Recognition and inhibition of SARS-CoV-2 by humoral innate immunity pattern recognition molecules.

The humoral arm of innate immunity includes diverse molecules with antibody-like functions, some of which serve as disease severity biomarkers in coronavirus disease 2019 (COVID-19). The present study was designed to conduct a systematic investigation of the interaction of human humoral fluid-phase pattern recognition molecules (PRMs) with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Of 12 PRMs tested, the long pentraxin 3 (PTX3) and mannose-binding lectin (MBL) bound the viral nucleocapsid and spike proteins, respectively. MBL bound trimeric spike protein, including that of variants of concern (VoC), in a glycan-dependent manner and inhibited SARS-CoV-2 in three in vitro models. Moreover, after binding to spike protein, MBL activated the lectin pathway of complement activation. Based on retention of glycosylation sites and modeling, MBL was predicted to recognize the Omicron VoC. Genetic polymorphisms at the MBL2 locus were associated with disease severity. These results suggest that selected humoral fluid-phase PRMs can play an important role in resistance to, and pathogenesis of, COVID-19, a finding with translational implications.

Fasciola hepatica is refractory to complement killing by preventing attachment of mannose binding lectin (MBL) and inhibiting MBL-associated serine proteases (MASPs) with serpins.

The complement system is a first-line innate host immune defence against invading pathogens. It is activated via three pathways, termed Classical, Lectin and Alternative, which are mediated by antibodies, carbohydrate arrays or microbial liposaccharides, respectively. The three complement pathways converge in the formation of C3-convertase followed by the assembly of a lethal pore-like structure, the membrane attack complex (MAC), on the pathogen surface. We found that the infectious stage of the helminth parasite Fasciola hepatica, the newly excysted juvenile (NEJ), is resistant to the damaging effects of complement. Despite being coated with mannosylated proteins, the main initiator of the Lectin pathway, the mannose binding lectin (MBL), does not bind to the surface of live NEJ. In addition, we found that recombinantly expressed serine protease inhibitors secreted by NEJ (rFhSrp1 and rFhSrp2) selectively prevent activation of the complement via the Lectin pathway. Our experiments demonstrate that rFhSrp1 and rFhSrp2 inhibit native and recombinant MBL-associated serine proteases (MASPs), impairing the primary step that mediates C3b and C4b deposition on the NEJ surface. Indeed, immunofluorescence studies show that MBL, C3b, C4b or MAC are not deposited on the surface of NEJ incubated in normal human serum. Taken together, our findings uncover new means by which a helminth parasite prevents the activation of the Lectin complement pathway to become refractory to killing via this host response, in spite of presenting an assortment of glycans on their surface.

Mannose-binding lectin and complement mediate follicular localization and enhanced immunogenicity of diverse protein nanoparticle immunogens.

Nanoparticle (NP) vaccine formulations promote immune responses through multiple mechanisms. We recently reported that mannose-binding lectin (MBL) triggers trafficking of glycosylated HIV Env-immunogen NPs to lymph node follicles. Here, we investigate effects of MBL and complement on NP forms of HIV and other viral antigens. MBL recognition of oligomannose on gp120 nanoparticles significantly increases antigen accumulation in lymph nodes and antigen-specific germinal center (GC) responses. MBL and complement also mediate follicular trafficking and enhance GC responses to influenza, HBV, and HPV particulate antigens. Using model protein nanoparticles bearing titrated levels of glycosylation, we determine that mannose patches at a minimal density of 2.1 × 10-3 mannose patches/nm2 are required to trigger follicular targeting, which increases with increasing glycan density up to at least ∼8.2 × 10-3 patches/nm2. Thus, innate immune recognition of glycans has a significant impact on humoral immunity, and these findings provide a framework for engineering glycan recognition to optimize vaccine efficacy.

Mannose-binding lectin promotes blood-brain barrier breakdown and exacerbates axonal damage after traumatic brain injury in mice.

