Hemagglutination Of Red Blood Cells Research Articles

A Quantitative Human Red Blood Cell Agglutination Assay for Characterisation of Galectin Inhibitors.

Galectins are a family of beta-galactoside-binding proteins that are characterised by their carbohydrate recognition domain (CRD) and include galectin-1 and galectin-3. These galectins have been implicated in numerous diseases due to their pleiotropic nature, including cancer and fibrosis, with therapeutic inhibitors being clinically developed to block the CRD. One of the early methods developed to characterise these galectins was the hemagglutination of red blood cells. Although it is insightful, this approach has been hampered by a lack of sensitivity and accurate quantification of the agglutination observed. In this study, we aimed to validate a more precise and quantitative method to enable the further investigation of differences between galectins in respect to agglutination induction in different blood groups, as well as the characterisation of small molecule inhibitors. Quantification of hemagglutination was shown to be optimal using U-bottom plates imaged and analysed with FIJI ImageJ rather than flat-bottom plates read for absorbance on an optical density plate reader. Galectin-3-induced red blood cell agglutination efficacy increased significantly from blood group O to A to B. However, for both the galectin-1 monomer and concatemer, a more comparable effect was observed between blood group B and O, but with more potent effects than in blood group A. Inhibition assays for both galectin-3 and galectin-1 induced-hemagglutination were able to demonstrate clear concentration responses and expected selectivity profiles for a set of small-molecule glycomimetics, confirming the historical profiles obtained in biochemical binding and functional cellular assays.

The Potent and Selective Antitumor Activity of CO-1 and Fully Humanized CO-1bi in Treatment of B Cell Non-Hodgkin's Lymphoma

Recent advancements have identified CD47 blockade as a promising approach for the treatment of B cell Non-Hodgkin's Lymphoma (B-NHL). CD47 blockade inhibits the CD47-SIRPα axis, which inhibits a “don't eat me” signal that facilitates the phagocytosis of tumor cells by macrophages [1,2]. While the effect of anti-CD47 monoclonal antibodies (mAbs) as monotherapy has been limited in B-NHL, combining CD47 mAbs with conventional treatment such as rituximab has demonstrated more encouraging outcomes. This combinatory approach may be attributed to the need for additional cytotoxic mechanisms to achieve clinically meaningful responses with CD47-mediated therapy in B-NHL [3]. Additionally, CD47 blockage has raised concerns about off-target effects and red blood cell depletion [4]. To address these challenges and improve treatment possibilities we developed a fully humanized CO-1bi based on the chimeric CO-1. The bifunctional CO-1 is an agonistic IgG4 anti-CD47 mAb designed to induce direct and rapid programmed cancer cell death (PCCD) and stimulate phagocytosis through blockage of CD47-SIRPα binding. CO-1bi is a humanized bivalent scFv-Fc fusion of CO-1 with superior safety (no red blood cell hemagglutination) while maintaining the functionality of CO-1. In this study, we evaluated the antitumor activity of CO-1 and CO-1bi on different B-NHL cell lines in vitro as well as in xenografts of B-NHL established in NOD- scid IL2Rγ null (NSG) mice. Both constructs demonstrated potent and fast cell death induction in lymphoma cell lines while sparing normal B cells. The robust antitumor activity demonstrated by CO-1 and CO-1bi as single agents in xenograft models of Burkitt's lymphoma underscores the significance of their additional agonistic PCCD capabilities compared to other anti-CD47 agents. The emergence of CO-1bi offers a unique strategy with dual functionality for the treatment of B-NHL and other malignancies without red blood cell depletion characterized by current anti-CD47 mAb in clinical trials. This innovative approach provides an improved and promising avenue for cancer treatment.

Glycosylation Contributes to Thermostability and Proteolytic Resistance of rFIP-nha (Nectria haematococca).

