4E10 anti-mper antigorputzaren optimizazioa: CDR-H3 begiztaren aldaketen ebaluazioa

VIH-ren fusioa inhibitzeko gai diren antigorputzen adierazpena eta purifikazioa bakterietan. Funtzionaltasunaren analisia (ITC eta pseudobirusak erabiliz egindako neutralizazio saioak) eta optimizazioa

Promotion of membrane interactions as a pathway for HIV antibody optimization

192 p.GIBak Env fusio glukoproteina erabiltzen du CD4 hartzailea adierazten duten zelulak infektatzeko. Immunitate sistemari eskuragarri dagoen antigeno bakarra izan arren, Env-ek honi ihes egiteko dituen mekanismoek erantzun egoki baten garapena izugarri zailtzen dute. Hala ere, azken urteetan GIBaren infekzioa blokeatzeko gaitasuna erakusten duten espektro zabaleko antigorputz neutralizatzaileak isolatu dira paziente batzuetatik, eta gaur egun erabiltzen den terapia antierretrobiralean oinarritzen den tratamenduaren osagarri edota ordezko aukera bezala proposatuak izan dira. Antigorputz neutralizatzaile hauen artean, mintz biralean txertatuta aurkitzen den MPER (ingelesetik, ¿membrane proximal external region¿) sekuentzia itu dutenak erakutsi dute espektro zabalena eta tesi lan honetan, hauen potentzia emendatzeko birusaren mintzarekin egiten dituzten elkarrekintzak bultzatu nahi izan dira. Anti-MPER antigorputzak mutagenesi konbentzionala erabiliz, zuzendutako konposatu aromatikoen konjugazioaren bidez eta espezifitate desberdineko bigarren batuketa-bloke bat erantsiz eraldatuak izan ondoren,antigorputz basatiak baino 10-100 aldiz potentzia altuagoa duten aldaerak eskuratu dira, antigorputzen eta mintzen interfasearen arteko elkarrekintzen sustapena potentzia emendatzeko estrategia egokia dela frogatuz

Promotion of membrane interactions as a pathway for HIV antibody optimization

192 p.GIBak Env fusio glukoproteina erabiltzen du CD4 hartzailea adierazten duten zelulak infektatzeko. Immunitate sistemari eskuragarri dagoen antigeno bakarra izan arren, Env-ek honi ihes egiteko dituen mekanismoek erantzun egoki baten garapena izugarri zailtzen dute. Hala ere, azken urteetan GIBaren infekzioa blokeatzeko gaitasuna erakusten duten espektro zabaleko antigorputz neutralizatzaileak isolatu dira paziente batzuetatik, eta gaur egun erabiltzen den terapia antierretrobiralean oinarritzen den tratamenduaren osagarri edota ordezko aukera bezala proposatuak izan dira. Antigorputz neutralizatzaile hauen artean, mintz biralean txertatuta aurkitzen den MPER (ingelesetik, ¿membrane proximal external region¿) sekuentzia itu dutenak erakutsi dute espektro zabalena eta tesi lan honetan, hauen potentzia emendatzeko birusaren mintzarekin egiten dituzten elkarrekintzak bultzatu nahi izan dira. Anti-MPER antigorputzak mutagenesi konbentzionala erabiliz, zuzendutako konposatu aromatikoen konjugazioaren bidez eta espezifitate desberdineko bigarren batuketa-bloke bat erantsiz eraldatuak izan ondoren,antigorputz basatiak baino 10-100 aldiz potentzia altuagoa duten aldaerak eskuratu dira, antigorputzen eta mintzen interfasearen arteko elkarrekintzen sustapena potentzia emendatzeko estrategia egokia dela frogatuz

Molecular Recognition of the Native HIV-1 MPER Revealed by STED Microscopy of Single Virions