Leukocyte infiltration and blood-brain barrier breakdown contribute to secondary brain damage after traumatic brain injury (TBI). TBI induces neuroimmune responses triggering pathogenic complement activation through different pathways, including the lectin pathway. We investigated mechanisms underlying mannose-binding lectin (MBL)-mediated brain damage focusing on neutrophil infiltration and blood-brain barrier breakdown in a TBI mouse model. Wild type mice and MBL-/- null mice were subjected to controlled cortical impact. We studied neutrophil infiltration and regional localization by confocal microscopy 1, 4 and 15 days post-trauma, and investigated neutrophil extracellular trap (NET) formation. By immunofluorescence and/or Western blotting in various brain regions we studied the presence of fibrin(ogen), pentraxin-3, albumin and immunoglobulin G. Finally, we studied neurofilament proteins, synaptophysin, and αII-spectrin, and assessed white matter content in the injured tissue. TBI triggered an acute wave of neutrophil infiltration at day 1 followed by a more discrete persistence of neutrophils in the injured tissue at least until day 15. We detected the presence of NETs and pentraxin-3 in the injured tissue, as well as accumulation of fibrin(ogen), increased blood-brain barrier permeability, and neurofilament, synaptophysin and white matter loss, and calpain-mediated αII spectrin breakdown. MBL-/- mice showed reduced number of Ly6G+ neutrophils 4 days after TBI, lower accumulation of pentraxin-3 and fibrin(ogen) in the injured tissue, reduced global plasma protein extravasation, and better preservation of axonal and white matter integrity. These results show that MBL participates in secondary neutrophil accumulation and blood-brain barrier breakdown, and promotes axonal and white matter damage after TBI in mice.

Dengue and the Lectin Pathway of the Complement System.

Dengue is a mosquito-borne viral disease causing significant health and economic burdens globally. The dengue virus (DENV) comprises four serotypes (DENV1-4). Usually, the primary infection is asymptomatic or causes mild dengue fever (DF), while secondary infections with a different serotype increase the risk of severe dengue disease (dengue hemorrhagic fever, DHF). Complement system activation induces inflammation and tissue injury, contributing to disease pathogenesis. However, in asymptomatic or primary infections, protective immunity largely results from the complement system's lectin pathway (LP), which is activated through foreign glycan recognition. Differences in N-glycans displayed on the DENV envelope membrane influence the lectin pattern recognition receptor (PRR) binding efficiency. The important PRR, mannan binding lectin (MBL), mediates DENV neutralization through (1) a complement activation-independent mechanism via direct MBL glycan recognition, thereby inhibiting DENV attachment to host target cells, or (2) a complement activation-dependent mechanism following the attachment of complement opsonins C3b and C4b to virion surfaces. The serum concentrations of lectin PRRs and their polymorphisms influence these LP activities. Conversely, to escape the LP attack and enhance the infectivity, DENV utilizes the secreted form of nonstructural protein 1 (sNS1) to counteract the MBL effects, thereby increasing viral survival and dissemination.

An inexpensive point-of-care immunochromatographic test for Talaromyces marneffei infection based on the yeast phase specific monoclonal antibody 4D1 and Galanthus nivalis agglutinin.

Talaromyces marneffei is a thermally dimorphic fungus that causes opportunistic systemic mycoses in patients with AIDS or other immunodeficiency syndromes. The purpose of this study was to develop an immunochromatographic strip test (ICT) based on a solid phase sandwich format immunoassay for the detection of T. marneffei antigens in clinical urine specimens. The T. marneffei yeast phase specific monoclonal antibody 4D1 (MAb4D1) conjugated with colloidal gold nanoparticle was used as a specific signal reporter. Galanthus nivalis Agglutinin (GNA) was adsorbed onto nitrocellulose membrane to serve as the test line. Similarly, a control line was created above the test line by immobilization of rabbit anti-mouse IgG. The immobilized GNA served as capturing molecule and as non-immune mediated anti-terminal mannose of T. marneffei antigenic mannoprotein. The MAb4D1-GNA based ICT showed specific binding activity with yeast phase antigen of T. marneffei, and it did not react with other common pathogenic fungal antigens. The limit of detection of this ICT for T. marneffei antigen spiked in normal urine was approximately 0.6 µg/ml. The diagnostic performance of the ICT was validated using 341 urine samples from patents with culture- confirmed T. marneffei infection and from a control group of healthy individuals and patients with other infections in an endemic area. The ICT exhibited 89.47% sensitivity, 100% specificity, and 97.65% accuracy. Our results demonstrate that the urine-based GNA-MAb4D1 based ICT produces a visual result within 30 minutes and that the test is highly specific for the diagnosis of T. marneffei infection. The findings validate the deployment of the ICT for clinical use.

Why does SARS-CoV-2 hit in different ways? Host genetic factors can influence the acquisition or the course of COVID-19.