Glycosylation is an important post-translational modification of proteins, contributing to protein function, stability and subcellular localization. Fungal immunomodulatory proteins (FIPs) are a group of small proteins with notable immunomodulatory activity, some of which are glycoproteins. In this study, the impact of glycosylation on the bioactivity and biochemical characteristics of FIP-nha (from Nectria haematococca) is described. Three rFIP-nha glycan mutants (N5A, N39A, N5+39A) were constructed and expressed in Pichia pastoris to study the functionality of the specific N-glycosylation on amino acid N5 and N39. Their protein characteristics, structure, stability and activity were tested. WT and mutants all formed tetramers, with no obvious difference in crystal structures. Their melting temperatures were 82.2 °C (WT), 81.4 °C (N5A), 80.7 °C (N39A) and 80.1 °C (N5+39A), indicating that glycosylation improves thermostability of rFIP-nha. Digestion assays showed that glycosylation on either site improved pepsin resistance, while 39N-glycosylation was important for trypsin resistance. Based on the 3D structure and analysis of enzyme cleavage sites, we conclude that glycosylation might interfere with hydrolysis via increasing steric hindrance. WT and mutants exerted similar bioactivity on tumor cell metabolism and red blood cells hemagglutination. Taken together, these findings indicate that glycosylation of FIP-nha impacts its thermostability and digestion resistance.

Electrostatic Surface Potential as a Key Parameter in Virus Transmission and Evolution: How to Manage Future Virus Pandemics in the Post-COVID-19 Era.

Virus-cell interactions involve fundamental parameters that need to be considered in strategies implemented to control viral outbreaks. Among these, the surface electrostatic potential can give valuable information to deal with new epidemics. In this article, we describe the role of this key parameter in the hemagglutination of red blood cells and in the co-evolution of synaptic receptors and neurotransmitters. We then establish the functional link between lipid rafts and the electrostatic potential of viruses, with special emphasis on gangliosides, which are sialic-acid-containing, electronegatively charged plasma membrane components. We describe the common features of ganglioside binding domains, which include a wide variety of structures with little sequence homology but that possess key amino acids controlling ganglioside recognition. We analyze the role of the electrostatic potential in the transmission and intra-individual evolution of HIV-1 infections, including gatekeeper and co-receptor switch mechanisms. We show how to organize the epidemic surveillance of influenza viruses by focusing on mutations affecting the hemagglutinin surface potential. We demonstrate that the electrostatic surface potential, by modulating spike-ganglioside interactions, controls the hemagglutination properties of coronaviruses (SARS-CoV-1, MERS-CoV, and SARS-CoV-2) as well as the structural dynamics of SARS-CoV-2 evolution. We relate the broad-spectrum antiviral activity of repositioned molecules to their ability to disrupt virus-raft interactions, challenging the old concept that an antibiotic or anti-parasitic cannot also be an antiviral. We propose a new concept based on the analysis of the electrostatic surface potential to develop, in real time, therapeutic and vaccine strategies adapted to each new viral epidemic.

Phenotypic Assessment of Clinical Escherichia coli Isolates as an Indicator for Uropathogenic Potential.

For women in the United States, urinary tract infections (UTIs) are the most frequent diagnosis in emergency departments, comprising 21.3% of total visits. Uropathogenic Escherichia coli (UPEC) causes ~80% of uncomplicated UTIs. To combat this public health issue, it is vital to characterize UPEC strains as well as to differentiate them from commensal strains to reduce the overuse of antibiotics. It has been challenging to determine a consistent genetic signature that clearly distinguishes UPEC from other E. coli strains. Therefore, we examined whether phenotypic data could be predictive of uropathogenic potential. We screened 13 clinical strains of UPEC, isolated from cases of uncomplicated UTI in young otherwise healthy women, in a series of microbiological phenotypic assays using UPEC prototype strain CFT073 and nonpathogenic E. coli strain MG1655 K-12 as controls. Phenotypes included adherence, iron acquisition, biofilm formation, human serum resistance, motility, and stress resistance. By use of a well-established experimental mouse model of UTI, these data were able to predict the severity of the bacterial burden in both the urine and bladders. Multiple linear regression using three different phenotypic assays, i.e., growth in minimal medium, siderophore production, and type 1 fimbrial expression, was predictive of bladder colonization (adjusted R2 = 0.6411). Growth in ex vivo human urine, hemagglutination of red blood cells, and motility modeled urine colonization (adjusted R2 = 0.4821). These results showcase the utility of phenotypic characterization to predict the severity of infection that these strains may cause. We predict that these methods will also be applicable to other complex, genetically redundant, pathogens. IMPORTANCE Urinary tract infections are the second leading infectious disease worldwide, occurring in over half of the female population during their lifetime. Most infections are caused by uropathogenic Escherichia coli (UPEC) strains. These strains can establish a reservoir in the gut, in which they do not cause disease but, upon introduction to the urinary tract, can infect the host and elicit pathogenesis. Clinically, it would be beneficial to screen patient E. coli strains to understand their pathogenic potential, which may lead to the administration of prophylactic antibiotic treatment for those with increased risk. Others have proposed the use of PCR-based genetic screening methods to detect UPEC strains and differentiate them from other E. coli pathotypes; however, this method has not yielded a consistent uropathogenic genetic signature. Here, we used phenotypic characteristics such as growth rate, siderophore production, and expression of fimbriae to better predict uropathogenic potential.

