Factor-inhibiting HIF (FIH) promotes lung cancer progression.

Factor-inhibiting HIF (FIH) is an asparagine hydroxylase that acts on hypoxia-inducible factors (HIFs) to control cellular adaptation to hypoxia. FIH is expressed in several tumor types, but its impact in tumor progression remains largely unexplored. We observed that FIH was expressed on human lung cancer tissue. Deletion of FIH in mouse and human lung cancer cells resulted in an increased glycolytic metabolism, consistent with increased HIF activity. FIH-deficient lung cancer cells exhibited decreased proliferation. Analysis of RNA-Seq data confirmed changes in the cell cycle and survival and revealed molecular pathways that were dysregulated in the absence of FIH, including the upregulation of angiomotin (Amot), a key component of the Hippo tumor suppressor pathway. We show that FIH-deficient tumors were characterized by higher immune infiltration of NK and T cells compared with FIH competent tumor cells. In vivo studies demonstrate that FIH deletion resulted in reduced tumor growth and metastatic capacity. Moreover, high FIH expression correlated with poor overall survival in non-small cell lung cancer (NSCLC). Our data unravel FIH as a therapeutic target for the treatment of lung cancer.

Structural insights into Siglec-15 reveal glycosylation dependency for its interaction with T cells through integrin CD11b.

Sialic acid-binding Ig-like lectin 15 (Siglec-15) is an immune modulator and emerging cancer immunotherapy target. However, limited understanding of its structure and mechanism of action restrains the development of drug candidates that unleash its full therapeutic potential. In this study, we elucidate the crystal structure of Siglec-15 and its binding epitope via co-crystallization with an anti-Siglec-15 blocking antibody. Using saturation transfer-difference nuclear magnetic resonance (STD-NMR) spectroscopy and molecular dynamics simulations, we reveal Siglec-15 binding mode to α(2,3)- and α(2,6)-linked sialic acids and the cancer-associated sialyl-Tn (STn) glycoform. We demonstrate that binding of Siglec-15 to T cells, which lack STn expression, depends on the presence of α(2,3)- and α(2,6)-linked sialoglycans. Furthermore, we identify the leukocyte integrin CD11b as a Siglec-15 binding partner on human T cells. Collectively, our findings provide an integrated understanding of the structural features of Siglec-15 and emphasize glycosylation as a crucial factor in controlling T cell responses.

Therapeutic Targeting of LIF Overcomes Macrophage-mediated Immunosuppression of the Local Tumor Microenvironment.

PURPOSE: Leukemia inhibitory factor (LIF) is a multifunctional cytokine with numerous reported roles in cancer and is thought to drive tumor development and progression. Characterization of LIF and clinical-stage LIF inhibitors would increase our understanding of LIF as a therapeutic target. EXPERIMENTAL DESIGN: We first tested the association of LIF expression with transcript signatures representing multiple processes regulating tumor development and progression. Next, we developed MSC-1, a high-affinity therapeutic antibody that potently inhibits LIF signaling and tested it in immune competent animal models of cancer. RESULTS: LIF was associated with signatures of tumor-associated macrophages (TAM) across 7,769 tumor samples spanning 22 solid tumor indications. In human tumors, LIF receptor was highly expressed within the macrophage compartment and LIF treatment drove macrophages to acquire immunosuppressive capacity. MSC-1 potently inhibited LIF signaling by binding an epitope that overlaps with the gp130 receptor binding site on LIF. MSC-1 showed monotherapy efficacy in vivo and drove TAMs to acquire antitumor and proinflammatory function in syngeneic colon cancer mouse models. Combining MSC-1 with anti-PD1 leads to strong antitumor response and a long-term tumor-free survival in a significant proportion of treated mice. CONCLUSIONS: Overall, our findings highlight LIF as a therapeutic target for cancer immunotherapy.

A flow cytometry-based neutralization assay for simultaneous evaluation of blocking antibodies against SARS-CoV-2 variants.

