A High-Affinity CDR-Grafted Antibody against Influenza A H5N1 Viruses Recognizes a Conserved Epitope of H5 Hemagglutinin

<div><p>Highly pathogenic avian influenza (HPAI) H5N1 virus infection is still a potential threat to public health worldwide. While vaccines and antiviral drugs are currently under development, neutralizing antibodies could offer an alternative strategy to prevent and treat H5N1 virus infection. In the present study, we had developed a humanized antibody against H5N1 viruses from mouse-derived hybridoma in order to minimize its immunogenicity for potential clinical application. The humanized antibody hH5M9 was generated by transferring the mouse complementarity determining region (CDR) residues together with four key framework region (FR) residues onto the FR of the human antibody. This humanized antibody exhibited high affinity and specificity comparable to the parental mouse or chimeric counterpart with broad and strong neutralization activity against all H5N1 clades and subclades except for Egypt clades investigated. Furthermore, through epitope mapping we identified a linear epitope on the top region of hemagglutinin (HA) that was H5N1 specific and conserved. Our results for the first time reported a humanized antibody against H5N1 viruses by CDR grafting method. With the expected lower immunogenicity, this humanized antibody was expected to be more efficacious than murine or human-mouse chimeric antibodies for future application in humans.</p></div

Amino acid residues in HA1 of A/Egypt/N05056/09 different from the other H5N1 viruses.

a<p>Amino acid numbering is based on H5 HA protein.</p

Epitope mapping of hH5M9 to site-directed mutant HAs from A/Anhui/1/05 by IFA.

a<p>The number represented amino acid position in H5 HA.</p>b<p>A rabbit polyclonal antibody against A/Anhui/1/05 was used as positive control (PC).</p>c<p>IFA were performed on 293T cells transfected with mutant HA constructions. (+) to (+++) indicated the relative intensity of fluorescence.</p

HI titers of three antibodies against Influenza A viruses.

<p>HI titers of three antibodies against Influenza A viruses.</p

The distribution of linear hH5M9 epitope among H5N1 virus strains from Influenza Research Database by Sep 2012.

<p>The distribution of linear hH5M9 epitope among H5N1 virus strains from Influenza Research Database by Sep 2012.</p

Enhancing the Magnetic Anisotropy of Linear Cr(II) Chain Compounds Using Heavy Metal Substitutions

Magnetic properties of the series of three linear, trimetallic chain compounds Cr₂Cr­(dpa)₄Cl₂, <b>1</b>, Mo₂Cr­(dpa)₄Cl₂, <b>2</b>, and W₂Cr­(dpa)₄Cl₂, <b>3</b> (dpa = 2,2′-dipyridylamido), have been studied using variable-temperature dc and ac magnetometry and high-frequency EPR spectroscopy. All three compounds possess an <i>S</i> = 2 electronic ground state arising from the terminal Cr²⁺ ion, which exhibits slow magnetic relaxation under an applied magnetic field, as evidenced by ac magnetic susceptibility and magnetization measurements. The slow relaxation stems from the existence of an easy-axis magnetic anisotropy, which is bolstered by the axial symmetry of the compounds and has been quantified through rigorous high-frequency EPR measurements. The magnitude of <i>D</i> in these compounds increases when heavier ions are substituted into the trimetallic chain; thus <i>D</i> = −1.640, −2.187, and −3.617 cm^–1 for Cr₂Cr­(dpa)₄Cl₂, Mo₂Cr­(dpa)₄Cl₂, and W₂Cr­(dpa)₄Cl₂, respectively. Additionally, the <i>D</i> value measured for W₂Cr­(dpa)₄Cl₂ is the largest yet reported for a high-spin Cr²⁺ system. While earlier studies have demonstrated that ligands containing heavy atoms can enhance magnetic anisotropy, this is the first report of this phenomenon using heavy metal atoms as “ligands”

Generation of chimeric cH5M9 and CDR-grafted hH5M9 antibodies.

<p>(A) Amino acid sequences of the VH regions of mouse antibody mH5M9, human antibody FabOX108 and CDR-grafted antibody hH5M9. Amino acid residues were listed according to the convention of Kabat et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088777#pone.0088777-Kabat1" target="_blank">[31]</a>. Residues shown by underline in frameworks were deemed essential for maintaining the combining sites of mH5M9. Dashes indicated residues that were identical in mH5M9, FabOX108 and hH5M9 whereas gaps denoted amino acid residues missing at those positions. (B) SDS-PAGE analysis of purified cH5M9 and hH5M9 under reducing conditions. mH5M9 was used as the positive control.</p

Epitope mapping of hH5M9.