Serological Characteristics and Gene Sequences of Congenital Blood Group Chimera

To identify the ABO positive and negative stereotypic inconsistencies in a dizygotic twin with positive stereotypic patterns and mixed agglutination, and explore the application of serological characteristics and gene sequence in congenital blood group chimeras. ABO blood group identification, Rh and MN typing were performed using the microcolumn gel method and ABO genotyping was performed using the PCR-SSP method. In this patient, both anti-A and anti-B tubes had mixed hemagglutination of red blood cells, and the anti-ABO tube was AB type. The Rh typing of the patient was CcDEe. Mixed agglutination of red blood cells was observed in both anti-M and anti-N tubes in MN typing. The patient's father and mother was normal Type O and AB, respectively. There were three alleles in the ABO gene of the patient, O0101 came from his father, while A102 and B01 came from his mother. The patient has two groups of red blood cells (type A and B). Because the patients is a dizygotic twin, these two groups of red blood cells can be chimeras formed by blood exchange between the twins. Through gene sequencing, it can be determined that the patient is a congenital A/B blood type chimera.

Abstract 5204: Optimization of novel anti-human CD47 antibody prodrugs as cancer therapeutics with low on-target toxicity

Abstract CD47 is a well-studied target for cancer immunotherapy. Blocking of CD47 binding to its receptor, SIRPα, on macrophages leads to inhibition of macrophage activation and phagocytosis. Several monoclonal anti-CD47 antibodies are currently in various stages of clinical development. However, since CD47 is also expressed on normal cells such as red blood cells and platelets, anti-CD47 antibodies also caused on-target side effects during clinical evaluations, such as anemia and red blood cell hemagglutination. To minimize the on-target side effects of anti-CD47 antibodies, we have generated anti-CD47 prodrugs. By screening a phage peptide library, we identified peptides that can specifically block the anti-CD47 antibody binding activity. These peptides were fused to the humanized anti-CD47 antibody via a linker containing protease substrate sequences to derive the anti-CD47 antibody prodrug. We demonstrated that these anti-CD47 antibody prodrugs had significantly diminished CD47 binding activity and were unable to block CD47:SIRPα interaction. Upon cleavage of the anti-CD47 antibody prodrug with proteases, the antibody regained full activities in both binding to CD47 and blocking CD47:SIRPα interaction. Importantly, we showed that the anti-CD47 antibody prodrug had minimal hemagglutination and RBC binding activities. Since different tumors may have different types of protease activities, anti-CD47 prodrugs with different types of protease substrates have been generated to demonstrate that the anti-CD47 prodrug can be used for treating various tumor types. These data suggest that novel anti-CD47 antibody prodrugs are promising candidates for the next generation anti-CD47 therapeutics with improved safety profile. Citation Format: Richard Zhang, Haishan Lin. Optimization of novel anti-human CD47 antibody prodrugs as cancer therapeutics with low on-target toxicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5204.

Abstract 2903: ISB 1442, a first-in-class CD38 and CD47 bispecific antibody innate cell modulator for the treatment of CD38+ malignancies