Vaccines against SARS-CoV-2 have alleviated infection rates, hospitalization and deaths associated with COVID-19. In order to monitor humoral immunity, several serology tests have been developed, but the recent emergence of variants of concern has revealed the need for assays that predict the neutralizing capacity of antibodies in a fast and adaptable manner. Sensitive and fast neutralization assays would allow a timely evaluation of immunity against emerging variants and support drug and vaccine discovery efforts. Here we describe a simple, fast, and cell-free multiplexed flow cytometry assay to interrogate the ability of antibodies to prevent the interaction of Angiotensin-converting enzyme 2 (ACE2) and the receptor binding domain (RBD) of the original Wuhan-1 SARS-CoV-2 strain and emerging variants simultaneously, as a surrogate neutralization assay. Using this method, we demonstrate that serum antibodies collected from representative individuals at different time-points during the pandemic present variable neutralizing activity against emerging variants, such as Omicron BA.1 and South African B.1.351. Importantly, antibodies present in samples collected during 2021, before the third dose of the vaccine was administered, do not confer complete neutralization against Omicron BA.1, as opposed to samples collected in 2022 which show significant neutralizing activity. The proposed approach has a comparable performance to other established surrogate methods such as cell-based assays using pseudotyped lentiviral particles expressing the spike of SARS-CoV-2, as demonstrated by the assessment of the blocking activity of therapeutic antibodies (i.e. Imdevimab) and serum samples. This method offers a scalable, cost effective and adaptable platform for the dynamic evaluation of antibody protection in affected populations against variants of SARS-CoV-2.

High-density binding to Plasmodium falciparum circumsporozoite protein repeats by inhibitory antibody elicited in mouse with human immunoglobulin repertoire.

Antibodies targeting the human malaria parasite Plasmodium falciparum circumsporozoite protein (PfCSP) can prevent infection and disease. PfCSP contains multiple central repeating NANP motifs; some of the most potent anti-infective antibodies against malaria bind to these repeats. Multiple antibodies can bind the repeating epitopes concurrently by engaging into homotypic Fab-Fab interactions, which results in the ordering of the otherwise largely disordered central repeat into a spiral. Here, we characterize IGHV3-33/IGKV1-5-encoded monoclonal antibody (mAb) 850 elicited by immunization of transgenic mice with human immunoglobulin loci. mAb 850 binds repeating NANP motifs with picomolar affinity, potently inhibits Plasmodium falciparum (Pf) in vitro and, when passively administered in a mouse challenge model, reduces liver burden to a similar extent as some of the most potent anti-PfCSP mAbs yet described. Like other IGHV3-33/IGKV1-5-encoded anti-NANP antibodies, mAb 850 primarily utilizes its HCDR3 and germline-encoded aromatic residues to recognize its core NANP motif. Biophysical and cryo-electron microscopy analyses reveal that up to 19 copies of Fab 850 can bind the PfCSP repeat simultaneously, and extensive homotypic interactions are observed between densely-packed PfCSP-bound Fabs to indirectly improve affinity to the antigen. Together, our study expands on the molecular understanding of repeat-induced homotypic interactions in the B cell response against PfCSP for potently protective mAbs against Pf infection.

Neddylation tunes peripheral blood mononuclear cells immune response in COVID-19 patients.

The COVID-19 pandemic caused by SARS-CoV-2 has reached 5.5 million deaths worldwide, generating a huge impact globally. This highly contagious viral infection produces a severe acute respiratory syndrome that includes cough, mucus, fever and pneumonia. Likewise, many hospitalized patients develop severe pneumonia associated with acute respiratory distress syndrome (ARDS), along an exacerbated and uncontrolled systemic inflammation that in some cases induces a fatal cytokine storm. Although vaccines clearly have had a beneficial effect, there is still a high percentage of unprotected patients that develop the pathology, due to an ineffective immune response. Therefore, a thorough understanding of the modulatory mechanisms that regulate the response to SARS-CoV-2 is crucial to find effective therapeutic alternatives. Previous studies describe the relevance of Neddylation in the activation of the immune system and its implications in viral infection. In this context, the present study postulates Neddylation, a reversible ubiquitin-like post-translational modification of proteins that control their stability, localization and activity, as a key regulator in the immune response against SARS-CoV-2. For the first time, we describe an increase in global neddylation levels in COVID-19 in the serum of patients, which is particularly associated with the early response to infection. In addition, the results showed that overactivation of neddylation controls activation, proliferation, and response of peripheral blood mononuclear cells (PBMCs) isolated from COVID-19 patients. Inhibition of neddylation, and the subsequent avoidance of activated PBMCs, reduces cytokine production, mainly IL-6 and MCP-1 and induce proteome modulation, being a critical mechanism and a potential approach to immunomodulate COVID-19 patients.

