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Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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At least 19 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2021

Scavenger receptor MARCO contributes to macrophage phagocytosis and clearance of tumor cells

Highlights: • The MARCO receptor on macrophages was positively correlated with the ability to phagocyte tumor cells. • Macrophage MARCO affects cytoskeleton rearrangement and pseudopodia formation through the SYK-PI3K-Rac1 pathway. • Integrin is the ligand of MARCO on the tumor cells. Macrophage receptor with collagenous structure (MARCO) is a member of the class A scavenger receptor family which is expressed on the cell surface of macrophages. It is well known that MARCO avidly binds to unopsonized pathogens, leading to its ingestion by macrophages. However, the role of MARCO in the recognition and phagocytosis of tumor cells by macrophages remains poorly understood. In this study, we used lentiviral technology to knockdown and overexpress MARCO and investigated the ability of macrophages to phagocytose tumor cells. Our results showed that MARCO expression was correlated with the ability of macrophages to carry on phagocytosis. MARCO knockdown led to significant decreases in the number of engulfment pseudopodia of macrophages and inhibition of the phagocytosis of tumor cells. On the other hand, MARCO overexpression elevated activity of SYK, PI3K and Rac1 in macrophages, which led to changes in macrophage morphology and enhanced phagocytosis by promoting formation of stress fibers and pseudopodia. By Co-IP analysis we showed that MARCO directly binds to the β5 integrin of SL4 tumor cells. In summary, these results demonstrated the important role for MARCO in demonstrated tumor cells uptake and clearance by macrophages.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

Antibody:CD47 ratio regulates macrophage phagocytosis through competitive receptor phosphorylation

Cancer immunotherapies often modulate macrophage effector function by introducing either targeting antibodies that activate Fcγ receptors (FcγRs) or blocking antibodies that disrupt inhibitory SIRPα-CD47 engagement. However, how these competing signals are integrated is poorly understood, raising questions about how to effectively titrate immune responses. Here, we find that macrophage phagocytic decisions are regulated by the ratio of activating ligand to inhibitory ligand over a broad range of absolute molecular densities. Using both endogenous and chimeric receptors, we show that activating:inhibitory ligand ratios of at least 10:1 are required to promote phagocytosis of model antibody-opsonized CD47-inhibited targets and that lowering that ratio reduces FcγR phosphorylation because of inhibitory phosphatases recruited to CD47-bound SIRPα. We demonstrate that ratiometric signaling is critical for phagocytosis of tumor cells and can be modified by blocking SIRPα, indicating that balancing targeting and blocking antibodies may be important for controlling macrophage phagocytosis in cancer immunotherapy.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Enhanced phagocytosis and complement-mediated killing of Mannheimia haemolytica serotype 1 following in-frame CMP-sialic acid synthetase ( neuA ) gene deletion

ABSTRACT Mannheimia haemolyticais the most significant bacterial pathogen associated with the bovine respiratory disease complex. Although sialic acid is a known virulence factor in other members of Pasteurellaceae, such asHistophilus somniandPasteurella multocida, the significance of sialic acid to the virulence ofM. haemolyticais currently unknown. Therefore, the role of sialic acid as a virulence determinant ofM. haemolyticawas investigated by constructing an in-frameneuA[CMP-N-acetylneuraminic acid (Neu5Ac/sialic) synthetase] mutant, which was shown by high-performance anion exchange chromatographic analysis (HPAEC) to be devoid of sialic acid on the lipopolysaccharide (LPS). Both theneuAmutant and wild-type parent strains exhibited similar growth rates in the growth curve assay. Real-time qPCR and ELISA evaluation showed no differences in proinflammatory cytokine expressions (IL-1β, IL-6, and IL-8) between theneuAmutant and parent strain when peripheral blood mononuclear cells were incubated with LPS. Interestingly, theneuAmutant was three to four logs more sensitive to a whole-blood bacterial killing assay than the parent strain. Similar results were also observed in plasma and serum bacterial killing assays. Flow cytometry analyses showed higher uptake ofneuAmutant by phagocytes, compared to the parent strain, in the whole-blood phagocytosis assay; however, no difference in reactive oxygen species production in neutrophils or monocytes was detected for either strain. Taken together, these results indicate that sialylation ofM. haemolyticaLPS plays a vital role in reducing complement-mediated and phagocytic killing. IMPORTANCE The Gram-negative coccobacillusMannheimia haemolyticais a natural inhabitant of the upper respiratory tract in ruminants and the most common bacterial agent involved in bovine respiratory disease complex development. Key virulence factors harbored byM. haemolyticaare leukotoxin, lipopolysaccharide, capsule, adhesins, and neuraminidase which are involved in evading innate and adaptive immune responses. In this study, we have shown that CMP-sialic acid synthetase (neuA) is necessary for the incorporation of sialic acid onto the membrane, and inactivation ofneuAresults in increased phagocytosis and complement-mediated killing ofM. haemolytica,thus demonstrating that sialylation contributes to the virulence ofM. haemolytica.

