Phagocytosis.

One hundred years have passed since the death of Élie Metchnikoff (1845-1916). He was the first to observe the uptake of particles by cells and realized the importance of this process, named phagocytosis, for the host response to injury and infection. He also was a strong advocate of the role of phagocytosis in cellular immunity, and with this, he gave us the basis for our modern understanding of inflammation and the innate immune response. Phagocytosis is an elegant but complex process for the ingestion and elimination of pathogens, but it is also important for the elimination of apoptotic cells and hence fundamental for tissue homeostasis. Phagocytosis can be divided into four main steps: (i) recognition of the target particle, (ii) signaling to activate the internalization machinery, (iii) phagosome formation, and (iv) phagolysosome maturation. In this chapter, we present a general view of our current knowledge on phagocytosis performed mainly by professional phagocytes through antibody and complement receptors and discuss aspects that remain incompletely understood.

Using machine learning to dissect host kinases required for Leishmania internalization and development.

The Leishmania life cycle alternates between promastigotes, found in the sandfly, and amastigotes, found in mammals. When an infected sandfly bites a host, promastigotes are engulfed by phagocytes (i.e., neutrophils, dendritic cells, and macrophages) to establish infection. When these phagocytes die or break down, amastigotes must be re-internalized to survive within the acidic phagolysosome and establish disease. To define host kinase regulators of Leishmania promastigote and amastigote uptake and survival within macrophages, we performed an image-based kinase regression screen using a panel of 38 kinase inhibitors with unique and overlapping kinase targets. We also targeted inert beads to complement receptor 3 (CR3) or Fcγ receptors (FcR) as controls by coating them with complement/C3bi or IgG respectively. Through this approach, we identified several host kinases that regulate receptor-mediated phagocytosis and/or the uptake of L. amazonensis. Findings included kinases previously implicated in Leishmania uptake (such as SRC family kinases (SFK), Abl family kinases (ABL1/c-Abl, ABL2/Arg), and spleen tyrosine kinase (SYK)); we also uncovered many novel kinases. These methods also predicted kinases necessary for promastigotes to convert to amastigotes or for amastigotes to survive within macrophages. Overall, our results suggest that the concerted action of multiple interconnected networks of host kinases are needed over the course of Leishmania infection, and that the kinases required for the parasite's life cycle substantially differ depending on which receptors are bound and the life cycle stage that is internalized. In addition, using our screen, we identified kinases that preferentially regulate the uptake of parasites over beads, indicating that the methods required for Leishmania to be internalized by macrophages differ significantly from generalized phagocytic mechanisms. Our findings are intended to be used as a hypothesis generation resource for the broader scientific community studying the roles of kinases in host-pathogen interactions.

Complement receptor 3-dependent engagement by Candida glabrata ß-glucan modulates dendritic cells to induce regulatory T-cell expansion.

Candida glabrata is an important pathogen causing invasive infection associated with a high mortality rate. One mechanism that causes the failure of Candida eradication is an increase in regulatory T cells (Treg), which play a major role in immune suppression and promoting Candida pathogenicity. To date, how C. glabrata induces a Treg response remains unclear. Dendritic cells (DCs) recognition of fungi provides the fundamental signal determining the fate of the T-cell response. This study investigated the interplay between C. glabrata and DCs and its effect on Treg induction. We found that C. glabrata ß-glucan was a major component that interacted with DCs and consequently mediated the Treg response. Blocking the binding of C. glabrata ß-glucan to dectin-1 and complement receptor 3 (CR3) showed that CR3 activation in DCs was crucial for the induction of Treg. Furthermore, a ligand-receptor binding assay showed the preferential binding of C. glabrata ß-glucan to CR3. Our data suggest that C. glabrata ß-glucan potentially mediates the Treg response, probably through CR3-dependent activation in DCs. This study contributes new insights into immune modulation by C. glabrata that may lead to a better design of novel immunotherapeutic strategies for invasive C. glabrata infection.

Complement receptor 4 mediates the clearance of extracellular tau fibrils by microglia.