Hypoxia reduces cell attachment of SARS-CoV-2 spike protein by modulating the expression of ACE2, neuropilin-1, syndecan-1 and cellular heparan sulfate.

A main clinical parameter of COVID-19 pathophysiology is hypoxia. Here we show that hypoxia decreases the attachment of the receptor-binding domain (RBD) and the S1 subunit (S1) of the spike protein of SARS-CoV-2 to epithelial cells. In Vero E6 cells, hypoxia reduces the protein levels of ACE2 and neuropilin-1 (NRP1), which might in part explain the observed reduction of the infection rate. In addition, hypoxia inhibits the binding of the spike to NCI-H460 human lung epithelial cells by decreasing the cell surface levels of heparan sulfate (HS), a known attachment receptor of SARS-CoV-2. This interaction is also reduced by lactoferrin, a glycoprotein that blocks HS moieties on the cell surface. The expression of syndecan-1, an HS-containing proteoglycan expressed in lung, is inhibited by hypoxia on a HIF-1α-dependent manner. Hypoxia or deletion of syndecan-1 results in reduced binding of the RBD to host cells. Our study indicates that hypoxia acts to prevent SARS-CoV-2 infection, suggesting that the hypoxia signalling pathway might offer therapeutic opportunities for the treatment of COVID-19.

Sensitive detection of SARS-CoV-2 seroconversion by flow cytometry reveals the presence of nucleoprotein-reactive antibodies in unexposed individuals.

There is an ongoing need of developing sensitive and specific methods for the determination of SARS-CoV-2 seroconversion. For this purpose, we have developed a multiplexed flow cytometric bead array (C19BA) that allows the identification of IgG and IgM antibodies against three immunogenic proteins simultaneously: the spike receptor-binding domain (RBD), the spike protein subunit 1 (S1) and the nucleoprotein (N). Using different cohorts of samples collected before and after the pandemic, we show that this assay is more sensitive than ELISAs performed in our laboratory. The combination of three viral antigens allows for the interrogation of full seroconversion. Importantly, we have detected N-reactive antibodies in COVID-19-negative individuals. Here we present an immunoassay that can be easily implemented and has superior potential to detect low antibody titers compared to current gold standard serology methods.

Hypoxia Promotes Syndecan-3 Expression in the Tumor Microenvironment.

The syndecan (Sdc) family is comprised of four members of cell surface molecules (Sdc-1 to 4) with different biological functions. Syndecan-3 (Sdc-3) is known to be mainly expressed in the brain and nervous tissue and plays a key role in development, cell adhesion, and migration. Recent studies point to important roles for Sdc-3 in inflammatory disease, but the patterns of expression and significance of Sdc-3 in cancer remains unexplored. Here we show that Sdc-3 expression is upregulated on several cancer types, especially in solid tumors that are known to be hypoxic. The Cancer Genome Atlas program (TCGA) data demonstrated that Sdc-3 expression in the tumor microenvironment positively correlates with a hypoxia gene signature. To confirm a potential cause-effect, we performed experiments with tumor cell lines showing increased expression upon in vitro exposure to 1% oxygen or dimethyloxalylglycine, an inhibitor of prolyl hydroxylases, indicating that Sdc-3 expression is promoted by hypoxia inducible factors (HIFs). HIF-1α was responsible for this upregulation as confirmed by CRISPR-engineered tumor cells. Using single-cell RNA sequencing data of melanoma patients, we show that Sdc-3 is expressed on tumor associated macrophages, cancer cells, and endothelial cells. Syndecan-3 expression positively correlated with a macrophage gene signature across several TCGA cancer types. In vitro experiments demonstrated that hypoxia (1% oxygen) or treatment with IFN-γ stimulate Sdc-3 expression on RAW-264.7 derived macrophages, linking Sdc-3 expression to a proinflammatory response. Syndecan-3 expression correlates with a better patient overall survival in hypoxic melanoma tumors.