Antibodies against the Membrane-Proximal External Region (MPER) of the Env gp41 subunit neutralize HIV-1 with exceptional breadth and potency. Due to the lack of knowledge on the MPER native structure and accessibility, different and exclusive models have been proposed for the molecular mechanism of MPER recognition by broadly neutralizing antibodies. Here, accessibility of antibodies to the native Env MPER on single virions has been addressed through STED microscopy. STED imaging of fluorescently labeled Fabs reveals a common pattern of native Env recognition for HIV-1 antibodies targeting MPER or the surface subunit gp120. In the case of anti-MPER antibodies, the process evolves with extra contribution of interactions with the viral lipid membrane to binding specificity. Our data provide biophysical insights into the recognition of the potent and broadly neutralizing MPER epitope on HIV virions, and as such is of importance for the design of therapeutic interventions.This study was supported by the Spanish MINECO (BIO2015-64421-R (MINECO/ FEDER UE) to J.L.N.) and the Basque Government (IT838-13 to J.L.N.). P.C., E.R., and S. I. received pre-doctoral fellowships from the Basque Government. P.C. would like to acknowledge the European Biophysical Societies’ Association (EBSA) for receiving an EBSA Bursary for a working visit to a laboratory in an EBSA country. J.C., D.W., and C. E. greatly acknowledge support by the MRC (grant number MC_UU_12010/unit programs G0902418 and MC_UU_12025), the Wellcome Trust (grant 104924/14/Z/14 and Strategic Award 091911 (Micron)), MRC/BBSRC/EPSRC (grant MR/K01577X/1), BBSRC (Deutsche Forschungsgemeinschaft (Research unit 1905 “Structure and function of the peroxisomal translocon”)), the Wolfson Foundation (for initial funding of the Wolfson Imaging Centre Oxford), the EPA Cephalosporin Fund and the John Fell Fund. T.S. is a recipient of a Canada Graduate Scholarship Master’s Award and a Vanier Canada Graduate Scholarship from the Canadian Institutes of Health Research. This work was supported by operating grant NIH-150414 (J.-P.J.) from the Canadian Institutes of Health Research. This research was undertaken, in part, thanks to funding from the Canada Research Chairs program (J.-P.J.). We acknowledge valuable technical assistance from Miguel García-Porra

Engineering pan–HIV-1 neutralization potency through multispecific antibody avidity