Abstract ISB 1442 is a fully human bispecific antibody (BsAb) using BEAT (Bispecific Engagement by Antibodies based on the T-cell receptor) platform to target CD38 and CD47 as treatment for CD38+ malignancies, including multiple myeloma (MM). ISB 1442 is designed with a bi-paratopic anti-CD38 arm that strongly binds to CD38+ tumor cells and an anti-CD47 arm made of a single Fab designed to block interaction between CD47 and the signal-regulatory protein alpha (SIRPα) with low affinity. This approach is expected to induce minimal unintended effects on red blood cells (RBC) compared to anti-CD47 monoclonal antibody (mAb) magrolimab as it enables the CD47 binding only upon avidity induced CD38 crosslinking. The Fc portion of ISB 1442 is engineered to enhance antibody dependent cell phagocytosis (ADCP), antibody dependent cell cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC). In vitro, ISB 1442 exhibited higher killing potency compared to daratumumab across a range of CD38-expressing tumor cells. Additionally, ISB 1442 showed in vitro tumor killing potency through phagocytosis comparable to magrolimab, acting mostly through ADCP. To assess the complex mechanisms of action of ISB 1442 in a single system, a multiple mode of action of killing (MMoAK) assay was established to allow for simultaneous killing by natural killer cells, autologous macrophages, as well as complement from human serum. In the MMoAK assay, ISB 1442 exhibited tumor cell killing that was twice as high as daratumumab. In vivo, in a therapeutic model of subcutaneously established Raji tumor xenograft in CB17/SCID mice, ISB 1442 induced higher tumor growth inhibition than daratumumab and comparable tumor regression to magrolimab. On-target specificity was evaluated in vitro in human and monkey whole blood assays. ISB 1442 did not cause any detectable hemolysis, RBC depletion or platelet aggregation and showed markedly lower RBC hemagglutination relative to magrolimab, suggesting a more favorable on-target specificity profile in humans. On the contrary, due to the higher expression of CD38 in monkey RBC compared to human, ISB 1442 showed more pronounced binding on RBC than magrolimab, suggesting that monkey is a more sensitive species than human for toxicological evaluation of CD38-targeted therapies. Finally, by integrating in vitro pharmacology data along with available clinical information on benchmark antibodies, a quantitative systems pharmacology model was developed to simulate potential efficacious dose range in MM. In summary, we report a novel approach for the treatment for CD38+ cancers by co-targeting CD38 and CD47. Based on its unique design and multiple mechanisms of action, ISB 1442 is anticipated to enhance antitumor activity by overcoming known primary and acquired tumor escape mechanisms of resistance relative to daratumumab. Citation Format: Stefano Sammicheli, Camille Grandclement, Elie Dheilly, Maria Panagopoulou, Evangelia Martini, Perrine Suere, Blandine Pouleau, Carole Estoppey, Julia Frei, Jeremy Loyau, Thierry Monney, Adam Drake, Alain Rubod, Marie Agnes Doucey, Vinu Menon, Venkatesha Udupa, Sunitha GN, Daniel Rasmussen, Jeppe Koch Olsen, Roberto Gionannini, Girish Gudi, Ankita Srivastava, Cyril Konto, Mario Perro. ISB 1442, a first-in-class CD38 and CD47 bispecific antibody innate cell modulator for the treatment of CD38+ malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2903.

Dual display hemagglutinin 1 and 5 on the surface of enveloped virus-like particles in silkworm expression system

Rous sarcoma virus-like particles (RSV-LPs) displaying hemagglutinins of H1N1 (A/New Caledonia/20/99) (H1) and H5N1 (A/Vietnam/1194/2004) (H5) of the influenza A virus were produced. The H1 has its transmembrane domain, but the H5 was fused with the transmembrane domain of glycoprotein 64 (BmGP64) from Bombyx mori nucleopolyhedrovirus (BmNPV). H1 and RSV Gag protein were coexpressed in the hemolymph of silkworm larvae, copurified, and confirmed RSV-LP displaying H1 (VLP/H1). Similarly, the RSV-LP displaying H5 (VLP/H5) production was also achieved. Using fetuin agarose column chromatography, RSV Gag protein-coexpressed H1 and H5 in silkworms were copurified from the hemolymph. By immuno-TEM, H1 and H5 were observed on the surface of an RSV-LP, indicating the formation of bivalent RSV-LP displaying two HAs (VLP/BivHA) in the hemolymph of silkworm larvae. VLP/H1 induced the hemagglutination of red blood cells (RBCs) of chicken and rabbit but not sheep, while VLP/H5 induced the hemagglutination of RBCs of chicken and sheep but not rabbit. Additionally, VLP/BivHA allowed the hemagglutination of RBCs of all three animals. Silkworm larvae can produce RSV-LPs displaying two HAs and is a promising tool to produce the bivalent enveloped VLPs for the vaccine platform.