The identification of high-risk factors for the infection by SARS-CoV-2 and the negative outcome of COVID-19 is crucial. The genetic background of the host might account for individual responses to SARS-CoV-2 infection besides age and comorbidities. A list of candidate polymorphisms is needed to drive targeted screens, given the existence of frequent polymorphisms in the general population. We carried out text mining in the scientific literature to draw up a list of genes referable to the term "SARS-CoV*". We looked for frequent mutations that are likely to affect protein function in these genes. Ten genes, mostly involved in innate immunity, and thirteen common variants were identified, for some of these the involvement in COVID-19 is supported by publicly available epidemiological data. We looked for available data on the population distribution of these variants and we demonstrated that the prevalence of five of them, Arg52Cys (rs5030737), Gly54Asp (rs1800450) and Gly57Glu (rs1800451) in MBL2, Ala59Thr (rs25680) in CD27, and Val197Met (rs12329760) in TMPRSS2, correlates with the number of cases and/or deaths of COVID-19 observed in different countries. The association of the TMPRSS2 variant provides epidemiological evidence of the usefulness of transmembrane protease serine 2 inhibitors for the cure of COVID-19. The identified genetic variants represent a basis for the design of a cost-effective assay for population screening of genetic risk factors in the COVID-19 pandemic.

Interindividual immunogenic variants: Susceptibility to coronavirus, respiratory syncytial virus and influenza virus.

The coronavirus disease (Covid-19) pandemic is the most serious event of the year 2020, causing considerable global morbidity and mortality. The goal of this review is to provide a comprehensive summary of reported associations between inter-individual immunogenic variants and disease susceptibility or symptoms caused by the coronavirus strains severe acute respiratory syndrome-associated coronavirus, severe acute respiratory syndrome-associated coronavirus-2, and two of the main respiratory viruses, respiratory syncytial virus and influenza virus. The results suggest that the genetic background of the host could affect the levels of proinflammatory and anti-inflammatory cytokines and might modulate the progression of Covid-19 in affected patients. Notably, genetic variations in innate immune components such as toll-like receptors and mannose-binding lectin 2 play critical roles in the ability of the immune system to recognize coronavirus and initiate an early immune response to clear the virus and prevent the development of severe symptoms. This review provides promising clues related to the potential benefits of using immunotherapy and immune modulation for respiratory infectious disease treatment in a personalized manner.

Deficiency of mannose-binding lectin is a risk of Pneumocystis jirovecii pneumonia in a natural history cohort of people living with HIV/AIDS in Northern Thailand.

BACKGROUND: Mannose-binding lectin (MBL) plays a pivotal role in innate immunity; however, its impact on susceptibility to opportunistic infections (OIs) has not yet been examined in a natural history cohort of people living with HIV/AIDS. METHODS: We used archived samples to analyze the association between MBL expression types and risk of major OIs including Pneumocystis jirovecii pneumonia (PCP), cryptococcosis, talaromycosis, toxoplasmosis, and tuberculosis in a prospective cohort in Northern Thailand conducted from 1 July 2000 to 15 October 2002 before the national antiretroviral treatment programme was launched. RESULTS: Of 632 patients, PCP was diagnosed in 96 (15.2%) patients, including 45 patients with new episodes during the follow-up period (1006.5 person-years). The total history of PCP was significantly associated with low MBL expression type: high/intermediate (81/587, 13.8%), low (10/33, 30.3%) and deficient (5/12, 41.7%) (p = 0.001), whereas the history of other OIs showed no relation with any MBL expression type. Kaplan-Meier analysis (n = 569; log-rank p = 0.011) and Cox's proportional hazards model revealed that deficient genotype dramatically increased the risk of PCP, which is independent upon sex, age, CD4 count, HIV-1 viral load and hepatitis B and C status (adjusted hazard ratio 7.93, 95% confidence interval 2.19-28.67, p = 0.002). CONCLUSIONS: Deficiency of MBL expression is a strong risk factor determining the incidence of PCP but not other major OIs.

The Influence of the Lectin Pathway of Complement Activation on Infections of the Respiratory System.