Tauopathies exhibit a characteristic accumulation of misfolded tau aggregates in the brain. Tau pathology shows disease-specific spatiotemporal propagation through intercellular transmission, which is closely correlated with the progression of clinical manifestations. Therefore, identifying molecular mechanisms that prevent tau propagation is critical for developing therapeutic strategies for tauopathies. The various innate immune receptors, such as complement receptor 3 (CR3) and complement receptor 4 (CR4), have been reported to play a critical role in the clearance of various extracellular toxic molecules by microglia. However, their role in tau clearance has not been studied yet. In the present study, we investigated the role of CR3 and CR4 in regulating extracellular tau clearance. We found that CR4 selectively binds to tau fibrils but not to tau monomers, whereas CR3 does not bind to either of them. Inhibiting CR4, but not CR3, significantly reduces the uptake of tau fibrils by BV2 cells and primary microglia. By contrast, inhibiting CR4 has no effect on the uptake of tau monomers by BV2 cells. Furthermore, inhibiting CR4 suppresses the clearance of extracellular tau fibrils, leading to more seed-competent tau fibrils remaining in the extracellular space relative to control samples. We also provide evidence that the expression of CR4 is upregulated in the brains of human Alzheimer's disease patients and the PS19 mouse model of tauopathy. Taken together, our data strongly support that CR4 is a previously undescribed receptor for the clearance of tau fibrils in microglia and may represent a novel therapeutic target for tauopathy.

X-Ray Crystallography for Macromolecular Complexes.

X-ray crystallography has for most of the last century been the standard technique to determine the high-resolution structure of biological macromolecules, including multi-subunit protein-protein and protein-nucleic acids as large as the ribosome and viruses. As such, the successful application of X-ray crystallography to many biological problems revolutionized biology and biomedicine by solving the structures of small molecules and vitamins, peptides and proteins, DNA and RNA molecules, and many complexes-affording a detailed knowledge of the structures that clarified biological and chemical mechanisms, conformational changes, interactions, catalysis and the biological processes underlying DNA replication, translation, and protein synthesis. Now reaching well into the first quarter of the twenty-first century, X-ray crystallography shares the structural biology stage with cryo-electron microscopy and other innovative structure determination methods, as relevant and central to our understanding of biological function and structure as ever. In this chapter, we provide an overview of modern X-ray crystallography and how it interfaces with other mainstream structural biology techniques, with an emphasis on macromolecular complexes.

Complement receptor 3 is required for maximum in vitro trogocytic killing of the parasite Trichomonas vaginalis by human neutrophil-like cells.

Trichomonas vaginalis (Tv) is a parasite that causes trichomoniasis, a prevalent sexually-transmitted infection. Neutrophils are found at the site of infection, and can rapidly kill the parasite in vitro, using trogocytosis. However, the specific molecular players in neutrophil killing of Tv are unknown. Here, we show that complement proteins play a role in Tv killing by human neutrophil-like cells (NLCs). Using CRISPR/Cas9, we generated NLCs deficient in each of three complement receptors (CRs) known to be expressed on human neutrophils: CR1, CR3, and CR4. Using in vitro trogocytosis assays, we found that CR3, but not CR1 or CR4 is required for maximum trogocytosis of the parasite by NLCs, with NLCs lacking CR3 demonstrating ~40% reduction in trogocytosis, on average. We also observed a reduction in NLC killing of Tv in CR3 knockout, but not CR1 or CR4 knockout NLCs. On average, NLCs lacking CR3 had ~50% reduction in killing activity. We also used a parallel approach of pre-incubating NLCs with blocking antibodies against CR3, which similarly reduced NLC killing of parasites. These data support a model in which Tv is opsonized by the complement protein iC3b, and bound by neutrophil CR3 receptor, to facilitate trogocytic killing of the parasite.

Triple-fusion protein (TriFu): A potent, targeted, enzyme-like inhibitor of all three complement activation pathways.