Abstract 1611: A novel anti-human CD47 antibody prodrug as cancer therapeutics to lower on-target side effects

Abstract CD47 is highly expressed in many types of cancers, including human acute myeloid leukemia (AML), non-Hodgkin's lymphoma (NHL), and solid tumors such as small-cell lung cancer (SCLC). Binding of CD47 to its receptor, SIRPα, on macrophages leads to inhibition of macrophage activation and phagocytosis. CD47 overexpression is one of the mechanisms cancer cells employ to evade immune surveillance and correlates with poor prognosis in cancers. Several strategies have been used to block CD47 binding to SIRPα, including anti-CD47 blocking antibodies and SIRPα-Fc fusion proteins. However, since CD47 is also expressed on normal cells, a major concern is that anti-CD47 antibodies could also cause on-target side effects, such as red blood cell hemagglutination and anemia. We have identified a panel of high affinity anti-human CD47 blocking antibodies. The lead antibodies bound to human CD47 with sub-nanomolar affinities and displayed a range of activities in blocking CD47:SIRPα interaction. These antibodies showed robust activities in stimulating macrophage-mediated phagocytosis of cancer cells. To minimize the on-target side effects of anti-CD47 antibodies, we have taken an antibody prodrug approach in which the antibody is inactive for normal cells while it becomes functional in the tumor microenvironment enriched with proteases. By screening a phage peptide library, we identified peptides that specifically block the anti-CD47 antibody binding activity. These peptides were fused to the humanized anti-CD47 antibody via a linker containing protease substrate sequences to derive the anti-CD47 antibody prodrug. We demonstrated that the anti-CD47 antibody prodrug lacked CD47 binding activity and was unable to block CD47:SIRPα interaction. Upon cleavage of the anti-CD47 antibody prodrug with proteases, the antibody regained full activities in both CD47 binding and blocking CD47:SIRPα interaction. Importantly, we showed that the anti-CD47 antibody prodrug had minimal hemagglutination and RBC binding activities. These data suggest that the novel anti-CD47 antibody prodrug is a promising candidate for the next generation anti-CD47 therapeutics with improved safety profile. Citation Format: Richard Zhang, Hunter Drobenaire, Huey Li, Veronica Pizzarella, Haishan Lin. A novel anti-human CD47 antibody prodrug as cancer therapeutics to lower on-target side effects [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1611.

Phenotypic characterization of trimeric autotransporter adhesin-defective bcaC mutant of Burkholderia cenocepacia: cross-talk towards the histidine kinase BCAM0218

Burkholderia cenocepacia is a virulent species belonging to the Burkholderia cepacia complex (Bcc) and one of the most problematic agents of chronic lung infection in cystic fibrosis patients. B. cenocepacia possesses a large panel of virulence traits that include trimeric autotransporter adhesins (TAAs). Such proteins are obligate homotrimeric anchored in the outer membrane. They are players in the adhesion events that occur between bacteria and biotic/abiotic surfaces. In this study, we constructed two insertional-mutants for TAA bcaC and Histidine kinase (HK) BCAM0218 genes, which are clustered together within the B. cenocepacia K56-2 TAA cluster. The bcaC-mutant affects B. cenocepacia adhesion to extracellular matrix proteins and red blood cells hemagglutination. BcaC contributes to enhancing B. cenocepacia K56-2 adhesion to bronchial epithelial cells. The expression of bcaC seems to affect biofilm formation negatively. Due to a BCAM0218 disruption, the bcaC expression increases significantly, indicating that they are functionally linked. The overexpression of bcaC in the BCAM0218-mutant background rescues at least part of the BcaC functions. Altogether, these findings reveal the multifunctionality of BcaC as a novel B. cenocepacia K56-2 virulence factor and postulate the involvement of a sensor HK (BCAM0218) in the control of this TAA gene.

Abstract A93: Development of blocking antibodies to human CD47 with lower side effects on RBCs and platelets