SARS-CoV-2 Infection Dysregulates the Metabolomic and Lipidomic Profiles of Serum.

COVID-19 is a systemic infection that exerts significant impact on the metabolism. Yet, there is little information on how SARS-CoV-2 affects metabolism. Using NMR spectroscopy, we measured the metabolomic and lipidomic serum profile from 263 (training cohort) + 135 (validation cohort) symptomatic patients hospitalized after positive PCR testing for SARS-CoV-2 infection. We also established the profiles of 280 persons collected before the coronavirus pandemic started. Principal-component analysis discriminated both cohorts, highlighting the impact that the infection has on overall metabolism. The lipidomic analysis unraveled a pathogenic redistribution of the lipoprotein particle size and composition to increase the atherosclerotic risk. In turn, metabolomic analysis reveals abnormally high levels of ketone bodies (acetoacetic acid, 3-hydroxybutyric acid, and acetone) and 2-hydroxybutyric acid, a readout of hepatic glutathione synthesis and marker of oxidative stress. Our results are consistent with a model in which SARS-CoV-2 infection induces liver damage associated with dyslipidemia and oxidative stress.

Structural Characterization of N-Linked Glycans in the Receptor Binding Domain of the SARS-CoV-2 Spike Protein and their Interactions with Human Lectins.

The glycan structures of the receptor binding domain of the SARS-CoV2 spike glycoprotein expressed in human HEK293F cells have been studied by using NMR. The different possible interacting epitopes have been deeply analysed and characterized, providing evidence of the presence of glycan structures not found in previous MS-based analyses. The interaction of the RBD 13 C-labelled glycans with different human lectins, which are expressed in different organs and tissues that may be affected during the infection process, has also been evaluated by NMR. In particular, 15 N-labelled galectins (galectins-3, -7 and -8 N-terminal), Siglecs (Siglec-8, Siglec-10), and C-type lectins (DC-SIGN, MGL) have been employed. Complementary experiments from the glycoprotein perspective or from the lectin's point of view have permitted to disentangle the specific interacting epitopes in each case. Based on these findings, 3D models of the interacting complexes have been proposed.

A high-affinity antibody against the CSP N-terminal domain lacks Plasmodium falciparum inhibitory activity.

Malaria is a global health concern, and research efforts are ongoing to develop a superior vaccine to RTS,S/AS01. To guide immunogen design, we seek a comprehensive understanding of the protective humoral response against Plasmodium falciparum (Pf) circumsporozoite protein (PfCSP). In contrast to the well-studied responses to the repeat region and the C-terminus, the antibody response against the N-terminal domain of PfCSP (N-CSP) remains obscure. Here, we characterized the molecular recognition and functional efficacy of the N-CSP-specific monoclonal antibody 5D5. The crystal structure at 1.85-Å resolution revealed that 5D5 binds an α-helical epitope in N-CSP with high affinity through extensive shape and charge complementarity and the unusual utilization of an antibody N-linked glycan. Nevertheless, functional studies indicated low 5D5 binding to live Pf sporozoites and lack of sporozoite inhibition in vitro and in vivo. Overall, our data do not support the inclusion of the 5D5 N-CSP epitope into the next generation of CSP-based vaccines.

Evolution of protective human antibodies against Plasmodium falciparum circumsporozoite protein repeat motifs.