The introduction of a therapeutic anti-C5 antibody into clinical practice in 2007 inspired a surge into the development of complement-targeted therapies. This has led to the recent approval of a C3 inhibitory peptide, an antibody directed against C1s and a full pipeline of several complement inhibitors in preclinical and clinical development. However, no inhibitor is available that efficiently inhibits all three complement initiation pathways and targets host cell surface markers as well as complement opsonins. To overcome this, we engineered a novel fusion protein combining selected domains of the three natural complement regulatory proteins decay accelerating factor, factor H and complement receptor 1. Such a triple fusion complement inhibitor (TriFu) was recombinantly expressed and purified alongside multiple variants and its building blocks. We analyzed these proteins for ligand binding affinity and decay acceleration activity by surface plasmon resonance. Additionally, we tested complement inhibition in several in vitro/ex vivo assays using standard classical and alternative pathway restricted hemolysis assays next to hemolysis assays with paroxysmal nocturnal hemoglobinuria erythrocytes. A novel in vitro model of the alternative pathway disease C3 glomerulopathy was established to evaluate the potential of the inhibitors to stop C3 deposition on endothelial cells. Next to the novel engineered triple fusion variants which inactivate complement convertases in an enzyme-like fashion, stoichiometric complement inhibitors targeting C3, C5, factor B, and factor D were tested as comparators. The triple fusion approach yielded a potent complement inhibitor that efficiently inhibits all three complement initiation pathways while targeting to surface markers.

Activation of leukotriene B4 receptor 1 is a prerequisite for complement receptor 3-mediated antifungal responses of neutrophils.

Invasive fungal infections are life-threatening, and neutrophils are vital cells of the innate immune system that defend against them. The role of LTA4H-LTB4-BLT1 axis in regulation of neutrophil responses to fungal infection remains poorly understood. Here, we demonstrated that the LTA4H-LTB4-BLT1 axis protects the host against Candida albicans and Aspergillus fumigatus, but not Cryptococcus neoformans infection, by regulating the antifungal activity of neutrophils. Our results show that deleting Lta4h or Blt1 substantially impairs the fungal-specific phagocytic capacity of neutrophils. Moreover, defective activation of the spleen tyrosine kinase (Syk) and extracellular signal-related kinase (ERK1/2) pathways in neutrophils accompanies this impairment. Mechanistically, BLT1 regulates CR3-mediated, ß-1,3-glucan-induced neutrophil phagocytosis, while a physical interaction with CR3 with slight influence on its dynamics is observed. Our findings thus demonstrate that the LTA4H-LTB4-BLT1 axis is essential for the phagocytic function of neutrophils in host antifungal immune response against Candida albicans and Aspergillus fumigatus.

Conformational variants of the ternary complex of C5a, C5aR1, and G-protein.

The complement component fragment 5a (C5a) binds and activates two complement receptors like C5aR1 and C5aR2, which play a significant role in orchestrating the proinflammatory function of C5a in tissues through the recruitment of heterotrimeric G-proteins and ß-arrestins. Dysregulation of the complement induces excessive production of C5a, which triggers aberrant activation of the C5a-C5aR1-G-protein and C5a-C5aR2-ß-arrestin signalling axes in tissues, contributing to the pathology of numerous immune-inflammatory diseases. Thus, understanding the interaction of C5a with C5aR1 and C5aR2, as well as the interaction of G-protein and ß-arrestins, respectively, with C5a-C5aR1 and C5a-C5aR2, holds tremendous therapeutic value. In the absence of structural data, we have previously elaborated the binary complexes of C5a-C5aR1 and C5a-C5aR2, as well as the ternary complex of C5a-C5aR2-ß-arrestin1, in highly refined model structures. While our ternary model complex of C5a-C5aR1-G-protein was in progress, two cryo-electron microscopy-based ternary structural complexes of C5aR1 were made available by others. However, it is observed that the interaction of the crucial NT-peptide of C5aR1 with C5a, including the portion of the G⍺i-subunit that harbors the switch-I region, is not fully resolved in both complexes. The current study addresses the issues and provides two highly refined alternative model ternary complexes of C5a-C5aR1-G-protein. The study highlights the conformational heterogeneity in C5aR1 by comparing the two conformational variants of the model ternary complex in the context of C5a-C5aR2-ß-arrestin1 for further devising methods and molecules targeting both surface and intracellular C5aR1/C5aR2 for effectively mitigating the proinflammatory role of C5a in various disease settings.Communicated by Ramaswamy H. Sarma.