Microbiology↗
DOE OSTI2023

Macrophage-derived MLKL in alcohol-associated liver disease: Regulation of phagocytosis

Mixed lineage kinase domain-like pseudokinase (MLKL), a key terminal effector of necroptosis, also plays a role in intracellular vesicle trafficking that is critical for regulating liver inflammation and injury in alcohol-associated liver disease (ALD). Although receptor interacting protein kinase 3 (Rip3) -/- mice are completely protected from ethanol-induced liver injury, Mlkl -/- mice are only partially protected. Therefore, we hypothesized that cell-specific functions of MLKL may contribute to ethanol-induced injury. Bone marrow transplants between Mlkl -/- mice and littermates were conducted to distinguish the role of myeloid versus nonmyeloid Mlkl in the Gao-binge model of ALD. Ethanol-induced hepatic injury, steatosis, and inflammation were exacerbated in Mlkl -/- →wild-type (WT) mice, whereas Mlkl deficiency in nonmyeloid cells (WT→Mlkl -/- ) had no effect on Gao-binge ethanol-induced injury. Importantly, Mlkl deficiency in myeloid cells exacerbated ethanol-mediated bacterial burden and accumulation of immune cells in livers. Mechanistically, challenging macrophages with lipopolysaccharide (LPS) induced signal transducer and activator of transcription 1–mediated expression and phosphorylation of MLKL, as well as translocation and oligomerization of MLKL to intracellular compartments, including phagosomes and lysosomes but not plasma membrane. Importantly, pharmacological or genetic inhibition of MLKL suppressed the phagocytic capability of primary mouse Kupffer cells (KCs) at baseline and in response to LPS with/without ethanol as well as peripheral monocytes isolated from both healthy controls and patients with alcohol-associated hepatitis. Further, in vivo studies revealed that KCs of Mlkl -/- mice phagocytosed fewer bioparticles than KCs of WT mice. Together, these data indicate that myeloid MLKL restricts ethanol-induced liver inflammation and injury by regulating hepatic immune cell homeostasis and macrophage phagocytosis.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2025

Cross-species analysis of FcγRIIa/b (CD32a/b) polymorphisms at position 131: structural and functional insights into the mechanism of IgG- mediated phagocytosis in human and macaque

