Abstract Dyslipidemia is characterized by elevated plasma levels of low‐density lipoprotein cholesterol (LDL‐C), triglycerides (TG), and TG‐rich lipoprotein (TGRLs) in circulation, and is closely associated with the incidence and development of cardiovascular disease. Angiopoietin‐like protein 3 (ANGPTL3) deficiency has been identified as a cause of familial combined hypolipidemia in humans, which allows it to be an important therapeutic target for reducing plasma lipids. Here, we report the discovery and characterization of a novel fully human antibody F1519‐D95aA against N‐terminal ANGPTL3 (NT‐ANGPTL3), which potently inhibits NT‐ANGPTL3 with a K D as low as 9.21 nM. In hyperlipidemic mice, F1519‐D95aA shows higher apolipoprotein B (ApoB) and TG‐lowering, and similar LDL‐C reducing activity as compared to positive control Evinacumab (56.50% vs 26.01% decrease in serum ApoB levels, 30.84% vs 25.28% decrease in serum TG levels, 23.32% vs 22.52% decrease in serum LDLC levels, relative to vehicle group). Molecular docking and binding energy calculations reveal that the F1519‐D95aA‐ANGPTL3 complex (10 hydrogen bonds, −65.51 kcal/mol) is more stable than the Evinacumab‐ANGPTL3 complex (4 hydrogen bonds, −63.76 kcal/mol). Importantly, F1519‐D95aA binds to ANGPTL3 with different residues in ANGPTL3 from Evinacumab, suggesting that F1519‐D95aA may be useful for the treatment of patients resistant to Evinacumab. In conclusion, F1519‐D95aA is a novel fully human anti‐NT‐ANGPTL3 antibody with potent plasma ApoB, TG, and LDL‐C lowering activities, which can potentially serve as a therapeutic agent for hyperlipidemia and relevant cardiovascular diseases.
BackgroundMaternal pertussis vaccination with Tdap vaccine is recommended to protect newborns from severe postnatal infection. HIV-exposed uninfected (HEU) infants have a higher incidence of pertussis infection and may particularly benefit from maternal immunization. The impact of HIV infection on the quality of IgG and memory B cell (MBC) responses to Tdap vaccination in pregnant women (PW) living with HIV (PWH) is unknown.MethodsIn this observational study, humoral immune responses to Tdap vaccination, including IgG levels, Fc-dependent effector functions, and MBC frequencies, were measured before and after vaccination in 40 PWH and 42 HIV-uninfected PW. Placental transfer of IgG and avidity were assessed in cord blood (CB). Soluble and cellular immune activation markers were quantified at baseline.FindingsOne month after vaccination, PWH had lower frequencies of MBC compared with HIV-uninfected PW. At delivery, PWH had attenuated pertussis-specific IgG levels and Fc-dependent effector functions. Reduced levels of maternal vaccine polyfunctional IgG and IgG avidity were transferred to HEU as compared to HIV-unexposed newborns. After adjustment with ethnicity, maternal antibody levels and gestational age at vaccination, HIV infection was independently associated with decreased levels of PT specific-IgG in CB. Both maternal and neonatal pertussis-specific IgG responses as well as PT-specific IgG avidity were inversely correlated with maternal sCD14 levels before vaccination among PWH.InterpretationMaternal HIV infection is associated with attenuated humoral immune responses to Tdap vaccination that correlate with sCD14. Suboptimal transfer of maternal immunity may further increase the risk of severe pertussis infection in HEU infants.FundingThis work was supported by IRIS Fund managed by the Foundation Roi Baudouin [2017J1820690206902], Association Vésale pour la Recherche Médicale and the Medical Council of CHU Saint-Pierre and has been funded in part with Federal funds from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, US Department of Health and Human Services, under Award No. U19AI145825. N.D. is a clinical researcher and A.M. is Research Director at the Fonds de la Recherche Scientifique (F.R.S.-FNRS), Belgium. M.E.A. was partially supported by NIH NIAID 1U19AI14825. This article is published with the support of the Fondation Universitaire of Belgium.
Topologically associating domains (TADs) are essential units of genome architecture, influencing transcriptional regulation and diseases. Despite numerous methods proposed for TAD identification, it remains challenging due to complex background and nested TAD structures. We introduce HTAD, a human-in-the-loop TAD caller that combines machine learning with human supervision to achieve high accuracy. HTAD begins with feature extraction for potential TAD border pairs, followed by an interactive labeling process through active learning. Performance assessments using public curation and synthetic datasets demonstrate HTAD's superiority over other state-of-the-art methods and reveal highly hierarchical TAD structures, offering a human-in-the-loop solution for detecting complex genomic patterns.