Deep mining of B cell repertoires of HIV-1-infected individuals has resulted in the isolation of dozens of HIV-1 broadly neutralizing antibodies (bNAbs). Yet, it remains uncertain whether any such bNAbs alone are sufficiently broad and potent to deploy therapeutically. Here, we engineered HIV-1 bNAbs for their combination on a single multispecific and avid molecule via direct genetic fusion of their Fab fragments to the human apoferritin light chain. The resulting molecule demonstrated a remarkable median IC50 value of 0.0009 g/mL and 100% neutralization coverage of a broad HIV-1 pseudovirus panel (118 isolates) at a 4 g/mL cutoff-a 32-fold enhancement in viral neutralization potency compared to a mixture of the corresponding HIV-1 bNAbs. Importantly, Fc incorporation on the molecule and engineering to modulate Fc receptor binding resulted in IgG-like bioavailability invivo. This robust plug-and-play antibody design is relevant against indications where multispecificity and avidity are leveraged simultaneously to mediate optimal biological activity.The following reagents were obtained through the NIH AIDS Reagent Program, Division of AIDS, National Institute of Allergy and Infectious Diseases: TZM-bl cells (ARP-8129; contributed by Dr. John C. Kappes and Dr. Xiaoyun Wu); anti–HIV-1 gp160 monoclonal antibody (N6/ PGDM1400x10E8v4) (ARP-13390; contributed by Drs. Ling Xu and Gary Nabel); HIV-1 NL4-3 ΔEnv Vpr luciferase reporter vector (pNL4-3.Luc.R-E-) (ARP-3418; contributed by Dr. Nathaniel Landau and Aaron Diamond); plasmids pcDNA3.1 D/V5-His TOPO-expressing HIV-1 Env/Rev (ARP-11017, ARP-11018, ARP-11024, and ARP-11022; contributed by Drs. David Montefiori, Feng Gao, and Ming Li); plasmid pcDNA3.1(+)-expressing HIV-1 Env/Rev (ARP-11037; contributed by Drs. B. H. Hahn and D. L. Kothe); plasmid pcDNA3.1 D/V5-His TOPO-expressing HIV-1 Env/Rev (ARP-11308; contributed by Drs. D. Montefiori, F. Gao, C. Wil- liamson, and S. Abdool Karim); plasmid pcDNA3.1 V5-His TOPO-expressing HIV-1 Env/Rev (ARP-11309; contributed by Drs. B. H. Hahn, Y. Li, and J. F. Sala- zar-Gonzalez); HIV-1 BG505 Env expression vector (BG505.W6M.ENV.C2) (ARP- 11518; contributed by Dr. Julie Overbaugh); HIV-1 Env expression vector (CRF02_AG clone 257) (ARP-11599; contributed by Drs. D. Ellenberger, B. Li, M. Callahan, and S. Butera); plasmid pcDNA3.1 V5-His TOPO-expressing HIV-1 CNE8 Env (ARP-12653; contributed by Drs. Linqi Zhang, Hong Shang, David Montefiori, Tsinghua University (Beijing, China), China Medical University (Bei- jing, China), and Duke University (Durham, NC); HIV-1 SF162 gp160 expression vector (ARP-10463; contributed by Drs. Leonidas Stamatatos and Cecilia Cheng- Mayer); plasmid pcDNA3.1 V5-His TOPO-expressing HIV-1 Env/Rev (ARP-11034; contributed by Drs. B. H. Hahn, X. Wei, and G. M. Shaw); plasmid pcDNA3.1/V5- His TOPO-expressing HIV Env/Rev (ARP-11038; contributed by Drs. B. H. Hahn and D. L. Kothe); plasmid pcDNA3.1 V5-His TOPO-expressing HIV-1 Env/Rev (ARP-11310; contributed by Drs. B. H. Hahn, Y. Li, and J. F. Salazar-Gonzalez); HIV-1 Env expression vector (p16845 env) (ARP-11503; contributed by Drs. R. Paranjape, S. Kulkarni, and D. Montefiori); HIV-1 1054 Env expression vector (p1054.TC4.1499) (ARP-11561) and 6244 Env expression vector (p6244_13.B5.4576) (ARP-11566; contributed by Drs. Beatrice H. Hahn, Brandon F. Keele, and George M. Shaw); HIV-1 ZM246F Env expression vector (pZM246F_C1G) (ARP-11830; contributed by Dr. Beatrice Hahn); HIV-1 Env expression vector (CRF02_AG clone 278) (ARP-11605; contributed by Drs. Michael Thomson, Ana Revilla, Elena Delgado, David Montefiori, Sonia P erez Castro, Centro Nacional de Microbiologia, Instituto de Salud Carlos III (Majada- honda, Madrid, Spain), Complejo Hospitalario Santa Mar ıa Madre (Orense, Spain), Duke University (Durham, NC), and the CAVD; and NL4-3 Env expression vector (pDOLHIVenv) (from Dr. Eric Freed and Dr. Rex Risser). The following reagents were kindly provided by CAVD: X2988, ZM106.9, and 3817. We thank S. Tabruyn and F. Arbogast for their assistance with in vivo studies. We thank the SickKids-University Health Network Flow Cytometry Facility. This work wassupported by Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grant 6280100058 (J.-P.J.) and by Operating Grant PJ4- 169662 from the Canadian Institutes of Health Research (CIHR; B.T. and J.-P.J.). This research was also supported by the European Union’s Horizon 2020 research and innovation program under Marie Sklodowska-Curie Grant 790012 (E.R.), a Hospital for Sick Children Restracomp Postdoctoral Fellowship (C.B.A.), an NSERC postgraduate doctoral scholarship (T.Z.), a predoctoral fel- lowship from the Basque Government (PRE_2019_2_0046) (S.I.), the Canadian Institute for Advanced Research (CIFAR) Azrieli Global Scholar program (J.-P.J.), the Ontario Early Researcher Awards program (J.-P.J.), and the CanadaResearch Chairs program (B.T. and J.-P.J.). This work was supported, in part, by NSERC Discovery Grant RGPIN-2019-06442 and CIHR Project Grant–Priority Announcement PJH-175379 to C.G., and a CIHR Canada Graduate Scholarship (CGS-M) to J.B. Further support was obtained from the Spanish Ministry of Sci- ence, Innovation and Universities (MCIU) with the support of the Spanish Research Agency/The European Regional Development Fund (AEI/FEDER) (RTI2018-095624-B-C21) (J.L.N.) and the Basque Government (IT1196-19) (J.L.N.). Biophysical data were collected at the Structural & Biophysical Core facility supported by the Canada Foundation for Innovation and Ontario Research Fun

Functional Delineation of a Protein–Membrane Interaction Hotspot Site on the HIV-1 Neutralizing Antibody 10E8