Complement receptor 1 is the human erythrocyte receptor for Plasmodium vivax erythrocyte binding protein.

The discovery that Africans were resistant to infection by Plasmodium vivax (P. vivax) led to the conclusion that P. vivax invasion relied on the P. vivax Duffy Binding Protein (PvDBP) interacting with the Duffy Antigen Receptor for Chemokines (DARC) expressed on erythrocytes. However, the recent reporting of P. vivax infections in DARC-negative Africans suggests that the parasite might use an alternate invasion pathway to infect DARC-negative reticulocytes. To identify the parasite ligands and erythrocyte receptors that enable P. vivax invasion of both DARC-positive and -negative erythrocytes, we expressed region II containing the Duffy Binding-Like (DBL) domain of P. vivax erythrocyte binding protein (PvEBP-RII) and verified that the DBL domain binds to both DARC-positive and -negative erythrocytes. Furthermore, an AVidity-based EXtracelluar Interaction Screening (AVEXIS) was used to identify the receptor for PvEBP among over 750 human cell surface receptor proteins, and this approach identified only Complement Receptor 1 (CR1, CD35, or C3b/C4b receptor) as a PvEBP receptor. CR1 is a well-known receptor for P. falciparum Reticulocyte binding protein Homology 4 (PfRh4) and is present on the surfaces of both reticulocytes and normocytes, but its expression decreases as erythrocytes age. Indeed, PvEBP-RII bound to a subpopulation of both reticulocytes and normocytes, and this binding was blocked by the addition of soluble CR1 recombinant protein, indicating that CR1 is the receptor of PvEBP. In addition, we found that the Long Homology Repeat A (LHR-A) subdomain of CR1 is the only subdomain responsible for mediating the interaction with PvEBP-RII.

Identification and structural analysis of dimeric chicken complement component 3d and its binding with chicken complement receptor 2.

Complement component 3d (C3d), the final cleavage product of complement component C3, interacts with CR2 and thus plays a crucial role in linking the innate and adaptive immune systems. Additionally, human C3d executes various functions in its dimeric form, which is more effective than its monomeric form. In this study, we aimed to explored whether chicken C3d (chC3d) exhibits similar characteristics, namely dimerization and binding of dimeric chC3d to chicken CR2 (chCR2). We investigated the interaction and co-localization of chC3d with itself using coimmunoprecipitation and confocal laser scanning microscopy, respectively. Then, dimeric chC3d was detected using native polyacrylamide gel electrophoresis and western blotting, and its equilibrium dissociation constant KD (827 nM) was determined using surface plasmon resonance. Finally, the interaction modes of dimeric chC3d were identified using molecular docking simulations, which revealed that dimeric chC3d could crosslink with chCR2 receptor. Overall, our findings will facilitate future explorations of the chicken complement system.

Impact of surface receptors TLR2, CR3, and FcγRIII on Rhodococcus equi phagocytosis and intracellular survival in macrophages.