Lung diseases are among the leading causes of morbidity and mortality. Complement activation may prevent a variety of respiratory infections, but on the other hand, could exacerbate tissue damage or contribute to adverse side effects. In this review, the associations of factors specific for complement activation via the lectin pathway (LP) with infections of the respiratory system, from birth to adulthood, are discussed. The most extensive data concern mannose-binding lectin (MBL) which together with other collectins (collectin-10, collectin-11) and the ficolins (ficolin-1, ficolin-2, ficolin-3) belong to pattern-recognition molecules (PRM) specific for the LP. Those PRM form complexes with MBL-associated serine proteases (MASP-1, MASP-2, MASP-3) and related non-enzymatic factors (MAp19, MAp44). Beside diseases affecting humanity for centuries like tuberculosis or neonatal pneumonia, some recently published data concerning COVID-19 are summarized.

Mannose-Binding Lectin Possesses Agglutination Activity and Promotes Opsonophagocytosis of Macrophages with Calreticulin Interaction in an Early Vertebrate.

The innate immune system is an ancient defense system in the process of biological evolution, which can quickly and efficiently resist pathogen infection. In mammals, mannose-binding lectin (MBL) is a key molecule in the innate immune and plays an essential role in the first line of host defense against pathogenic bacteria. However, the evolutionary origins and ancient roles of immune defense of MBL and its mechanism in clearance of microbial pathogens are still unclear, especially in early vertebrates. In this study, Oreochromis niloticus MBL (OnMBL) was successfully isolated and purified from the serum of Nile tilapia (O. niloticus). The OnMBL was able to bind and agglutinate with two important pathogens of tilapia, Streptococcus agalactiae and Aeromonas hydrophila Interestingly, the OnMBL was able to significantly inhibit the proliferation of pathogenic bacteria and reduce the inflammatory response. Upon bacterial challenge, the downregulation of OnMBL expression by RNA interference could lead to rapid proliferation of the pathogenic bacteria, ultimately resulting in tilapia death. However, the phenotype was rescued by reinjection of the OnMBL, which restored the healthy status of the knockdown tilapia. Moreover, a mechanistic analysis revealed that the OnMBL could clear pathogenic bacteria by collaborating with cell-surface calreticulin to facilitate phagocytosis in a complement activation-independent manner. To our knowledge, these results provide the first evidence on the antibacterial response mechanism of MBL performing evolutionary conserved function to promote opsonophagocytosis of macrophages in early vertebrates and reveals new insights into the understanding of the evolutionary origins and ancient roles basis of the C-type lectins in the innate immune defense.

MAp34 Regulates the Non-specific Cell Immunity of Monocytes/Macrophages and Inhibits the Lectin Pathway of Complement Activation in a Teleost Fish.

The lectin pathway of the complement system is one of the main components of innate immunity, which plays a pivotal role in the defense against infectious microorganisms and maintains immune homeostasis. However, its control mechanisms remain unclear in teleost fish. In this study, we described the identification and functional characterization of a mannose-binding lectin associated protein MAp34 (OnMAp34) from Nile tilapia (Oreochromis niloticus) at molecular, cellular, and protein levels. The open reading frame (ORF) of OnMAp34 is 918 bp of nucleotide sequence encoding a polypeptide of 305 amino acids. The deduced amino acid sequence has three characteristic structures, including two C1r/C1s-Uegf-BMP domains (CUB) and one epidermal growth factor domain (EGF). Expression analysis revealed that the OnMAp34 was highly expressed in the liver and widely existed in other examined tissues. In addition, the mRNA and protein expression levels of OnMAp34 were remarkably altered upon infection with Streptococcus agalactiae and Aeromonas hydrophila in vivo and in vitro. Further, we found that the OnMAp34 could participate in the non-specific cellular immune defense, including the regulation of inflammation, migration, and enhancement of phagocytosis of monocytes/macrophages. Moreover, the OnMAp34 could compete with OnMASPs to combine OnMBL and inhibit the lectin pathway of complement activation. Overall, our results provide new insights into the understanding of MAp34 as a potent regulator in the lectin complement pathway and non-specific cell immunity in an early vertebrate.

Complement Activation and Thrombin Generation by MBL Bound to ß2-Glycoprotein I.