The circumsporozoite protein of the human malaria parasite Plasmodium falciparum (PfCSP) is the main target of antibodies that prevent the infection and disease, as shown in animal models. However, the limited efficacy of the PfCSP-based vaccine RTS,S calls for a better understanding of the mechanisms driving the development of the most potent human PfCSP antibodies and identification of their target epitopes. By characterizing 200 human monoclonal PfCSP antibodies induced by sporozoite immunization, we establish that the most potent antibodies bind around a conserved (N/D)PNANPN(V/A) core. High antibody affinity to the core correlates with protection from parasitemia in mice and evolves around the recognition of NANP motifs. The data suggest that the rational design of a next-generation PfCSP vaccine that elicits high-affinity antibody responses against the core epitope will promote the induction of protective humoral immune responses.

Potent antibody lineage against malaria transmission elicited by human vaccination with Pfs25.

Transmission-blocking vaccines have the potential to be key contributors to malaria elimination. Such vaccines elicit antibodies that inhibit parasites during their development in Anopheles mosquitoes, thus breaking the cycle of transmission. To date, characterization of humoral responses to Plasmodium falciparum transmission-blocking vaccine candidate Pfs25 has largely been conducted in pre-clinical models. Here, we present molecular analyses of human antibody responses generated in a clinical trial evaluating Pfs25 vaccination. From a collection of monoclonal antibodies with transmission-blocking activity, we identify the most potent transmission-blocking antibody yet described against Pfs25; 2544. The interactions of 2544 and three other antibodies with Pfs25 are analyzed by crystallography to understand structural requirements for elicitation of human transmission-blocking responses. Our analyses provide insights into Pfs25 immunogenicity and epitope potency, and detail an affinity maturation pathway for a potent transmission-blocking antibody in humans. Our findings can be employed to guide the design of improved malaria transmission-blocking vaccines.

Antihomotypic affinity maturation improves human B cell responses against a repetitive epitope.

Affinity maturation selects B cells expressing somatically mutated antibody variants with improved antigen-binding properties to protect from invading pathogens. We determined the molecular mechanism underlying the clonal selection and affinity maturation of human B cells expressing protective antibodies against the circumsporozoite protein of the malaria parasite Plasmodium falciparum (PfCSP). We show in molecular detail that the repetitive nature of PfCSP facilitates direct homotypic interactions between two PfCSP repeat-bound monoclonal antibodies, thereby improving antigen affinity and B cell activation. These data provide a mechanistic explanation for the strong selection of somatic mutations that mediate homotypic antibody interactions after repeated parasite exposure in humans. Our findings demonstrate a different mode of antigen-mediated affinity maturation to improve antibody responses to PfCSP and presumably other repetitive antigens.

Rare PfCSP C-terminal antibodies induced by live sporozoite vaccination are ineffective against malaria infection.

Antibodies against the central repeat of the Plasmodium falciparum (Pf) circumsporozoite protein (CSP) inhibit parasite activity and correlate with protection from malaria. However, the humoral response to the PfCSP C terminus (C-PfCSP) is less well characterized. Here, we describe B cell responses to C-PfCSP from European donors who underwent immunization with live Pf sporozoites (PfSPZ Challenge) under chloroquine prophylaxis (PfSPZ-CVac), and were protected against controlled human malaria infection. Out of 215 PfCSP-reactive monoclonal antibodies, only two unique antibodies were specific for C-PfCSP, highlighting the rare occurrence of C-PfCSP-reactive B cells in PfSPZ-CVac-induced protective immunity. These two antibodies showed poor sporozoite binding and weak inhibition of parasite traversal and development, and did not protect mice from infection with PfCSP transgenic Plasmodium berghei sporozoites. Structural analyses demonstrated that one antibody interacts with a polymorphic region overlapping two T cell epitopes, suggesting that variability in C-PfCSP may benefit parasite escape from humoral and cellular immunity. Our data identify important features underlying C-PfCSP shortcomings as a vaccine target.

Natural Parasite Exposure Induces Protective Human Anti-Malarial Antibodies.