Antibody engagement with the membrane-proximal external region (MPER) of the envelope glycoprotein (Env) of HIV-1 constitutes a distinctive molecular recognition phenomenon, the full appreciation of which is crucial for understanding the mechanisms that underlie the broad neutralization of the virus. Recognition of the HIV-1 Env antigen seems to depend on two specific features developed by antibodies with MPER specificity: (i) a large cavity at the antigen-binding site that holds the epitope amphipathic helix; and (ii) a membrane-accommodating Fab surface that engages with viral phospholipids. Thus, besides the main Fab–peptide interaction, molecular recognition of MPER depends on semi-specific (electrostatic and hydrophobic) interactions with membranes and, reportedly, on specific binding to the phospholipid head groups. Here, based on available cryo-EM structures of Fab–Env complexes of the anti-MPER antibody 10E8, we sought to delineate the functional antibody–membrane interface using as the defining criterion the neutralization potency and binding affinity improvements induced by Arg substitutions. This rational, Arg-based mutagenesis strategy revealed the position-dependent contribution of electrostatic interactions upon inclusion of Arg-s at the CDR1, CDR2 or FR3 of the Fab light chain. Moreover, the contribution of the most effective Arg-s increased the potency enhancement induced by inclusion of a hydrophobic-at-interface Phe at position 100c of the heavy chain CDR3. In combination, the potency and affinity improvements by Arg residues delineated a protein–membrane interaction site, whose surface and position support a possible mechanism of action for 10E8-induced neutralization. Functional delineation of membrane-interacting patches could open new lines of research to optimize antibodies of therapeutic interest that target integral membrane epitopes.This study was supported by the Spanish MCIN (Grants PID2021-126014OB-I00 MCIN/AEI/FEDER, UE to JLN and BA; and PID2021-122212OA-I00 MCIN/AEI/FEDER, UE to ER), Basque Government (Grant: IT1449-22) and JSPS KAKENHI 20H03228 (to J.M.M.C.)

Affinity for the Interface Underpins Potency of Antibodies Operating In Membrane Environments

The contribution of membrane interfacial interactions to recognition of membrane-embedded antigens by antibodies is currently unclear. This report demonstrates the optimization of this type of antibodies via chemical modification of regions near the membrane but not directly involved in the recognition of the epitope. Using the HIV-1 antibody 10E8 as a model, linear and polycyclic synthetic aromatic compounds are introduced at selected sites. Molecular dynamics simulations predict the favorable interactions of these synthetic compounds with the viral lipid membrane, where the epitope of the HIV-1 glycoprotein Env is located. Chemical modification of 10E8 with aromatic acetamides facilitates the productive and specific recognition of the native antigen, partially buried in the crowded environment of the viral membrane, resulting in a dramatic increase of its capacity to block viral infection. These observations support the harnessing of interfacial affinity through site-selective chemical modification to optimize the function of antibodies that target membrane-proximal epitopes.We are grateful to Professor Ueda (Kyushu University) for valuable advice. C.D. acknowledges RES (Red Espanola de Supercomputacio ' n) for providing computational resources. S.I. received a pre-doctoral fellowship from the Basque Government. P.C. acknowledges a research associate contract from the University of the Basque Country (DOCREC18/01) and a postdoctoral fellowship from the Basque Government (POS_2018_1_0066).This study was supported by the following grants: European Commission (790012 SI H2020MSCA-IF-2017 to E.R., J.-P.J., and J.L.N.); US NIAID (NIH) (R01 AI143563 to M.B.Z.); James B. Pendleton Charitable Trust (to M.B.Z.); Grant-in-Aid for Scientific Research on Innovative Areas "Chemistry for Multimolecular Crowding Biosystems, JSPS KAKENHI (JP17H06349 to A.O.); JSPS KAKENHI (15K06962 and 20H03228 to J.M.M.C.); Spanish MINECO (BIO2015-64421R and MINECO/AEI/FEDER, UE to J.L.N.); Spanish MCIU (RTI2018-095624B-C21 and MCIU/AEI/FEDER, UE to J.L.N.); and the Basque Government (IT1196-19) (to J.L.N.). C.E. acknowledges funding from Medical Research Council (MC_UU_12010/unit programs G0902418 and MC_UU_12025), Wolfson Foundation, Deutsche Forschungsgemeinschaft (Research unit 1905, Excellence Cluster Balance of the Microverse, Collaborative Research Centre 1278 Polytarget), Wellcome Institutional Strategic Support Fund, Oxford internal funds (EPA Cephalosporin Fund and John Fell Fund), and support from the Micron Oxford Advanced Bioimaging Unit (Wellcome Trust funding 107457/Z/15/Z). This research was undertaken, in part, thanks to funding from the CIFAR Azrieli Global Scholar program (to J.-P.J.) and the Canada Research Chairs program (950-231604 to J.-P.J.). This work was also supported by the Platform Project for Supporting Drug Discovery and Life Science Research (Basis for Supporting Innovative Drug Discovery and Life Science Research [BINDS] from AMED JP19am0101091)

Focal accumulation of aromaticity at the CDRH3 loop mitigates 4E10 polyreactivity without altering its HIV neutralization profile