Abstract CD47 is a major immune “checkpoint” molecule for macrophage activation. It is highly expressed on many types of cancers, including human acute myeloid leukemia (AML), non-Hodgkin’s lymphoma (NHL), and solid tumors such as small-cell lung cancer (SCLC). Binding of CD47 to its receptor, SIRPα, on macrophages leads to inhibition of macrophage activation and phagocytosis. CD47 overexpression is considered to be one of the mechanisms cancer cells employ to evade immune surveillance and correlates with poor prognosis in cancers. Several strategies have been used to block CD47 binding to SIRPα, including anti-CD47 blocking antibodies and SIRPα Fc proteins. However, since CD47 is also expressed on normal cells, a major concern is that these anti-CD47 antibodies may also induce platelet aggregation and red blood cell hemagglutination. In addition, since anti-CD47 antibodies bind to many different cell types in the peripheral circulation, the half-life for these antibody drugs could be lower. Here we report the generation and characterization of a panel of high-affinity anti-human CD47 blocking antibodies. The lead antibodies bound to human CD47 with sub-nanomolar affinities and displayed a range of activities in blocking CD47/SIRPα interaction. These antibodies showed robust activities in stimulating macrophage-mediated phagocytosis of cancer cells. Importantly, our antibodies did not induce red blood cell hemagglutination in in vitro assays. One of the lead antibodies demonstrates pH- dependent binding to human CD47. It has lower binding affinity to human CD47 at neutral pH but higher binding affinity to CD47 at more acidic pH, suggesting that this antibody may preferentially bind to CD47 in tumor microenvironment. Currently we are evaluating these anti-CD47 antibodies in preclinical efficacy and safety models. Citation Format: Richard Zhang, Haishan Lin. Development of blocking antibodies to human CD47 with lower side effects on RBCs and platelets [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A93.

Anti-CD47 Antibody, CC-90002, in Combination with Rituximab in Subjects with Relapsed and/or Refractory Non-Hodgkin Lymphoma (R/R NHL)

Anti-CD47 Antibody, CC-90002, in Combination with Rituximab in Subjects with Relapsed and/or Refractory Non-Hodgkin Lymphoma (R/R NHL)

A Randomized, Double-Blind, Placebo-Controlled Pilot Study to Assess the Urinary Anti-Adhesion Activity Following Consumption of Cranberry + health™ Cranberry Supplement (P06-114-19)

ObjectivesUrinary tract infections (UTIs) are the second most common bacterial infections and are often treated with antibiotics. The North American cranberry is recommended for UTI prophylaxis in women with recurrent UTI as a nutritional alternative approach. The ability of cranberry components or their metabolites to inhibit the adhesion of uropathogenic E.coli to the bladder is considered as an important mechanism by which cranberry mitigates UTIs. The objective of this study was to evaluate the anti-adhesion activity of urine from healthy individuals against both type I pili and P-fimbriae uropathogenic E.coli, after the consumption of Cranberry + health™ Cranberry Supplement. MethodsIn this randomized, double-blind, placebo-controlled pilot trial (n = 20), subjects consumed two cranberry chews, one in the morning and one in the evening. Clean-catch urine samples collected at baseline and post-intervention (0–3 h, 3–6 h, 6–9 h, 9–12 h, 12–24 h, 24–30 h, 30–36 h) were tested for anti-adhesion activity utilizing both a mannose-resistant human red blood cell hemagglutination assay specific for P-fimbriae E. coli and a T24 cell line model for type I pili E.coli (UTI89). ResultsUrinary ex vivo anti-adhesion activity against P-fimbriae E. coli after consuming cranberry chew was significantly greater (p < 0.05) than placebo chew at 0–3 h, 3–6 h, 6–9 h, 9–12, and 12–24 time point. Urinary anti-adhesion activity against type I pili E.coli (UTI89) following cranberry chew was significantly greater (p < 0.1) than placebo chew at 3–6 h, 6–9 h, and 12–24 h time point. ConclusionsAcute consumption of Cranberry + health™ Cranberry Supplement provides ex vivo anti-adhesion activity, which may help to improve urinary tract health. Funding SourcesOcean Spray Cranberries, Inc.

A randomized, double-blind, placebo-controlled pilot study to assess bacterial anti-adhesive activity in human urine following consumption of a cranberry supplement.