ß2-Glycoprotein I (ß2-GPI) is an abundant plasma glycoprotein with unknown physiological function and is currently recognized as the main target of antiphospholipid Abs responsible for complement activation and vascular thrombosis in patients with antiphospholipid syndrome (APS). In this study, we provide evidence that mannose-binding lectin (MBL) binds to ß2-GPI in Ca++ and a dose-dependent manner and that this interaction activates complement and promotes complement-dependent thrombin generation. Surprisingly, a significant binding was observed between MBL and isolated domains II and IV of ß2-GPI, whereas the carbohydrate chains, domain I and domain V, were not involved in the interaction, documenting a noncanonical binding mode between MBL and ß2-GPI. Importantly, this interaction may occur on endothelial cells because binding of MBL to ß2-GPI was detected on the surface of HUVECs, and colocalization of MBL with ß2-GPI was observed on the endothelium of a biopsy specimen of a femoral artery from an APS patient. Because ß2-GPI-mediated MBL-dependent thrombin generation was increased after priming the endothelium with TNF-α, our data suggests that this mechanism could play an important yet unrecognized role under physiological conditions and may be upregulated in pathological situations. Moreover, the complement activation and the procoagulant effects of the ß2-GPI/MBL complex may contribute to amplify similar activities of anti-ß2-GPI Abs in APS and possibly act independently of Abs, raising the issue of developing appropriate therapies to avoid recurrences and disability in patients at risk for these clinical conditions.

Functional prediction and comparative population analysis of variants in genes for proteases and innate immunity related to SARS-CoV-2 infection.

New coronavirus SARS-CoV-2 is capable to infect humans and cause a novel disease COVID-19. Aiming to understand a host genetic component of COVID-19, we focused on variants in genes encoding proteases and genes involved in innate immunity that could be important for susceptibility and resistance to SARS-CoV-2 infection. Analysis of sequence data of coding regions of FURIN, PLG, PRSS1, TMPRSS11a, MBL2 and OAS1 genes in 143 unrelated individuals from Serbian population identified 22 variants with potential functional effect. In silico analyses (PolyPhen-2, SIFT, MutPred2 and Swiss-Pdb Viewer) predicted that 10 variants could impact the structure and/or function of proteins. These protein-altering variants (p.Gly146Ser in FURIN; p.Arg261His and p.Ala494Val in PLG; p.Asn54Lys in PRSS1; p.Arg52Cys, p.Gly54Asp and p.Gly57Glu in MBL2; p.Arg47Gln, p.Ile99Val and p.Arg130His in OAS1) may have predictive value for inter-individual differences in the response to the SARS-CoV-2 infection. Next, we performed comparative population analysis for the same variants using extracted data from the 1000 Genomes project. Population genetic variability was assessed using delta MAF and Fst statistics. Our study pointed to 7 variants in PLG, TMPRSS11a, MBL2 and OAS1 genes with noticeable divergence in allelic frequencies between populations worldwide. Three of them, all in MBL2 gene, were predicted to be damaging, making them the most promising population-specific markers related to SARS-CoV-2 infection. Comparing allelic frequencies between Serbian and other populations, we found that the highest level of genetic divergence related to selected loci was observed with African, followed by East Asian, Central and South American and South Asian populations. When compared with European populations, the highest divergence was observed with Italian population. In conclusion, we identified 4 variants in genes encoding proteases (FURIN, PLG and PRSS1) and 6 in genes involved in the innate immunity (MBL2 and OAS1) that might be relevant for the host response to SARS-CoV-2 infection.

A novel complement inhibitor sMAP-FH targeting both the lectin and alternative complement pathways.

Inhibition of the complement activation has emerged as an option for treatment of a range of diseases. Activation of the lectin and alternative pathways (LP and AP, respectively) contribute to the deterioration of conditions in certain diseases such as ischemia-reperfusion injuries and age-related macular degeneration (AMD). In the current study, we generated dual complement inhibitors of the pathways MAp44-FH and sMAP-FH by fusing full-length MAp44 or small mannose-binding lectin-associated protein (sMAP), LP regulators, with the N-terminal five short consensus repeat (SCR) domains of complement factor H (SCR1/5-FH), an AP regulator. The murine forms of both fusion proteins formed a complex with endogenous mannose-binding lectin (MBL) or ficolin A in the circulation when administered in mice intraperitoneally. Multiple complement activation assays revealed that sMAP-FH had significantly higher inhibitory effects on activation of the LP and AP in vivo as well as in vitro compared to MAp44-FH. Human form of sMAP-FH also showed dual inhibitory effects on LP and AP activation in human sera. Our results indicate that the novel fusion protein sMAP-FH inhibits both the LP and AP activation in mice and in human sera, and could be an effective therapeutic agent for diseases in which both the LP and AP activation are significantly involved.

Antimicrobial activity of mannose binding lectin in grass carp (Ctenopharyngodon idella) in vivo and in vitro.