Background: Proprotein convertase subtilisin/kexin type 9 (PCSK9) regulates serum LDL cholesterol (LDL-C) levels by facilitating the degradation of the LDL receptor (LDLR) and is an attractive therapeutic target for hypercholesterolemia intervention. Herein, we generated a novel fully human antibody with favorable druggability by utilizing phage display-based strategy. Methods: A potent single-chain variable fragment (scFv) named AP2M21 was obtained by screening a fully human phage display library with hPCSK9, and performing twoin vitro affinity maturation processes including CDR-targeted tailored mutagenesis and cross-cloning. Thereafter, it was transformed to a full-length Fc-silenced anti-PCSK9 antibody FAP2M21 by fusing to a modified human IgG1 Fc fragment with L234A/L235A/N297G mutations and C-terminal lysine deletion, thus eliminating its immune effector functions and mitigating mAb heterogeneity. Findings: Our data showed that the generated full-length anti-PCSK9 antibody FAP2M21 binds to hPCSK9 with a KD as low as 1.42 nM, and a dramatically slow dissociation rate (koff, 4.68 × 10−6s-1), which could be attributed to its lower binding energy (-47.51 kcal/mol) than its parent counterpart FAP2 (-30.39 kcal/mol). We verified that FAP2M21 potently inhibited PCSK9-induced reduction of LDL-C uptake in HepG2 cells, with an EC50 of 43.56 nM. Further, in hPCSK9 overexpressed C57BL/6 mice, a single tail i.v. injection of FAP2M21 at 1, 3 and 10 mg/kg, dose-dependently up-regulated hepatic LDLR levels, and concomitantly reduced serum LDL-C by 3.3% (P=0.658), 30.2% (P=0.002) and 37.2% (P=0.002), respectively. Interpretation: FAP2M21 with potent inhibitory effect on PCSK9 may serve as a promising therapeutic agent for treating hypercholesterolemia and associated cardiovascular diseases.Funding Statement: This work was funded by National Fund for Major Projects of China (2009ZX09103-653; 2013ZX09301303-006; 2018ZX09301035), The Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), National Fund for Fostering Talents of Basic Science (NFFTBS, 3050040016), China Pharmaceutical University “Double First-Class” project (CPU2018GY15).Declaration of Interests: The authors declare no conflict of interest concerning this article.Ethics Approval Statement: The experimental procedures for the in vivo studies were approved by the Animal Ethics Committees of China Pharmaceutical University (No. 201601179, 19 October 2016) and conformed to the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health.
Background: Maternal pertussis vaccination with Tdap vaccine is recommended to protect newborns from severe postnatal infection. HIV-exposed uninfected (HEU) newborns have a higher incidence of pertussis infection and may particularly benefit from maternal immunization. The impact of HIV infection on the quality of IgG and memory B cell (MBC) responses to Tdap vaccination in pregnant women (PW) living with HIV (PWH) is unknown.Methods: Humoral immune responses to Tdap vaccination, including IgG levels, Fc-dependent effector functions, and MBC frequencies, were measured before and after vaccination in 40 PWH and 42 HIV-uninfected PW. Placental transfer of IgG and avidity were assessed in cord blood (CB). Soluble and cellular immune activation (IA) markers were quantified at baseline.Findings: Following Tdap immunization, PWH had lower pertussis-specific IgG levels and Fc-dependent effector functions, as well as lower frequencies of MBC compared with HIV-uninfected PW. Reduced levels of maternal polyfunctional IgG and IgG avidity were transferred to HEU as compared to HIV-unexposed newborns. IgG responses to pertussis vaccination as well as maternal IgG levels and quality in HEU newborns were inversely correlated with IA in PWH.Interpretation: Maternal HIV infection is associated with defective humoral immune responses to Tdap vaccination that correlate with IA. Suboptimal transfer of maternal immunity may further increase the risk of severe pertussis infection in HEU newborns.Trial Registration: Registered on ClinicalTrials.Gov (NCT03519373).Funding: This work was supported by IRIS Fund managed by the Foundation Roi Baudouin [2017J1820690206902], Association Vésale pour la Recherche Médicale and the Medical Council of CHU Saint-Pierre.Declaration of Interest: The authors have declared that no conflict of interest exists. N.D. is a post-doctorate clinical master specialist and A.M. is Research Director at the Fonds de la Recherche Scientifique (F.R.S.-FNRS), Belgium. M.E.A. was partially supported by NIH NIAID 1U19AI14825.Ethical Approval: The study was conducted in accordance with the Helsinki Declaration of the World Medical Association, approved by the local ethic committee (#B076201630448). Written informed consent of all participating subjects was received prior to participation.
