INTRODUCTION: Imprinting broadly neutralizing antibody (bNAb) paratopes by shape complementary protein mimotopes represents a potential alternative for developing vaccine immunogens. This approach, designated as a Non-Cognate Ligand Strategy (NCLS), has recently been used for the identification of protein variants mimicking CD4 binding region epitope or membrane proximal external region (MPER) epitope of HIV-1 envelope (Env) glycoprotein. However, the potential of small binding proteins to mimic viral glycan-containing epitopes has not yet been verified. METHODS: In this work, we employed a highly complex combinatorial Myomedin scaffold library to identify variants recognizing paratopes of super candidate bNAbs, PGT121 and PGT126, specific for HIV-1 V3 loop epitopes. RESULTS: In the collection of Myomedins called MLD variants targeted to PGT121, three candidates competed with gp120 for binding to this bNAb in ELISA, thus suggesting an overlapping binding site and epitope-mimicking potential. Myomedins targeted to PGT126 designated MLB also provided variants that competed with gp120. Immunization of mice with MLB or MLD binders resulted in the production of anti-gp120 and -Env serum antibodies. Mouse hyper-immune sera elicited with MLB036, MLB041, MLB049, and MLD108 moderately neutralized 8-to-10 of 22 tested HIV-1-pseudotyped viruses of A, B, and C clades in vitro. DISCUSSION: Our data demonstrate that Myomedin-derived variants can mimic particular V3 glycan epitopes of prominent anti-HIV-1 bNAbs, ascertain the potential of particular glycans controlling neutralizing sensitivity of individual HIV-1 pseudoviruses, and represent promising prophylactic candidates for HIV-1 vaccine development.

Department of Immunology Palacky University Olomouc Hnevotinska Olomouc Czechia

Laboratory of Ligand Engineering Institute of Biotechnology of the Czech Academy of Sciences BIOCEV Research Center Prumyslova Vestec Czechia

Laboratory of Structural Bioinformatics of Proteins Institute of Biotechnology of the Czech Academy of Sciences BIOCEV Research Center Prumyslova Vestec Czechia

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Wainhobson S. The fastest genome evolution ever described - hiv variation PubMed DOI

Rambaut A, Posada D, Crandall KA, Holmes EC. The causes and consequences of HIV evolution. Nat Rev Genet (2004) 5:52–61. doi: 10.1038/nrg1246 PubMed DOI

Robertson DL, Anderson JP, Bradac JA, Carr JK, Foley B, Funkhouser RK, et al. HIV-1 nomenclature proposal. Science (2000) 288:55–6. doi: 10.1126/science.288.5463.55d PubMed DOI

Plantier JC, Leoz M, Dickerson JE, De Oliveira F, Cordonnier F, Lemee V, et al. A new human immunodeficiency virus derived from gorillas. Nat Med (2009) 15:871–2. doi: 10.1038/nm.2016 PubMed DOI

Vallari A, Holzmayer V, Harris B, Yamaguchi J, Ngansop C, Makamche F, et al. Confirmation of putative HIV-1 group p in Cameroon. J Virol (2011) 85:1403–7. doi: 10.1128/JVI.02005-10 PubMed DOI PMC

Garcia-Knight MA, Slyker J, Payne BL, Pond SL, De Silva TI, Chohan B, et al. Viral evolution and cytotoxic T cell restricted selection in acute infant HIV-1 infection. Sci Rep (2016) 6:29536. doi: 10.1038/srep29536 PubMed DOI PMC

Zhu X, Borchers C, Bienstock RJ, Tomer KB. Mass spectrometric characterization of the glycosylation pattern of HIV-gp120 expressed in CHO cells. Biochemistry (2000) 39:11194–204. doi: 10.1021/bi000432m PubMed DOI

Mathys L, Balzarini J. The role of n-glycans of HIV-1 gp41 in virus infectivity and susceptibility to the suppressive effects of carbohydrate-binding agents. Retrovirology (2014) 11:107. doi: 10.1186/s12977-014-0107-7 PubMed DOI PMC

Julien JP, Cupo A, Sok D, Stanfield RL, Lyumkis D, Deller MC, et al. Crystal structure of a soluble cleaved HIV-1 envelope trimer. Science (2013) 342:1477–83. doi: 10.1126/science.1245625 PubMed DOI PMC

Zhang M, Gaschen B, Blay W, Foley B, Haigwood N, Kuiken C, et al. Tracking global patterns of n-linked glycosylation site variation in highly variable viral glycoproteins: HIV, SIV, and HCV envelopes and influenza hemagglutinin. Glycobiology (2004) 14:1229–46. doi: 10.1093/glycob/cwh106 PubMed DOI

Tomaras GD, Yates NL, Liu P, Qin L, Fouda GG, Chavez LL, et al. Initial b-cell responses to transmitted human immunodeficiency virus type 1: Virion-binding immunoglobulin m (IgM) and IgG antibodies followed by plasma anti-gp41 antibodies with ineffective control of initial viremia. J Virol (2008) 82:12449–63. doi: 10.1128/JVI.01708-08 PubMed DOI PMC