Mannose-binding lectin (MBL) is a crucial pattern recognition receptor in the host innate immune system. Previously, we reported the biological function of Ctenopharyngodon idella MBL (CiMBL) in initiating the lectin pathway of the complement system. In the present study, we further explored its biological function including the agglutinating ability, binding capacity and protective role in vitro and in vivo. After Aeromonas hydrophila infection, western blot analysis revealed that the CiMBL were fluctuated and expressed in the serum and major immune-related tissues. The result of quantitative PCR (qPCR) showed that the recombinant CiMBL (rCiMBL) significantly inhibited the mRNA expression of interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF-α) in liver, spleen and hepatic cells. Due to rCiMBL bound to d-mannose, d-galactose, d-glucose, N-acetyl-d-glucosamine (GlcNAc), lipopolysaccharide (LPS), peptidoglycan (PGN) and Agar in the presence of Ca2+, herein gram-positive (Staphylococcus aureus and Micrococcus luteus) and gram-negative (A. hydrophila and Vibrio anguillarum) bacteria were agglutinated by rCiMBL in a Ca2+-dependent manner. More importantly, rCiMBL enhanced the survival rate of grass carp following bacterial infection. Overall, the results provide an evidence that CiMBL can protect grass carp against A. hydrophila infection in aquaculture.

Specific contribution of mannose-binding lectin murine isoforms to brain ischemia/reperfusion injury.

Mannose-binding lectin (MBL), an initiator of the lectin pathway (LP) of complement activation, is detrimental in ischemic stroke, as shown in clinical studies and rodent models. Whereas humans have one functional MBL protein, rodents have two isoforms, MBL-A and MBL-C, whose functions relative to that of human MBL are unknown. To permit the clinical translation of preclinical data, we aimed to define the specific contributions of MBL-A and MBL-C to brain ischemia. We subjected mice with double (MBL-/-) or single (MBL-A-/- or MBL-C-/-) MBL isoform depletion to transient middle cerebral artery occlusion (tMCAo). MBL-/- mice had fewer neurological deficits and smaller ischemic lesions than WT mice. MBL-A-/- mice had smaller lesions than WT mice and exhibited no significant behavioral defects, whereas MBL-C-/- mice did not differ from WT mice. The induction of Mbl1 and Mbl2 (the MBL-A and MBL-C genes) expression 48 h after tMCAo was similar across genotypes. The time course of Mbl1 and Mbl2 expression in WT ischemic mice showed that Mbl1 activation occurred earlier (24 h) than Mbl2 activation (48 h). The plasma levels of MBL-A and MBL-C in MBL-C-/- and MBL-A-/- mice, respectively, were similar to those in WT mice both at baseline and at 48 h after tMCAo. At 48 h, MBL-A-/- ischemic mice showed higher MBL-C levels in the brain than WT mice. WT and MBL-C-/- ischemic mice had higher LP activity in plasma and, accordingly, higher levels of C3 deposition in the brain than MBL-A-/- and MBL-/- mice. In conclusion, mice with depletion of both MBL isoforms exhibited strong protection from ischemia/reperfusion injury. MBL-A was the main contributor to injury, likely owing to its earlier activation after ischemia and more efficient activation of the complement system than MBL-C.

Neutralizing Antibody Induction by HIV-1 Envelope Glycoprotein SOSIP Trimers on Iron Oxide Nanoparticles May Be Impaired by Mannose Binding Lectin.