Proprotein convertase subtilisin/kexin type 9 (PCSK9) regulates serum LDL cholesterol (LDL-C) levels by facilitating the degradation of the LDL receptor (LDLR) and is an attractive therapeutic target for hypercholesterolemia intervention. Herein, we generated a novel fully human antibody with favourable druggability by utilizing phage display-based strategy.A potent single-chain variable fragment (scFv) named AP2M21 was obtained by screening a fully human scFv phage display library with hPCSK9, and performing two in vitro affinity maturation processes including CDR-targeted tailored mutagenesis and cross-cloning. Thereafter, it was transformed to a full-length Fc-silenced anti-PCSK9 antibody FAP2M21 by fusing to a modified human IgG1 Fc fragment with L234A/L235A/N297G mutations and C-terminal lysine deletion, thus eliminating its immune effector functions and mitigating mAb heterogeneity.Our data showed that the generated full-length anti-PCSK9 antibody FAP2M21 binds to hPCSK9 with a KD as low as 1.42 nM, and a dramatically slow dissociation rate (koff, 4.68 × 10-6 s-1), which could be attributed to its lower binding energy (-47.51 kcal/mol) than its parent counterpart FAP2 (-30.39 kcal/mol). We verified that FAP2M21 potently inhibited PCSK9-induced reduction of LDL-C uptake in HepG2 cells, with an EC50 of 43.56 nM. Further, in hPCSK9 overexpressed C57BL/6 mice, a single tail i.v. injection of FAP2M21 at 1, 3 and 10 mg/kg, dose-dependently up-regulated hepatic LDLR levels, and concomitantly reduced serum LDL-C by 3.3% (P = 0.658, unpaired Student's t-test), 30.2% (P = 0.002, Mann-Whitney U-test) and 37.2% (P = 0.002, Mann-Whitney U-test), respectively.FAP2M21 with potent inhibitory effect on PCSK9 may serve as a promising therapeutic agent for treating hypercholesterolemia and associated cardiovascular diseases.
We have been investigating modulation strategies tailored around the selective stimulation of the host's immune system as an alternative to direct targeting of microbial pathogens by antibiotics. One such approach is the use of a group of small cationic peptides (BT) produced by a Gram-positive soil bacterium, Brevibacillus texasporus. These peptides have immune modulatory properties that enhance both leukocyte functional efficiency and leukocyte proinflammatory cytokine and chemokine mRNA transcription activities in vitro. In addition, when provided as a feed additive for just 4 days posthatch, BT peptides significantly induce a concentration-dependent protection against cecal and extraintestinal colonization by Salmonella enterica serovar Enteritidis. In the present studies, we assessed the effects of feeding BT peptides on transcriptional changes on proinflammatory cytokines, inflammatory chemokines, and Toll-like receptors (TLR) in the ceca of broiler chickens with and without S. Enteritidis infection. After feeding a BT peptide-supplemented diet for the first 4 days posthatch, chickens were then challenged with S. Enteritidis, and intestinal gene expression was measured at 1 or 7 days postinfection (p.i.) (5 or 11 days of age). Intestinal expression of innate immune mRNA transcripts was analyzed by quantitative real-time PCR (qRT-PCR). Analysis of relative mRNA expression showed that a BT peptide-supplemented diet did not directly induce the transcription of proinflammatory cytokine, inflammatory chemokine, type I/II interferon (IFN), or TLR mRNA in chicken cecum. However, feeding the BT peptide-supplemented diet primed cecal tissue for increased (P ≤ 0.05) transcription of TLR4, TLR15, and TLR21 upon infection with S. Enteritidis on days 1 and 7 p.i. Likewise, feeding the BT peptides primed the cecal tissue for increased transcription of proinflammatory cytokines (interleukin 1β [IL-1β], IL-6, IL-18, type I and II IFNs) and inflammatory chemokine (CxCLi2) in response to S. Enteritidis infection 1 and 7 days p.i. compared to the chickens fed the basal diet. These small cationic peptides may prove useful as alternatives to antibiotics as local immune modulators in neonatal poultry by providing prophylactic protection against Salmonella infections.
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