Urinary tract infections (UTIs) are one of the common bacterial infections treated with antibiotics. The North American cranberry is recommended for prophylaxis in women with recurrent UTIs as a nutritional alternative. The ability of cranberry components and their metabolites to inhibit adhesion of uropathogenic Escherichia coli (E. coli) is an important mechanism by which cranberry mitigates UTIs. The objective of this study was to evaluate urinary anti-adhesion activity against type 1 and P-type uropathogenic E. coli after consumption of cranberry +health™ cranberry supplement (cranberry chew). In this randomized, double-blind, placebo-controlled, crossover design pilot trial (n = 20), subjects consumed two cranberry or placebo chews, one in the morning and one in the evening. Clean-catch urine samples collected at the baseline and post-intervention (0-3, 3-6, 6-9, 9-12, 12-24, 24-30, 30-36 h) were tested for anti-adhesion effects with a mannose-resistant human red blood cell hemagglutination assay specific for P-type E. coli, or a T24 cell line model for type 1 E. coli. Urinary anti-adhesion activity against P-type E. coli after consumption of the cranberry chew was significantly greater (p < 0.05) than that observed with placebo chew at all time points except 24-36 h. Ex vivo anti-adhesion effects on type 1 E. coli were greater (p < 0.05) after cranberry chew consumption than placebo chew at 3-6 and 6-9 h urine collections. In conclusion, consumption of cranberry +health™ cranberry supplement exhibited greater ex vivo urinary anti-adhesion activity compared to placebo, suggesting that it may have the potential to help promote urinary tract health.

Human Group C Rotavirus VP8*s Recognize Type A Histo-Blood Group Antigens as Ligands.

Group/species C rotaviruses (RVCs) have been identified as important pathogens of acute gastroenteritis (AGE) in children, family-based outbreaks, as well as animal infections. However, little is known regarding their host-specific interaction, infection, and pathogenesis. In this study, we performed serial studies to characterize the function and structural features of a human G4P[2] RVC VP8* that is responsible for the host receptor interaction. Glycan microarrays demonstrated that the human RVC VP8* recognizes type A histo-blood group antigens (HBGAs), which was confirmed by synthetic glycan-/saliva-based binding assays and hemagglutination of red blood cells, establishing a paradigm of RVC VP8*-glycan interactions. Furthermore, the high-resolution crystal structure of the human RVC VP8* was solved, showing a typical galectin-like structure consisting of two β-sheets but with significant differences from cogent proteins of group A rotaviruses (RVAs). The VP8* in complex with a type A trisaccharide displays a novel ligand binding site that consists of a particular set of amino acid residues of the C-D, G-H, and K-L loops. RVC VP8* interacts with type A HBGAs through a unique mechanism compared with that used by RVAs. Our findings shed light on the host-virus interaction and the coevolution of RVCs and will facilitate the development of specific antivirals and vaccines.IMPORTANCE Group/species C rotaviruses (RVCs), members of Reoviridae family, infect both humans and animals, but our knowledge about the host factors that control host susceptibility and specificity is rudimentary. In this work, we characterized the glycan binding specificity and structural basis of a human RVC that recognizes type A HBGAs. We found that human RVC VP8*, the rotavirus host ligand binding domain that shares only ∼15% homology with the VP8* domains of RVAs, recognizes type A HBGA at an as-yet-unknown glycan binding site through a mechanism distinct from that used by RVAs. Our new advancements provide insights into RVC-cell attachment, the critical step of virus infection, which will in turn help the development of control and prevention strategies against RVs.

An extract from Taxodium distichum targets hemagglutinin- and neuraminidase-related activities of influenza virus in vitro.

Influenza virus remains an emerging virus and causes pandemics with high levels of fatality. After screening different plant extracts with potential anti-influenza activity, a water extract of Taxodium distichum stems (TDSWex) showed excellent activity against influenza viruses. The EC50 of TDSWex was 0.051 ± 0.024 mg/mL against influenza virus A/WSN/33. TDSWex had excellent antiviral efficacy against various strains of human influenza A and B viruses, particularly oseltamivir-resistant clinical isolates and a swine-origin influenza strain. We observed that the synthesis of viral RNA and protein were inhibited in the presence of TDSWex. The results of the time-of-addition assay suggested that TDSWex inhibited viral entry and budding. In the hemagglutination inhibition assay, TDSWex inhibited the hemagglutination of red blood cells, implying that the extract targeted hemagglutin-related functions such as viral entry. In the attachment and penetration assay, TDSWex showed antiviral activity with EC50s of 0.045 ± 0.026 and 0.012 ± 0.003 mg/mL, respectively. In addition, TDSWex blocked neuraminidase activity. We conclude that TDSWex has bimodal activities against both hemagglutinin and neuraminidase during viral replication.