<p>(A) Western blotting analysis of hH5M9 with HA. Purified A/Anhui/1/05 HA was applied to SDS-PAGE under reducing conditions. The hH5M9 was used as primary antibody. A rabbit polyclonal antibody against A/Anhui/1/05 was used as positive control (PC). (B) Binding analysis of synthesized epitope “KPNDAINF” with hH5M9 was measured by an indirect ELISA. (C) Schematic representation of the epitope recognized by hH5M9. The linear epitope (KPNDAINF, amino acids 234–241) on the three dimensional structure of the H5 HA (VN1194) mono-structure (PDB No. 2IBX) was colored in green while the receptor binding domain (RBD) was highlighted in yellow. (D) Cartoon illustration of the three dimensional structure of the linear epitope for hH5M9.</p

Table3_Application of circulating tumour DNA in terms of prognosis prediction in Chinese follicular lymphoma patients.XLSX

Background: Follicular lymphoma (FL), an indolent non-Hodgkin lymphoma (NHL), is generally incurable. Favourable prognosis and durable remission are crucial for FL patients. The genetic mutation spectrum provides novel biomarkers for determining the prognosis of FL patients, but its detection is easily affected by the collection of tumour tissue biopsies. In this study, we aimed to describe the mutational landscape of FL using circulating tumour DNA (ctDNA) samples and to explore the relationship between mutations and prognostic indicators of clinical outcome in patients with newly diagnosed follicular lymphoma and the prognostic value of such mutations.Methods: A total of 28 patients with newly diagnosed FL were included in this study. A targeted NGS-based 59-gene panel was used to assess the ctDNA mutation profiles. Differences in clinical factors between patients carrying mutations and those without mutations were analysed. We also explored the relationship between gene mutation status, mean VAFs (variant allele frequencies) and clinical factors. The Kaplan‒Meier method was applied to analyse the overall survival (OS) and progression-free survival (PFS) of patients carrying mutations and those without mutations.Results: ctDNA mutations were detectable in 21 (75%) patients. The most commonly mutated genes were CREBBP (54%, 15/28), KMT2D (50%, 14/28), STAT6 (29%, 8/28), CARD11 (18%, 5/28), PCLO (14%, 4/28), EP300 (14%, 4/28), BCL2 (11%, 3/28), and TNFAIP3 (11%, 3/28), with a mutation frequency of >10%. Patients with detectable ctDNA mutation tended to present with advanced Ann Arbor stage (III-IV) (p = 0.009), high FLIPI risk (3–5) (p = 0.023) and severe lymph node involvement (No. of involved areas ≥5) (p = 0.02). In addition, we found that the mean VAF was significantly higher in patients with advanced Ann Arbor stage, high-risk FLIPI, elevated lactate dehydrogenase (LDH: 0–248U/L), advanced pathology grade, bone marrow involvement (BMI) and lymph node involvement. Additionally, KMT2D, EP300, and STAT6 mutations were associated with inferior PFS (p Conclusion: We described the ctDNA mutation landscapes in Chinese patients with newly diagnosed FL and found that ctDNA VAF means reflect tumour burden. Moreover, PFS was shorter in patients with KMT2D, EP300 and STAT6 mutations.</p

Table1_Application of circulating tumour DNA in terms of prognosis prediction in Chinese follicular lymphoma patients.XLSX

Background: Follicular lymphoma (FL), an indolent non-Hodgkin lymphoma (NHL), is generally incurable. Favourable prognosis and durable remission are crucial for FL patients. The genetic mutation spectrum provides novel biomarkers for determining the prognosis of FL patients, but its detection is easily affected by the collection of tumour tissue biopsies. In this study, we aimed to describe the mutational landscape of FL using circulating tumour DNA (ctDNA) samples and to explore the relationship between mutations and prognostic indicators of clinical outcome in patients with newly diagnosed follicular lymphoma and the prognostic value of such mutations.Methods: A total of 28 patients with newly diagnosed FL were included in this study. A targeted NGS-based 59-gene panel was used to assess the ctDNA mutation profiles. Differences in clinical factors between patients carrying mutations and those without mutations were analysed. We also explored the relationship between gene mutation status, mean VAFs (variant allele frequencies) and clinical factors. The Kaplan‒Meier method was applied to analyse the overall survival (OS) and progression-free survival (PFS) of patients carrying mutations and those without mutations.Results: ctDNA mutations were detectable in 21 (75%) patients. The most commonly mutated genes were CREBBP (54%, 15/28), KMT2D (50%, 14/28), STAT6 (29%, 8/28), CARD11 (18%, 5/28), PCLO (14%, 4/28), EP300 (14%, 4/28), BCL2 (11%, 3/28), and TNFAIP3 (11%, 3/28), with a mutation frequency of >10%. Patients with detectable ctDNA mutation tended to present with advanced Ann Arbor stage (III-IV) (p = 0.009), high FLIPI risk (3–5) (p = 0.023) and severe lymph node involvement (No. of involved areas ≥5) (p = 0.02). In addition, we found that the mean VAF was significantly higher in patients with advanced Ann Arbor stage, high-risk FLIPI, elevated lactate dehydrogenase (LDH: 0–248U/L), advanced pathology grade, bone marrow involvement (BMI) and lymph node involvement. Additionally, KMT2D, EP300, and STAT6 mutations were associated with inferior PFS (p Conclusion: We described the ctDNA mutation landscapes in Chinese patients with newly diagnosed FL and found that ctDNA VAF means reflect tumour burden. Moreover, PFS was shorter in patients with KMT2D, EP300 and STAT6 mutations.</p