Introduction Antibodies play a critical role in immunity in part by mediating clearance of pathogens and infected cells by antibody-dependent cellular phagocytosis (ADCP) through engagement of Fc gamma receptors (FcγRs) on innate immune cells. Among these, FcγRIIa (CD32a) is a key activating receptor expressed on macrophages, dendritic cells, and other antigen-presenting cells. Its affinity for IgG and ability to mediate ADCP is influenced by allelic polymorphisms. In humans, a single amino acid polymorphism at position 131, where histidine (H) is substituted with arginine (R), leads to decreased IgG1 and IgG2 subclass binding affinity and, consequently, lower efficiency of phagocytic responses. Rhesus macaques ( Macaca mulatta ), which are widely used as nonhuman primate models, exhibit a similar polymorphism at position 131 of FcγRIIa, but with arginine replaced by proline (P). Here, we investigated structure-function relationships associated with the FcγRIIa polymorphism at position 131 in both species, specifically with respect to IgG1 and IgG2. Methods We determined the structures of complexes formed by each variant with IgG1 Fc and those formed by the higher affinity variant with IgG2 Fc for both species by x-ray crystallography and linked these structures to affinity and activity using SPR and an ADCP assay. We also determined the structure of human inhibitory FcγRIIb (CD32b) in complex with IgG1 Fc by x-ray crystallography. Results Through analysis of these structures, our studies reveal that FcγRIIa engagement is minimally influenced by Fc glycan composition, distinguishing it from FcγRIIIa whose affinity is strongly influenced by glycan-composition. Comparative structures of human and macaque FcγRIIa variants demonstrate species- and allele-specific differences in Fc binding, but our functional assays showed only minimal allele-specific effects in humans. In contrast, allele-specific effects in macaques were highly significant; the macaque P 131 variant showing uniformly reduced IgG affinity. Conclusion These insights highlight fundamental interspecies and allelic distinctions that are critical for interpreting FcγRIIa-mediated effector functions in macaque models and for optimizing translational antibody and vaccine design.

Tolbert, William D.↗
DOE OSTI2022

Polyunsaturated Fatty Acids Mend Macrophage Transcriptome, Glycome, and Phenotype in the Patients with Neurodegenerative Diseases, Including Alzheimer’s Disease

Macrophages of healthy subjects have a pro-resolution phenotype, upload amyloid-β (Aβ) into endosomes, and degrade Aβ, whereas macrophages of patients with Alzheimer’s disease (AD) generally have a pro-inflammatory phenotype and lack energy for brain clearance of Aβ. To clarify the pathogenesis of sporadic AD and therapeutic effects of polyunsaturated fatty acids (PUFA) with vitamins B and D and antioxidants on monocyte/macrophage (MM) migration in the AD brain, MM transcripts in energy and Aβ degradation, MM glycome, and macrophage clearance of Aβ. We followed for 31.3 months (mean) ten PUFA-supplemented neurodegenerative patients: 3 with subjective cognitive impairment (SCI), 2 with mild cognitive impairment (MCI), 3 MCI/vascular cognitive impairment, 2 with dementia with Lewy bodies, and 7 non-supplemented caregivers. We examined: monocyte migration in the brain and a blood-brain barrier model by immunochemistry and electron microscopy; macrophage transcriptome by RNAseq; macrophage glycome by N-glycan profiling and LTQ-Orbitrap mass spectrometry; and macrophage phenotype and phagocytosis by immunofluorescence. MM invade Aβ plaques, upload but do not degrade Aβ, and release Aβ into vessels, which develop cerebrovascular amyloid angiopathy (CAA); PUFA upregulate energy and Aβ degradation enzyme transcripts in macrophages; PUFA enhance sialylated N-glycans in macrophages; PUFA reduce oxidative stress and increase pro-resolution MM phenotype, mitochondrial membrane potential, and Aβ phagocytosis (p < 0.001). Macrophages of SCI, MCI, and AD patients have interrelated defects in the transcriptome, glycome, Aβ phagocytosis, and Aβ degradation. PUFA mend macrophage transcriptome, enrich glycome, enhance Aβ clearance, and benefit the cognition of early-stage AD patients.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

CD47 blockade enhances therapeutic efficacy of cisplatin against lung carcinoma in a murine model