The virulence-associated protein A (VapA) produced by virulent Rhodococcus equi allows it to replicate in macrophages and cause pneumonia in foals. It is unknown how VapA interacts with mammalian cell receptors, but intracellular replication of avirulent R. equi lacking vapA can be restored by supplementation with recombinant VapA (rVapA). Our objectives were to determine whether the absence of the surface receptors Toll-like receptor 2 (TLR2), complement receptor 3 (CR3), or Fc gamma receptor III (FcγRIII) impacts R. equi phagocytosis and intracellular replication in macrophages, and whether rVapA restoration of virulence in R. equi is dependent upon these receptors. Wild-type (WT) murine macrophages with TLR2, CR3, or FcγRIII blocked or knocked out (KO) were infected with virulent or avirulent R. equi, with or without rVapA supplementation. Quantitative bacterial culture and immunofluorescence imaging were performed. Phagocytosis of R. equi was not affected by blockade or KO of TLR2 or CR3. Intracellular replication of virulent R. equi was not affected by TLR2, CR3, or FcγRIII blockade or KO; however, avirulent R. equi replicated in TLR2-/- and CR3-/- macrophages but not in WT and FcγRIII-/-. rVapA supplementation did not affect avirulent R. equi phagocytosis but promoted intracellular replication in WT and all KO cells. By demonstrating that TLR2 and CR3 limit replication of avirulent but not virulent R. equi and that VapA-mediated virulence is independent of TLR2, CR3, or FcγRIII, our study provides novel insights into the role of these specific surface receptors in determining the entry and intracellular fate of R. equi.

Gene expression analysis revealed downregulation of complement receptor 1 in clonal B cells in cold agglutinin disease.

Cold agglutinin disease (CAD) is a rare B-cell lymphoproliferative disorder of the bone marrow, manifested by autoimmune hemolytic anemia caused by binding of monoclonal IgM autoantibodies to the I antigen. Underlying genetic changes have previously been reported, but their impact on gene expression profile has been unknown. Here, we define differentially expressed genes in CAD B cells. To unravel downstream alteration in cellular pathways, gene expression by RNA sequencing was undertaken. Clonal B-cell samples from 12 CAD patients and IgM-expressing memory B cells from 4 healthy individuals were analyzed. Differential expression analysis and filtering resulted in 93 genes with significant differential expression. Top upregulated genes included SLC4A1, SPTA1, YBX3, TESC, HBD, AHSP, TRAF1, HBA2, RHAG, CA1, SPTB, IL10, UBASH3B, ALAS2, HBA1, CRYM, RGCC, KANK2, and IGHV4-34. They were upregulated at least 8-fold, while complement receptor 1 (CR1/CD35) was downregulated 11-fold in clonal CAD B cells compared to control B cells. Flow cytometry analyses further confirmed reduced CR1 (CD35) protein expression by clonal CAD IgM+ B cells compared to IgM+ memory B cells in controls. CR1 (CD35) is an important negative regulator of B-cell activation and differentiation. Therefore, reduced CR1 (CD35) expression may increase activation, proliferation, and antibody production in CAD-associated clonal B cells.

Functional evaluation of complement factor I variants by immunoassays and SDS-PAGE.

Factor I (FI) is an essential regulator of the complement system. Together with co-factors, FI degrades C3b, which inhibits further complement activation. Genetic mutations in FI are associated with pathological conditions like age-related macular degeneration and atypical hemolytic uremic syndome. Here, we evaluated eight recombinant FI genetic variants found in patients. We assessed FI's co-factor activity in the presence of two co-factors; Factor H and soluble CR1. Different analytical assays were employed; SDS-PAGE to evaluate the degradation of C3b, ELISA to measure the generation of fluid phase iC3b and the degradation of surface-bound C3b using a novel Luminex bead-based assay. We demonstrate that mutations in the FIMAC and SP domains of FI led to significantly reduced protease activity, whereas the two analyzed mutations in the LDLRA2 domain did not result in any profound changes in FI's function. The different assays employed displayed a strong positive correlation, but differences in the activity of the genetic variants Ile55Phe and Gly261Asp could only be observed by combining different methods and co-factors for evaluating FI activity. In conclusion, our results provide a new perspective regarding available diagnostic tools for assessing the impact of mutations in FI.

Complement receptor 3-mediated neurotoxic glial activation contributes to rotenone-induced cognitive decline in mice.