Legrand E, Pellegrin I, Neau D, Pellegrin JL, Ragnaud JM, Dupon M, et al. Course of specific T lymphocyte cytotoxicity, plasma and cellular viral loads, and neutralizing antibody titers in 17 recently seroconverted HIV type 1-infected patients. AIDS Res Hum Retroviruses (1997) 13:1383–94. doi: 10.1089/aid.1997.13.1383 PubMed DOI

Overbaugh J, Morris L. The antibody response against HIV-1. Cold Spring Harb Perspect Med (2012) 2:a007039. doi: 10.1101/cshperspect.a007039 PubMed DOI PMC

Zolla-Pazner S, Cardozo T. Structure-function relationships of HIV-1 envelope sequence-variable regions refocus vaccine design. Nat Rev Immunol (2010) 10:527–35. doi: 10.1038/nri2801 PubMed DOI PMC

Cardozo T, Kimura T, Philpott S, Weiser B, Burger H, Zolla-Pazner S. Structural basis for coreceptor selectivity by the HIV type 1 V3 loop. AIDS Res Hum Retroviruses (2007) 23:415–26. doi: 10.1089/aid.2006.0130 PubMed DOI

Cao J, Sullivan N, Desjardin E, Parolin C, Robinson J, Wyatt R, et al. Replication and neutralization of human immunodeficiency virus type 1 lacking the V1 and V2 variable loops of the gp120 envelope glycoprotein. J Virol (1997) 71:9808–12. doi: 10.1128/jvi.71.12.9808-9812.1997 PubMed DOI PMC

Davis KL, Bibollet-Ruche F, Li H, Decker JM, Kutsch O, Morris L, et al. Human immunodeficiency virus type 2 (HIV-2)/HIV-1 envelope chimeras detect high titers of broadly reactive HIV-1 V3-specific antibodies in human plasma. J Virol (2009) 83:1240–59. doi: 10.1128/JVI.01743-08 PubMed DOI PMC

Jiang X, Burke V, Totrov M, Williams C, Cardozo T, Gorny MK, et al. Conserved structural elements in the V3 crown of HIV-1 gp120. Nat Struct Mol Biol (2010) 17:955–61. doi: 10.1038/nsmb.1861 PubMed DOI

Pritchard LK, Vasiljevic S, Ozorowski G, Seabright GE, Cupo A, Ringe R, et al. Structural constraints determine the glycosylation of HIV-1 envelope trimers. Cell Rep (2015) 11:1604–13. doi: 10.1016/j.celrep.2015.05.017 PubMed DOI PMC

Bonomelli C, Doores KJ, Dunlop DC, Thaney V, Dwek RA, Burton DR, et al. The glycan shield of HIV is predominantly oligomannose independently of production system or viral clade. PloS One (2011) 6(8):e23521. doi: 10.1371/journal.pone.0023521 PubMed DOI PMC

Coss KP, Vasiljevic S, Pritchard LK, Krumm SA, Glaze M, Madzorera S, et al. HIV-1 glycan density drives the persistence of the mannose patch within an infected individual. J Virol (2016) 90:11132–44. doi: 10.1128/JVI.01542-16 PubMed DOI PMC

Trkola A, Purtscher M, Muster T, Ballaun C, Buchacher A, Sullivan N, et al. Human monoclonal antibody 2G12 defines a distinctive neutralization epitope on the gp120 glycoprotein of human immunodeficiency virus type 1. J Virol (1996) 70:1100–8. doi: 10.1128/jvi.70.2.1100-1108.1996 PubMed DOI PMC

Walker LM, Huber M, Doores KJ, Falkowska E, Pejchal R, Julien JP, et al. Broad neutralization coverage of HIV by multiple highly potent antibodies. Nature (2011) 477:466–70. doi: 10.1038/nature10373 PubMed DOI PMC

Kosztyu P, Kuchar M, Cerny J, Barkocziova L, Maly M, Petrokova H, et al. Proteins mimicking epitope of HIV-1 virus neutralizing antibody induce virus-neutralizing sera in mice. EBioMedicine (2019) 47:247–56. doi: 10.1016/j.ebiom.2019.07.015 PubMed DOI PMC

Klasse PJ. Non-cognate ligands of procrustean paratopes as potential vaccine components. Ebiomedicine (2019) 47:6–7. doi: 10.1016/j.ebiom.2019.07.040 PubMed DOI PMC

Kuchar M, Kosztyu P, Daniel Liskova V, Cerny J, Petrokova H, Vroblova E, et al. Myomedin scaffold variants targeted to 10E8 HIV-1 broadly neutralizing antibody mimic gp41 epitope and elicit HIV-1 virus-neutralizing sera in mice. Virulence (2021) 12:1271–87. doi: 10.1080/21505594.2021.1920251 PubMed DOI PMC