Cisplatin (CDDP) is the first generation of platinum-based drug and is widely used to treat many cancers due to its potency. The present study aims to explore the effects of CDDP on lung carcinoma and its relationship with macrophage phagocytosis. In in vitro study, murine and human lung cancer cell lines were applied and treated with CDDP, CD47 antibody (aCD47), or CDDP plus aCD47. In in vivo study, a tumor xenograft animal model was treated with CDDP, aCD47, or CDDP plus aCD47. Real-time PCR was applied to determine the mRNA expressions. Enzyme-linked immunosorbent assay (ELISA), Western blotting, and Immunofluorescent staining were applied to determine the protein expressions. Flow cytometry was applied to analyze cell apoptosis, phagocytosis, and specific cell populations. CDDP enhanced the expressions of CD47 in lung cancer cells. Interestingly, the blockage of CD47 enhanced the macrophages' phagocytic activity on the CDDP-treated tumor cells. The treatment of CDDP and aCD47 exhibited anti-tumor effects and prolonged the LLC tumor-bearing mice survival time. Mechanistic studies revealed that the treatment of CDDP and aCD47 regulated the phagocytic activity of macrophage, percentage of CD8{sup +} T cells, and cytokines (tumor growth factor (TGF)-β, interleukin (IL)12p70, and interferon (IFN)-γ) in the tumor-bearing model. CD47 blockade enhanced therapeutic efficacy of cisplatin against lung carcinoma in vivo and in vitro.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

The protein 4.1R downregulates VEGFA in M2 macrophages to inhibit colon cancer metastasis

Highlights: • Protein 4.1R knockout reduced the phagocytosis of M2 macrophages. • Co-culture with 4.1R knockout M2 macrophages enhances the malignancy of colon cancer. • Protein 4.1R knockout upregulates vascular endothelial growth factor A secreted by M2 macrophages. • Protein 4.1R knockout activates the PI3K/AKT signaling pathway and promotes the progression of colon cancer. M2 macrophages are crucial components of the tumour microenvironment and have been shown to be closely related to tumour progression. Co-culture with 4.1R{sup −/−} M2 macrophages enhances the malignancy of colon cancer (CC), but the mechanism remains unclear. Here, we report that protein 4.1R knockout reduced the phagocytosis of M2 macrophages (M-CSF/IL-4-treated bone marrow cells) and promoted MC38 colon cancer cell proliferation, migration, invasion, tumour formation and epithelial-mesenchymal transition (EMT), which are regulated by M2 macrophages. Further mechanistic dissection revealed that the 4.1R knockout upregulated vascular endothelial growth factor A (VEGFA) secreted by M2 macrophages and promoted colon cancer progression by activating the PI3K/AKT signalling pathway. In summary, our present study identified that 4.1R downregulates VEGFA secretion in M2 macrophages and delays the malignant potential of colon cancer by inhibiting the PI3K/AKT signalling pathway.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2020

Horizontal gene transfer in Fungi

Horizontal gene transfer (HGT), the sharing of genes between species outside of inheritance, is a common feature among prokaryotes, but a growing body of evidence supports HGT in eukaryotic genomes. The driving forces behind horizontal gene transfer occur within a shared environment, and are selective—meaning they improve survivability of stress, increase nutrient access, or provide an evolutionary advantage. The development of toxic and pathogenic properties was one of the first indications of phenotypic transfer among fungal species. An growing species diversity of genome sequences has brought a new set of tools to tracking HGT. The detection of HGT based on sequence similarity utilizes species tree phylogenies, which compare similarity of core conserved genes to determine lineage. Divergence of an HGT candidate gene from a phylogenetic background tree can show inter-species DNA transfer. It is by following phenotypes and analysis of DNA sequence context and content that we can uncover previous transfer events, and track lab fostered and recorded HGT. Evidence of HGT is found heavily in parasitic and pathogenic fungi; both of these lifestyles rely on phagocytosis and assimilation of host parts and pieces, suggesting phagocytosis is implicated in DNA uptake mechanisms. Our understanding of the molecular mechanisms of DNA uptake and incorporation of foreign DNA into fungal genomes, i.e. natural competence, remain based on a set of laboratory conditions used in molecular transformation. The mechanism of DNA uptake in the natural world and new DNA incorporation remain under investigation.