Broadly neutralizing antibodies (bnAbs) against HIV-1 are frequently associated with the presence of autoreactivity/polyreactivity, a property that can limit their use as therapeutic agents. The bnAb 4E10, targeting the conserved Membrane proximal external region (MPER) of HIV-1, displays almost pan-neutralizing activity across globally circulating HIV-1 strains but exhibits nonspecific off-target interactions with lipid membranes. The hydrophobic apex of the third complementarity-determining region of the heavy chain (CDRH3) loop, which is essential for viral neutralization, critically contributes to this detrimental effect. Here, we have replaced the aromatic/hydrophobic residues from the apex of the CDRH3 of 4E10 with a single aromatic molecule through chemical modification to generate a variant that preserves the neutralization potency and breadth of 4E10 but with reduced autoreactivity. Collectively, our study suggests that the localized accumulation of aromaticity by chemical modification provides a pathway to ameliorate the adverse effects triggered by the CDRH3 of anti-HIV-1 MPER bnAbs.This study was supported by the following Grants: European Commission (790012 SI H2020-MSCA-IF-2017) (E.R.); US NIAID, NIH grant R01 AI143563 (M.B. Z.); James B. Pendleton Charitable Trust (M.B.Z.); JSPS grant 20H03228 (J. M.M.C.); Spanish MCIU (RTI2018-095624-B-C21; MCIU/AEI/FEDER, UE) (J.L.N.), Basque Government (IT1196-19) (J.L.N.). C.E. acknowledges funding from Medical Research Council (grant number MC_UU_12010/unit programs G0902418 and MC_UU_12025), Wolfson Foundation, Deutsche Forschungsgemeinschaft (Excellence Cluster Balance of the Microverse, Collaborative Research Center 1278 Polytarget), Leibniz Association (Leibniz Campus Infectooptics), Wellcome Institutional Strategic Support Fund, Oxford internal funds (EPA Cephalosporin Fund and John Fell Fund), and support from the Micron Oxford Advanced Bioimaging Unit (Wellcome Trust funding 107457/Z/15/Z). This work was also supported by the Platform Project for Supporting Drug Discovery and Life Science Research [Basis for Supporting Innovative Drug Discovery and Life Science Research (BINDS)] from AMED (JP21am0101091). S.I. received a predoctoral fellowship from the BasqueGovernment. P.C. would like to acknowledge the University of the Basque Country (DOCREC18/01), the Basque Government (POS_2018_1_0066) and the European Commission (H2020-MSCA-IF-2019-ST project 892232 FILM-HIV) for funding his position. This research was also supported by the CIFAR Azrieli Global Scholar program (J-P.J.), the Ontario Early Researcher Awards program (J-P.J.), and the Canada Research Chairs program (J-P.J.). Part of the biophysical data presented in this manuscript were collected at the Hospital for Sick Children Structural & Biophysical Core facility supported by the Canada Foundation for Innovation and Ontario Research Fund

4E10 anti-mper antigorputzaren optimizazioa: CDR-H3 begiztaren aldaketen ebaluazioa

VIH-ren fusioa inhibitzeko gai diren antigorputzen adierazpena eta purifikazioa bakterietan. Funtzionaltasunaren analisia (ITC eta pseudobirusak erabiliz egindako neutralizazio saioak) eta optimizazioa

The Error of Estimated GFR in Type 2 Diabetes Mellitus

Type 2 diabetes mellitus represents 30–50% of the cases of end stage renal disease worldwide. Thus, a correct evaluation of renal function in patients with diabetes is crucial to prevent or ameliorate diabetes-associated kidney disease. The reliability of formulas to estimate renal function is still unclear, in particular, those new equations based on cystatin-C or the combination of creatinine and cystatin-C. We aimed to assess the error of the available formulas to estimate glomerular filtration rate in diabetic patients. We evaluated the error of creatinine and/or cystatin-C based formulas in reflecting real renal function over a wide range of glomerular filtration rate (from advanced chronic kidney disease to hyperfiltration). The error of estimated glomerular filtration rate by any equation was common and wide averaging 30% of real renal function, and larger in patients with measured glomerular filtration rate below 60 mL/min. This led to chronic kidney disease stages misclassification in about 30% of the individuals and failed to detect 25% of the cases with hyperfiltration. Cystatin-C based formulas did not outperform creatinine based equations, and the reliability of more modern algorithms proved to be as poor as older equations. Formulas failed in reflecting renal function in type 2 diabetes mellitus. Caution is needed with the use of these formulas in patients with diabetes, a population at high risk for kidney disease. Whenever possible, the use of a gold standard method to measure renal function is recommended