Microglia-mediated chronic neuroinflammation has been associated with cognitive decline induced by rotenone, a well-known neurotoxic pesticide used in agriculture. However, the mechanisms remain unclear. This work aimed to elucidate the role of complement receptor 3 (CR3), a highly expressed receptor in microglia, in cognitive deficits induced by rotenone. Rotenone up-regulated the expression of CR3 in the hippocampus and cortex area of mice. CR3 deficiency markedly ameliorated rotenone-induced cognitive impairments, neurodegeneration and phosphorylation (Ser129) of α-synuclein in mice. CR3 deficiency also attenuated rotenone-stimulated microglial M1 activation. In microglial cells, siRNA-mediated knockdown of CR3 impeded, while CR3 activation induced by LL-37 exacerbated, rotenone-induced microglial M1 activation. Mechanistically, CR3 deficiency blocked rotenone-induced activation of nuclear factor κB (NF-κB), signal transducer and activator of transcription 1 (STAT1) and STAT3 signaling pathways. Pharmacological inhibition of NF-κB or STAT3 but not STAT1 was confirmed to suppress microglial M1 activation elicited by rotenone. Further study revealed that CR3 deficiency or knockdown also reduced rotenone-induced expression of C3, an A1 astrocyte marker, and production of microglial C1q, TNFα and IL-1α, a cocktail for activated microglia to induce neurotoxic A1 astrocytes, via NF-κB and STAT3 pathways. Finally, a small molecule modulator of CR3 efficiently mitigated rotenone-elicited cognitive deficits in mice even administered after the establishment of cognitive dysfunction. Taken together, our findings demonstrated that CR3 is a key factor in mediating neurotoxic glial activation and subsequent cognitive impairments in rotenone-treated mice, giving novel insights into the immunopathogenesis of cognitive impairments in pesticide-related Parkinsonism.

[The Functional Roles and the Potential as Drug Targets of Glycoproteins Regulating Complement and Coagulation Pathways].

Complement (C) activation occurs via three pathways, namely the classical, lectin, and alternative pathways. Intercommunication occurs between the complement and coagulation systems, which can trigger tissue injury and inflammation. Disseminated intravascular coagulation (DIC) is a life-threatening disease characterized by disordered coagulation and systemic inflammation; here, the intercommunication between the complement and coagulation systems contributes to the development of DIC. Extracellular histones, which are contributors to the damage-associated molecular pattern, induce severe thrombosis. C5 is a key molecule in the intercommunication between the complement and coagulation systems and is associated with the development of lethal histone-induced thrombosis. Heparin and chondroitin sulfate (CS) are negatively charged, allowing them to bind to extracellular histones. As the coagulation system is less affected by CS than heparin, CS shows potential as an effective drug for the treatment of patients with DIC who have a high risk of bleeding. Complement receptor type-1-related gene Y (Crry) inhibits the complement pathway via binding to C3b and C4b. Hence, Crry is a potent inhibitor of the classical and alternative C pathways. The expression of Crry is decreased by the endothelial damage induced by extracellular histones. Crry dysfunction promotes the activation of C on the surface of endothelial cells. The prevention of C3 cleavage on endothelial cells might be a useful therapy targeting acute lung injury.

Alzheimer's disease-associated complement gene variants influence plasma complement protein levels.

BACKGROUND: Alzheimer's disease (AD) has been associated with immune dysregulation in biomarker and genome-wide association studies (GWAS). GWAS hits include the genes encoding complement regulators clusterin (CLU) and complement receptor 1 (CR1), recognised as key players in AD pathology, and complement proteins have been proposed as biomarkers. MAIN BODY: To address whether changes in plasma complement protein levels in AD relate to AD-associated complement gene variants we first measured relevant plasma complement proteins (clusterin, C1q, C1s, CR1, factor H) in a large cohort comprising early onset AD (EOAD; n = 912), late onset AD (LOAD; n = 492) and control (n = 504) donors. Clusterin and C1q were significantly increased (p < 0.001) and sCR1 and factor H reduced (p < 0.01) in AD plasma versus controls. ROC analyses were performed to assess utility of the measured complement biomarkers, alone or in combination with amyloid beta, in predicting AD. C1q was the most predictive single complement biomarker (AUC 0.655 LOAD, 0.601 EOAD); combining C1q with other complement or neurodegeneration makers through stepAIC-informed models improved predictive values slightly. Effects of GWS SNPs (rs6656401, rs6691117 in CR1; rs11136000, rs9331888 in CLU; rs3919533 in C1S) on protein concentrations were assessed by comparing protein levels in carriers of the minor vs major allele. To identify new associations between SNPs and changes in plasma protein levels, we performed a GWAS combining genotyping data in the cohort with complement protein levels as endophenotype. SNPs in CR1 (rs6656401), C1S (rs3919533) and CFH (rs6664877) reached significance and influenced plasma levels of the corresponding protein, whereas SNPs in CLU did not influence clusterin levels. CONCLUSION: Complement dysregulation is evident in AD and may contribute to pathology. AD-associated SNPs in CR1, C1S and CFH impact plasma levels of the encoded proteins, suggesting a mechanism for impact on disease risk.