Bredeweg, Erin L.↗
DOE OSTI2020

Staphylococcus pseudintermedius 5'-nucleotidase suppresses canine phagocytic activity

Staphylococcus pseudintermedius is a major opportunistic bacterial pathogen and the leading cause of pyoderma in dogs. In canines it is also often associated with infections of the urinary system and wounds and occasionally infects people. Widespread antimicrobial resistance has made the development of alternative treatments a high priority. The development of a staphylococcal vaccine, however, has proven challenging. Identification of virulence factors that inhibit phagocytosis and avoid innate immunity may play a significant role in preventing or treating infection with S. pseudintermedius. In this study, we identified a putative 5'-nucleotidase provisionally named SpAdsA, a S. pseudintermedius cell- wall protein encoded by SpAdsA. SpAdsA shares approximately 52% identity with the orthologous protein of Staphylococcus aureus and 14.8% identity with that of Streptococcus suis type2. It catalyzes the dephosphorylation of adenosine triphosphate and attenuation of this enzyme with critical amino acid substitutions nearly eliminated its hydrolytic activity. Furthermore, exogenous adenosine inhibited phagocytosis of S. pseudintermedius by canine neutrophils and monocytes. Conversely, the addition of SpAdsA inhibitor or A2A adenosine receptor antagonist impaired the capacity of S. pseudintermedius to escape from killing by phagocytic cells. The neutralizing ability of canine antibody produced against SpAdsA-M was determined. Taken together, these results suggest that SpAdsA likely plays an important role in S. pseudintermedius virulence and that attenuated SpAdsA may be a good candidate for inclusion in a vaccine against S. pseudintermedius.

5'-nucleotidase↗
DOE OSTI2024

Characterization of Histophilus somni sialic acid uptake mutant (Δ nanP -Δ nanU ) using a mouse septicemia and mortality model

Histophilus somni is an important pathogen of the bovine respiratory disease complex, yet the mechanisms underlying its virulence remain poorly understood. It is known that H. somni can incorporate sialic acid into lipooligosaccharide (LOS), and sialylated H. somni is more resistant to phagocytosis and complement-mediated killing by serum compared to non-sialylated bacteria in vitro. However, the virulence of non-sialylated H. somni has not been evaluated in vivo using an animal model. In this study, we investigated the contribution of sialic acid to virulence by constructing an H. somni sialic acid uptake mutant (ΔnanP-ΔnanU) and comparing the parent and mutant strains in a mouse septicemia and mortality model. Intraperitoneal challenge of mice with wildtype H. somni (1 × 10 8 colony forming units/mouse, CFU) was lethal to all animals. Mice challenged with three different doses (1, 2, or 5 × 108 CFU/mouse) of an H. somni ΔnanP-ΔnanU sialic acid uptake mutant exhibited survival rates of 90 %, 60 %, and 0 % respectively. High-performance anion exchange chromatography analyses revealed that LOS prepared from both parent and the ΔnanP-ΔnanU mutant strains of H. somni were sialylated. These findings suggest the presence of de novo sialic acid synthesis pathway, although the genes associated with de novo sialic acid synthesis (neuB and neuC) were not identified by genomic analysis. The lower attenuation in mice is most likely attributed to the sialylated LOS of H. somni nanPU mutant.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Nicotinamide-Loaded Peptoid Nanotubes for Energy Regeneration in Acute Brain Injury