433. DNA Monoclonal Antibodies Target Influenza Virus In Vivo

Despite promising innovations, influenza vaccines and antiviral drugs fail to provide full protection from seasonal infection, and provide little defense against novel and potentially pandemic viral strains. Broadly cross-protective monoclonal antibodies have been developed with the aim of providing protection against highly divergent influenza viruses. However, the utility of delivering purified protein antibody as therapy or prophylaxis against influenza is limited, especially in pandemic settings. Use of gene therapy to generate monoclonal antibodies in vivo provides a simplified, flexible, and relatively inexpensive alternative to protein antibody treatment.In this study, we used intramuscular electroporation of plasmid DNA encoding immunoglobulin to express DNA monoclonal antibodies (DMAb) against influenza hemagglutinin (HA) surface protein in mice. Multiple aspects of plasmid construction, antibody design, and delivery were optimized to enhance expression of DMAb from muscle cells in vivo. We investigated multiple antibody clones, including the broadly-neutralizing anti-influenza-H1 antibody 5J8. The 5J8 DMAb was expressed at µg/mL levels in serum of both nude and immune-competent mice. Serum DMAb produced from muscle in vivo were functional in vitro - with the ability to bind influenza HA, block hemagglutination of red blood cells, and neutralize influenza virus. Serum DMAb expression levels approximate those required for protection. Influenza challenge studies of mice treated with 5J8 DMAb are underway.DMAb provide an important new approach to immune therapy. DNA has an excellent safety profile and averts challenges of pre-existing serology associated with many viral vectors. Here, we demonstrate that DNA can be used to deliver consistently high levels of potent monoclonal antibodies for protection against a viral pathogen.

A DNA Aptamer Against Influenza A Virus: An Effective Inhibitor to the Hemagglutinin-Glycan Interactions.

Most therapeutical nucleic acid aptamers tend to inhibit protein-protein interactions and thereby function as antagonists. Attachment of the influenza virus surface glycoprotein hemagglutinin (HA) to sialic acid-containing host cell receptors (glycan) facilitates the initial stage of viral infection. Inhibition of the attachment may result in an antiviral effect on the proliferation of the influenza virus. To develop therapeutically interesting agents, we selected two single-stranded DNA (ssDNA) aptamers specific to the HA protein of H1N1 influenza virus (A/Puerto Rico/8/1934) through a procedure of systematic evolution of ligands by exponential enrichment. As it showed a higher binding affinity for HA protein (Kd = 78 ± 1 nM), aptamer 1 was tested for its ability to interfere with HA-glycan interactions using chicken red blood cell hemagglutination and microneutralization assays, which demonstrated that it significantly suppressed the viral infection in host cells. These results indicate that the isolated ssDNA aptamer may be developed as an antiviral agent against influenza through appropriate therapeutic formulation.

Llama nanoantibodies with therapeutic potential against human norovirus diarrhea.

Noroviruses are a major cause of acute gastroenteritis, but no vaccines or therapeutic drugs are available. Llama-derived single chain antibody fragments (also called VHH) are small, recombinant monoclonal antibodies of 15 kDa with several advantages over conventional antibodies. The aim of this study was to generate recombinant monoclonal VHH specific for the two major norovirus (NoV) genogroups (GI and GII) in order to investigate their potential as immunotherapy for the treatment of NoV diarrhea. To accomplish this objective, two llamas were immunized with either GI.1 (Norwalk-1968) or GII.4 (MD2004) VLPs. After immunization, peripheral blood lymphocytes were collected and used to generate two VHH libraries. Using phage display technology, 10 VHH clones specific for GI.1, and 8 specific for GII.4 were selected for further characterization. All VHH recognized conformational epitopes in the P domain of the immunizing VP1 capsid protein, with the exception of one GII.4 VHH that recognized a linear P domain epitope. The GI.1 VHHs were highly specific for the immunizing GI.1 genotype, with only one VHH cross-reacting with GI.3 genotype. The GII.4 VHHs reacted with the immunizing GII.4 strain and showed a varying reactivity profile among different GII genotypes. One VHH specific for GI.1 and three specific for GII.4 could block the binding of homologous VLPs to synthetic HBGA carbohydrates, saliva, and pig gastric mucin, and in addition, could inhibit the hemagglutination of red blood cells by homologous VLPs. The ability of Nov-specific VHHs to perform well in these surrogate neutralization assays supports their further development as immunotherapy for NoV treatment and immunoprophylaxis.