We covalently attached human immunodeficiency virus type 1 (HIV-1) Env SOSIP trimers to iron oxide nanoparticles (IO-NPs) to create a particulate immunogen for neutralizing antibody (NAb) induction. The attached trimers, ∼20 per particle, retained native-like antigenicity, judged by reactivity with NAbs and non-NAbs. Bivalent (BG505 and B41) trimer IO-NPs were made, as were IO-NPs displaying B41 trimers carrying a PADRE T-cell helper epitope (TCHE). We immunized mice with B41 soluble or IO-NP trimers after PADRE peptide priming. After two immunizations, IO-NP presentation and the TCHE tag independently and substantially increased anti-trimer antibody responses, but titer differences waned after two further doses. Notable and unexpected findings were that autologous NAbs to the N289 glycan hole epitope were consistently induced in mice given soluble but not IO-NP trimers. Various recombinant mannose binding lectins (MBLs) and MBLs in sera of both murine and human origin bound to soluble and IO-NP trimers. MBL binding occluded the autologous NAb epitope on the B41 IO-NP trimers, which may contribute to its poor immunogenicity. The exposure of a subset of broadly active NAb epitopes was also impaired by MBL binding, which could have substantial implications for the utility of trimer-bearing nanoparticles in general and perhaps also for soluble Env proteins.IMPORTANCE Recombinant trimeric SOSIP proteins are vaccine components intended to induce neutralizing antibodies (NAbs) that prevent cells from infection by human immunodeficiency virus type 1 (HIV-1). A way to increase the strength of antibody responses to these proteins is to present them on the surface of nanoparticles (NPs). We chemically attached about 20 SOSIP trimers to NPs made of iron oxide (IO). The resulting IO-NP trimers had appropriate properties when we studied them in the laboratory but, unexpectedly, were less able to induce NAbs than nonattached trimers when used to immunize mice. We found that mannose binding lectins, proteins naturally present in the serum of mice and other animals, bound strongly to the soluble and IO-NP trimers, blocking access to antibody epitopes in a way that may impede the development of NAb responses. These findings should influence how trimer-bearing NPs of various designs are made and used.

Platelet thrombus formation in eHUS is prevented by anti-MBL2.

E. coli associated Hemolytic Uremic Syndrome (epidemic hemolytic uremic syndrome, eHUS) caused by Shiga toxin-producing bacteria is characterized by thrombocytopenia, microangiopathic hemolytic anemia, and acute kidney injury that cause acute renal failure in up to 65% of affected patients. We hypothesized that the mannose-binding lectin (MBL) pathway of complement activation plays an important role in human eHUS, as we previously demonstrated that injection of Shiga Toxin-2 (Stx-2) led to fibrin deposition in mouse glomeruli that was blocked by co-injection of the anti-MBL-2 antibody 3F8. However, the markers of platelet thrombosis in affected mouse glomeruli were not delineated. To investigate the effect of 3F8 on markers of platelet thrombosis, we used kidney sections from our mouse model (MBL-2+/+ Mbl-A/C-/-; MBL2 KI mouse). Mice in the control group received PBS, while mice in a second group received Stx-2, and those in a third group received 3F8 and Stx-2. Using double immunofluorescence (IF) followed by digital image analysis, kidney sections were stained for fibrin(ogen) and CD41 (marker for platelets), von-Willebrand factor (marker for endothelial cells and platelets), and podocin (marker for podocytes). Electron microscopy (EM) was performed on ultrathin sections from mice and human with HUS. Injection of Stx-2 resulted in an increase of both fibrin and platelets in glomeruli, while administration of 3F8 with Stx-2 reduced both platelet and fibrin to control levels. EM studies confirmed that CD41-positive objects observed by IF were platelets. The increases in platelet number and fibrin levels by injection of Stx-2 are consistent with the generation of platelet-fibrin thrombi that were prevented by 3F8.

The fungal mycobiome promotes pancreatic oncogenesis via activation of MBL.

Bacterial dysbiosis accompanies carcinogenesis in malignancies such as colon and liver cancer, and has recently been implicated in the pathogenesis of pancreatic ductal adenocarcinoma (PDA)1. However, the mycobiome has not been clearly implicated in tumorigenesis. Here we show that fungi migrate from the gut lumen to the pancreas, and that this is implicated in the pathogenesis of PDA. PDA tumours in humans and mouse models of this cancer displayed an increase in fungi of about 3,000-fold compared to normal pancreatic tissue. The composition of the mycobiome of PDA tumours was distinct from that of the gut or normal pancreas on the basis of alpha- and beta-diversity indices. Specifically, the fungal community that infiltrated PDA tumours was markedly enriched for Malassezia spp. in both mice and humans. Ablation of the mycobiome was protective against tumour growth in slowly progressive and invasive models of PDA, and repopulation with a Malassezia species-but not species in the genera Candida, Saccharomyces or Aspergillus-accelerated oncogenesis. We also discovered that ligation of mannose-binding lectin (MBL), which binds to glycans of the fungal wall to activate the complement cascade, was required for oncogenic progression, whereas deletion of MBL or C3 in the extratumoral compartment-or knockdown of C3aR in tumour cells-were both protective against tumour growth. In addition, reprogramming of the mycobiome did not alter the progression of PDA in Mbl- (also known as Mbl2) or C3-deficient mice. Collectively, our work shows that pathogenic fungi promote PDA by driving the complement cascade through the activation of MBL.