Acute brain injuries such as perinatal asphyxia, stroke, and traumatic brain injury result in ischemia, oxidative stress, excitotoxicity, and inflammation, leading to a depletion of ATP. Nicotinamide adenine dinucleotide (NAD+) is crucial for ATP regeneration and DNA repair during postinjury recovery. However, the therapeutic benefits of NAD+ and its precursors, such as nicotinamide (NAM), are limited by challenges in achieving effective cell-specific intracellular delivery. In this study, we use a nanopeptoid delivery strategy to replenish the cellular redox state and increase energy production in the acutely injured brain. By self-assembling peptoids into tubular structures, we created biocompatible NAM-conjugated peptoid nanotubes (NAM-PNTs) that vary in tubular length. NAM-PNTs demonstrated significant therapeutic benefits by enhancing cell viability and replenishing intracellular ATP levels within 24 h of treatment in oxygen–glucose-deprived (OGD) BV-2 cells. In organotypic brain slices, NAM-PNT treatment promoted glial proliferation, reduced proinflammatory cytokines, and increased anti-inflammatory cytokines after OGD, an ex vivo model of hypoxia-ischemia. The effect of NAM-PNTs is associated with their uptake into microglia via fluid-phase phagocytosis and caveolae-mediated endocytosis. A single systemic dose of NAM-PNTs localized in microglia in the injured hemisphere and reduced brain tissue loss and improved neuropathology after hypoxia-ischemia in term-equivalent rats. These findings highlight the therapeutic potential of NAM-PNTs for cell-specific targeted delivery and energy restoration in the acutely injured neonatal brain. In the neonatal brain injury field, this work demonstrates the development of an innovative nanoparticle platform from first-principles design and synthesis to in vitro screening and then demonstration of efficacy in vivo .

ATP↗
DOE OSTI2025

Imaging and speciation of intracellular metallic implant debris using synchrotron-based X-ray fluorescence micro-spectroscopy: a study of two cases

Debris generated from total hip arthroplasty (THA) components made from metal alloys can cause, in some cases, inflammatory cell (e.g., macrophages) responses that lead to adverse local tissue reactions (ALTR) and implant failure. The lack of information on intracellular chemical alterations of metal debris has hindered the understanding of the pathogenesis of ALTR. The goal of this study was to characterize intracellular debris within macrophages using Synchrotron imaging and spectroscopy. We studied periprosthetic tissues of two retrieved THAs with (1) a metal-on-metal (MoM) articulation and (2) a metal-on-polyethylene (MoP) articulation exhibiting corrosion of the metal femoral head. The MoM-THA exhibited different valence states of chromium- and cobalt-containing debris, suggesting three different moieties: Cr 2 O 3 , CrPO 4 , and an alloy-oxide mixture. The findings further suggest that Cr 2 O 3 formed in the tribological interfaces of the implant, while CrPO 4 is a by-product of the phagocytosis process of cobalt alloy-containing debris. Titanium debris appeared to occur in a mixed crystalline/amorphous oxide state. It remains unclear if this chemical state results from the tribochemical processes at the implant surface or intracellular alterations. The MoP-THA specimen exhibited no intracellular particulate debris associated with macrohpages, indicating that the ALTR may be entirely triggered by metal ionic species in this case. A better understanding of in vivo chemical alteration of implant debris will aid in assessing the risk for ALTR during implant design and material choice. However, various techniques are needed to accurately determine the interaction between metal particles and the inta- and extra-cellular environment.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2020

Fc-mediated effector function contributes to the in vivo antiviral effect of an HIV neutralizing antibody

Significance Neutralizing antibodies (NAbs) against HIV-1 are under clinical development. Despite demonstrated antiviral activity when used as treatment in HIV-infected people, the mechanism of action of NAbs remains controversial. Quantification of the relative contribution of Fab-mediated (neutralization of free virus) and Fc-mediated functions such as antibody-dependent cellular cytotoxicity (ADCC), phagocytosis, and complement fixation is essential to develop better therapies. Here, we treated chronically SHIV-infected rhesus macaques with a single dose of NAb VRC07-523LS with or without Fc modifications. Fc function contributes 21% to the antiviral activity of VRC07-523LS with an unmodified Fc region, when compared to NAb with Fc knockout mutations. However, NAbs with improved ADCC function in vitro did not show enhanced antiviral activity in vivo.