Successful treatment with avacopan (CCX168) in a pediatric patient with C3 glomerulonephritis.

BACKGROUND: C3 glomerulonephritis (C3GN) is a subtype of C3 glomerulopathy (C3G), characterized by dysregulation of the alternative pathway of complement and by dominant C3 by immunofluorescence on the kidney biopsy. There is no approved treatment for patients with C3G. Immunosuppressive drugs as well as biologics have been used with limited success. In recent decades, substantial advances in the understanding of the complement system have led to the development of new complement inhibitors. Avacopan (CCX168) is an orally administered small-molecule C5aR antagonist that blocks the effects of C5a, one of the most potent pro-inflammatory mediators of the complement system. CASE REPORT: We describe a child with biopsy-proven C3GN treated with avacopan. She was enrolled in the ACCOLADE double-blind placebo-controlled Phase 2 study (NCT03301467), where during the first 26 weeks she was randomized to receive an avacopan-matching placebo orally twice daily, while in the following 26 weeks, the study was open-label and she received avacopan. After a wash-out period, she was restarted on avacopan through an expanded access program. CONCLUSIONS: In this case, use of avacopan in a pediatric patient with C3GN was safe and well tolerated. On avacopan, the patient was able to discontinue mycophenolate mofetil (MMF) while maintaining remission.

A self-binding immune complex vaccine elicits strong neutralizing responses against herpes simplex virus in mice.

Introduction: It has been known for over half a century that mixing an antigen with its cognate antibody in an immune complex (IC) can enhance antigen immunogenicity. However, many ICs produce inconsistent immune responses, and the use of ICs in the development new vaccines has been limited despite the otherwise widespread success of antibody-based therapeutics. To address this problem, we designed a self-binding recombinant immune complex (RIC) vaccine which mimics the larger ICs generated during natural infection. Materials and methods: In this study, we created two novel vaccine candidates: 1) a traditional IC targeting herpes simplex virus 2 (HSV-2) by mixing glycoprotein D (gD) with a neutralizing antibody (gD-IC); and 2) an RIC consisting of gD fused to an immunoglobulin heavy chain and then tagged with its own binding site, allowing self-binding (gD-RIC). We characterized the complex size and immune receptor binding characteristics in vitro for each preparation. Then, the in vivo immunogenicity and virus neutralization of each vaccine were compared in mice. Results: gD-RIC formed larger complexes which enhanced C1q receptor binding 25-fold compared to gD-IC. After immunization of mice, gD-RIC elicited up to 1,000-fold higher gD-specific antibody titers compared to traditional IC, reaching endpoint titers of 1:500,000 after two doses without adjuvant. The RIC construct also elicited stronger virus-specific neutralization against HSV-2, as well as stronger cross-neutralization against HSV-1, although the proportion of neutralizing antibodies to total antibodies was somewhat reduced in the RIC group. Discussion: This work demonstrates that the RIC system overcomes many of the pitfalls of traditional IC, providing potent immune responses against HSV-2 gD. Based on these findings, further improvements to the RIC system are discussed. RIC have now been shown to be capable of inducing potent immune responses to a variety of viral antigens, underscoring their broad potential as a vaccine platform.