Antibodies against the NANP repeat of circumsporozoite protein (CSP), the major surface antigen of Plasmodium falciparum (Pf) sporozoites, can protect from malaria in animal models but protective humoral immunity is difficult to induce in humans. Here we cloned and characterized rare affinity-matured human NANP-reactive memory B cell antibodies elicited by natural Pf exposure that potently inhibited parasite transmission and development in vivo. We unveiled the molecular details of antibody binding to two distinct protective epitopes within the NANP repeat. NANP repeat recognition was largely mediated by germline encoded and immunoglobulin (Ig) heavy-chain complementarity determining region 3 (HCDR3) residues, whereas affinity maturation contributed predominantly to stabilizing the antigen-binding site conformation. Combined, our findings illustrate the power of exploring human anti-CSP antibody responses to develop tools for malaria control in the mammalian and the mosquito vector and provide a molecular basis for the structure-based design of next-generation CSP malaria vaccines.

Molecular definition of multiple sites of antibody inhibition of malaria transmission-blocking vaccine antigen Pfs25.

The Plasmodium falciparum Pfs25 protein (Pfs25) is a leading malaria transmission-blocking vaccine antigen. Pfs25 vaccination is intended to elicit antibodies that inhibit parasite development when ingested by Anopheles mosquitoes during blood meals. The Pfs25 three-dimensional structure has remained elusive, hampering a molecular understanding of its function and limiting immunogen design. We report six crystal structures of Pfs25 in complex with antibodies elicited by immunization via Pfs25 virus-like particles in human immunoglobulin loci transgenic mice. Our structural findings reveal the fine specificities associated with two distinct immunogenic sites on Pfs25. Importantly, one of these sites broadly overlaps with the epitope of the well-known 4B7 mouse antibody, which can be targeted simultaneously by antibodies that target a non-overlapping site to additively increase parasite inhibition. Our molecular characterization of inhibitory antibodies informs on the natural disposition of Pfs25 on the surface of ookinetes and provides the structural blueprints to design next-generation immunogens.

HLA Epitopes and Shared Molecular Eplet Characterization and Their Implication on Transplant Outcome: The Experience of One Center.

We report three interesting cases concerning antibody-mediated rejection (AMR), associated or not with anti-donor-specific antibodies, and detection of implicated molecular epitopes. The first report presents a case of intra-allele sensitization. The second case presents an interesting case concerning Luminex mean fluorescence intensity (MFI) levels considered to be low risk antibodies (<1000), but producing AMR. The third case occurred after a second kidney transplantation mediated by antibodies directed against HLA-C antigens (MFI<1000) in the previous transplantation (which was considered to be an indicator of low-risk of AMR).

HLA-C antibodies are associated with irreversible rejection in kidney transplantation: Shared molecular eplets characterization.

We report an interesting case concerning an irreversible antibody-mediated rejection (AMR), associated with anti-HLA-C DSA, which occurred after a second kidney transplantation despite having determined a low number of antibodies directed against HLA-C antigens (MFI<1000) in the previous transplantation (which was then considered to be an indicator of low risk of AMR). A 63-year-old woman was re-transplanted with pre-transplant (PrT) sensitization. On day 7 post-transplantation, oligoanuria occurred and increased MFIs for the detected PrT antibodies and other antibodies (non-detected or detected with very low PrT MFI) were observed. SAB assay also showed antibodies against the second donor HLA-C mismatches and other HLA-C antigens. Nephrologists suspected AMR and the patient was therefore treated with methylprednisolone/plasmapheresis/IVIG/anti-CD20 without improvement, which led to transplantectomy. Histologic analysis confirmed acute AMR. Interestingly, it was possible to define exactly the potential immunizing epitopes whose recognition determines the specific antibody production. So, 1st donor DSAs (detected PrT with low MFI), 2nd donor DSAs (detected PTP), and non-DSA detected PTP have several shared eplets, being the 11AVR eplet the only one present on all alleles. Thus, the recognition of 11AVR eplet in the first transplant modeled the patient's antibody response. Therefore, we propose that donor HLA-C typing should always be performed for recipients with anti-HLA-C antibodies, and specific shared-eplets should be investigated in order to determine previous transplant mismatches.