compared to PBS, BCG, and EH groups. Although in all the immunized groups, the ratio of IFN-γ/IL-4 was in favor of Th1 responses, the highest Th1/Th2 balance was observed in EF immunized group. Fc fragment of mouse IgG2a may induce a selective uptake of APCs towards the cross-presentation and formation of Th1 responses in favor of an appropriate protective anti-tuberculosis reaction. Thus, further research on Fc-fusion proteins is required to develop Fc-based TB vaccines.

Highlights from the latest articles on vaccine development using nanoparticles.

2014 Designer aluminum salt-based nanoparticles as vaccine adjuvants Evaluation of: Sun B, Ji Z, Liao Y et al. Engineering an effective immune adjuvant by designed control of shape and crystallinity of aluminum oxyhydroxide nanoparticles. ACS Nano. 7(12), 10834–10849 (2013). Aluminum salts, such as aluminum hydroxide, are widely used in vaccines to improve antigen-specific immune responses [1]. Despite their widespread usage, the exact mechanism of their action is still not fully elucidated. Recently, NLPR3 inflammasome, also known as NALP3 or cryopyrin, has been implicated in the immunostimulatory effects observed with aluminum salts [2]. Structural studies using x-ray diffraction and infrared spectroscopy have shown that aluminum hydroxide adjuvant is actually crystalline aluminum oxyhydroxide, as opposed to aluminum hydroxide or Al(OH) 3 . The geometrical shape of the adjuvant in general has also been implicated in its uptake by antigen-presenting cells (APCs). In this study, Sun et al. designed aluminum oxyhydroxide nanoparticles with different morphologies, shapes and hydroxide concentrations and investigated their adjuvant activities [3]. They studied the effect of these parameters on aluminum oxyhydroxide’s ability to trigger the NLPR3 inflammasome and APC uptake, relative to a commercial alum preparation, using in vitro assays and in vivo studies. An aluminum oxyhydroxide library of nanorods, nanoplates and nano polyhedra was synthesized using a hydrothermal method and structures confirmed by transmission electron microscopy, x-ray diffraction, thermogravimetric analysis and Fourier transform infrared spectroscopy analyses. All the nanostructures were able to activate the NLRP3 inflammasome through generation of oxidative stress in THP-1 human monocytes and induce maturation of mouse bone marrow derived dendritic cells and IL-1β cytokine production by them, but the nanorods were more effective than the nanoplates, nanopolyhedra and the commercial alum. The nanorods were hence used in an in vivo immunization study using ovalbumin (OVA) as a model antigen. The OVA-specific immune responses induced by the OVA-adsorbed aluminum oxyhydroxide nanorods tended to be stronger than that induced by OVA-adsorbed on the commercial alum preparation. This study opens the possibility of engineering designer aluminum salt-based adjuvants with a stronger adjuvant activity.

A double‐blind placebo‐controlled birch allergy vaccination study II: correlation between inhibition of IgE binding, histamine release and facilitated allergen presentation

The pathogenesis of IgE-mediated allergic disease is closely related to the production of T-helper type 2 (Th2) cytokines, which lead to IgE production pivotal for activation of mast cells and basophils. Proliferating T cells along with eosinophils expanded and attracted by Th2 cytokines are major contributors to the late-phase reaction. The activation of these Th2 cells is strongly enhanced by CD23-mediated IgE facilitated allergen presentation (FAP). The present study aims to investigate the effect of specific immunotherapy (SIT)-induced allergen-specific non-IgE antibodies (blocking antibodies) on IgE binding to allergen, histamine release (HR) and CD23-mediated allergen uptake in antigen-presenting cells. Competition between IgE and non-IgE for allergen binding was studied by Advia Centaur antibody measurements, passively sensitized basophils were used to study HR and IgE-facilitated binding of allergen to B cells (FAP) was studied by flow cytometry. FAP measurements were performed both with and without the addition of a reference IgE serum, which was included to obtain optimal complex formation. The serum samples were obtained from birch pollen immunotherapy (n=21) or placebo control patients (n=21) before and after 1 and 2 years of treatment. Statistically significant reduction of all parameters investigated was observed after 1 year of treatment and the effect was maintained during the second year of treatment. There was a clear correlation between the two FAP measurements and between each of them and the level of T cell activation reported upon previously. Moreover, strong correlations were found between changes in FAP, IgE binding and HR. The present study clearly demonstrates that SIT induces changes in the composition of serum antibodies that inhibit IgE binding, HR and FAP to a similar extent. This suggests that these measurements, individually or in combination, may be used to monitor the immunological effect of SIT, even though direct correlations to changes in clinical parameters could not be demonstrated.

Individual cathepsins degrade immune complexes internalized by antigen-presenting cells via Fcγ receptors

We have analyzed the intracellular degradation of an immune complex after its FcgammaR-mediated uptake in antigen-presenting cells (APC). Mice that lack the cathepsins (Cat) S, L, B and D allowed us to assess the direct contribution of these individual proteases to the processing events observed. CatS and CatB mediate the bulk of degradation of the Ig-125I-labeled F(ab')2 immune complex delivered via FcgammaR, while CatL and CatD are dispensable. CatS and CatB are involved in independent processing pathways and can substitute in part for each other's absence. The combined ablation of both proteases reduces the rate of degradation observed by > 80 %. CatB is required for the generation of F(ab')23, a predominant degradation intermediate smaller by approximately 3 kDa than the 125I-labeled F(ab')2 itself. In addition, absence of CatB in vivo significantly affects the activity pattern of the remaining cysteine proteases. Thus, we conclude that CatB is a key enzyme for the proper degradation of an immune complex taken up by FcgammaR and for the control of protease activity in the endocytic pathway of APC.