Asokan, Mangaiarkarasi↗
DOE OSTI2024

Supramolecular assembly of polycation/mRNA nanoparticles and in vivo monocyte programming

Size-dependent phagocytosis is a well-characterized phenomenon in monocytes and macrophages. However, this size effect for preferential gene delivery to these important cell targets has not been fully exploited because commonly adopted stabilization methods for electrostatically complexed nucleic acid nanoparticles, such as PEGylation and charge repulsion, typically arrest the vehicle size below 200 nm. Here, we bridge the technical gap in scalable synthesis of larger submicron gene delivery vehicles by electrostatic self-assembly of charged nanoparticles, facilitated by a polymer structurally designed to modulate internanoparticle Coulombic and van der Waals forces. Specifically, our strategy permits controlled assembly of small poly(β-amino ester)/messenger ribonucleic acid (mRNA) nanoparticles into particles with a size that is kinetically tunable between 200 and 1,000 nm with high colloidal stability in physiological media. We found that assembled particles with an average size of 400 nm safely and most efficiently transfect monocytes following intravenous administration and mediate their differentiation into macrophages in the periphery. When a CpG adjuvant is co-loaded into the particles with an antigen mRNA, the monocytes differentiate into inflammatory dendritic cells and prime adaptive anticancer immunity in the tumor-draining lymph node. This platform technology offers a unique ligand-independent, particle-size-mediated strategy for preferential mRNA delivery and enables therapeutic paradigmsviamonocyte programming.

Science & Technology - Other Topics↗
DOE OSTI2024

Plant‐produced SARS ‐ CoV ‐2 antibody engineered towards enhanced potency and in vivo efficacy

Summary Prevention of severe COVID‐19 disease by SARS‐CoV‐2 in high‐risk patients, such as immuno‐compromised individuals, can be achieved by administration of antibody prophylaxis, but producing antibodies can be costly. Plant expression platforms allow substantial lower production costs compared to traditional bio‐manufacturing platforms depending on mammalian cells in bioreactors. In this study, we describe the expression, production and purification of the originally human COVA2‐15 antibody in plants. Our plant‐produced mAbs demonstrated comparable neutralizing activity with COVA2‐15 produced in mammalian cells. Furthermore, they exhibited similar capacity to prevent SARS‐CoV‐2 infection in a hamster model. To further enhance these biosimilars, we performed three glyco‐ and protein engineering techniques. First, to increase antibody half‐life, we introduced YTE‐mutation in the Fc tail; second, optimization ofN‐linked glycosylation by the addition of a C‐terminal ER‐retention motif (HDEL), and finally; production of mAb in plant production lines lacking β‐1,2‐xylosyltransferase and α‐1,3‐fucosyltransferase activities (FX‐KO). These engineered biosimilars exhibited optimized glycosylation, enhanced phagocytosis and NK cell activation capacity compared to conventional plant‐produced S15 and M15 biosimilars, in some cases outperforming mammalian cell produced COVA2‐15. These engineered antibodies hold great potential for enhancingin vivoefficacy of mAb treatment against COVID‐19 and provide a platform for the development of antibodies against other emerging viruses in a cost‐effective manner.

Biotechnology & Applied Microbiology↗
DOE OSTI2022

Engineered ACE2-Fc counters murine lethal SARS-CoV-2 infection through direct neutralization and Fc-effector activities

Soluble angiotensin-converting enzyme 2 (ACE2) constitutes an attractive antiviral capable of targeting a wide range of coronaviruses using ACE2 as their receptor. Using structure-guided approaches, we developed a series of bivalent ACE2-Fcs harboring functionally and structurally validated mutations that enhance severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) receptor binding domain recognition by up to ~12-fold and remove angiotensin enzymatic activity. The lead variant M81 potently cross-neutralized SARS-CoV-2 variants of concern (VOCs), including Omicron, at subnanomolar half-maximal inhibitory concentration and was capable of robust Fc-effector functions, including antibody-dependent cellular cytotoxicity, phagocytosis, and complement deposition. When tested in a stringent K18-hACE2 mouse model, Fc-enhanced ACE2-Fc delayed death by 3 to 5 days or effectively resolved lethal SARS-CoV-2 infection in both prophylactic and therapeutic settings via the combined effects of neutralization and Fc-effector functions. These data add to the demonstrated utility of soluble ACE2 as a valuable SARS-CoV-2 antiviral and indicate that Fc-effector functions may constitute an important component of ACE2-Fc therapeutic activity.

60 APPLIED LIFE SCIENCES↗