Raska M, Moldoveanu Z, Novak J, Hel Z, Novak L, Bozja J, et al. Delivery of DNA HIV-1 vaccine to the liver induces high and long-lasting humoral immune responses. Vaccine (2008) 26:1541–51. doi: 10.1016/j.vaccine.2008.01.035 PubMed DOI PMC

Raska M, Takahashi K, Czernekova L, Zachova K, Hall S, Moldoveanu Z, et al. Glycosylation patterns of HIV-1 gp120 depend on the type of expressing cells and affect antibody recognition. J Biol Chem (2010) 285:20860–9. doi: 10.1074/jbc.M109.085472 PubMed DOI PMC

Raska M, Czernekova L, Moldoveanu Z, Zachova K, Elliott MC, Novak Z, et al. Differential glycosylation of envelope gp120 is associated with differential recognition of HIV-1 by virus-specific antibodies and cell infection. AIDS Res Ther (2014) 11:23. doi: 10.1186/1742-6405-11-23 PubMed DOI PMC

Sali A, Blundell TL. Comparative protein modelling by satisfaction of spatial restraints. J Mol Biol (1993) 234:779–815. doi: 10.1006/jmbi.1993.1626 PubMed DOI

Pinotsis N, Chatziefthimiou SD, Berkemeier F, Beuron F, Mavridis IM, Konarev PV, et al. Superhelical architecture of the myosin filament-linking protein myomesin with unusual elastic properties. PloS Biol (2012) 10:e1001261. doi: 10.1371/journal.pbio.1001261 PubMed DOI PMC

Mouquet H, Scharf L, Euler Z, Liu Y, Eden C, Scheid JF, et al. Complex-type n-glycan recognition by potent broadly neutralizing HIV antibodies. Proc Natl Acad Sci USA (2012) 109:E3268–3277. doi: 10.1073/pnas.1217207109 PubMed DOI PMC

Garces F, Lee JH, De Val N, de la Pena AT, Kong L, Puchades C, et al. Affinity maturation of a potent family of HIV antibodies is primarily focused on accommodating or avoiding glycans. Immunity (2015) 43:1053–63. doi: 10.1016/j.immuni.2015.11.007 PubMed DOI PMC

Lee JH, De Val N, Lyumkis D, Ward AB. Model building and refinement of a natively glycosylated HIV-1 env protein by high-resolution cryoelectron microscopy. Structure (2015) 23:1943–51. doi: 10.1016/j.str.2015.07.020 PubMed DOI PMC

Kozakov D, Brenke R, Comeau SR, Vajda S. PIPER: An FFT-based protein docking program with pairwise potentials. Proteins (2006) 65:392–406. doi: 10.1002/prot.21117 PubMed DOI

Kozakov D, Beglov D, Bohnuud T, Mottarella SE, Xia B, Hall DR, et al. How good is automated protein docking? Proteins (2013) 81:2159–66. doi: 10.1002/prot.24403 PubMed DOI PMC

Kilkenny C, Browne WJ, Cuthill IC, Emerson M, Altman DG. Improving bioscience research reporting: the ARRIVE guidelines for reporting animal research. PloS Biol (2010) 8:e1000412. doi: 10.1002/0471142735.im1211s64 PubMed DOI PMC

Montefiori DC. Evaluating neutralizing antibodies against HIV, SIV, and SHIV in luciferase reporter gene assays. Curr Protoc Immunol (2005) 12. doi: 10.1016/j.chom.2018.12.001 PubMed DOI

Garces F, Sok D, Kong L, Mcbride R, Kim HJ, Saye-Francisco KF, et al. Structural evolution of glycan recognition by a family of potent HIV antibodies. Cell (2014) 159:69–79. doi: 10.1016/j.cell.2014.09.009 PubMed DOI PMC

Lee JH, Ozorowski G, Ward AB. Cryo-EM structure of a native, fully glycosylated, cleaved HIV-1 envelope trimer. Science (2016) 351:1043–8. doi: 10.1126/science.aad2450 PubMed DOI PMC

Ozorowski G, Pallesen J, De Val N, Lyumkis D, Cottrell CA, Torres JL, et al. Open and closed structures reveal allostery and pliability in the HIV-1 envelope spike. Nature (2017) 547:360–3. doi: 10.1038/nature23010 PubMed DOI PMC

Zolla-Pazner S. Improving on nature: Focusing the immune response on the V3 loop. Hum Antibodies (2005) 14:69–72. doi: 10.3233/HAB-2005-143-403 PubMed DOI

Hessell AJ, Mcburney S, Pandey S, Sutton W, Liu L, Li LZ, et al. Induction of neutralizing antibodies in rhesus macaques using V3 mimotope peptides. Vaccine (2016) 34:2713–21. doi: 10.1016/j.vaccine.2016.04.027 PubMed DOI PMC

Barnes CO, Gristick HB, Freund NT, Escolano A, Lyubimov AY, Hartweger H, et al. Structural characterization of a highly-potent V3-glycan broadly neutralizing antibody bound to natively-glycosylated HIV-1 envelope. Nat Commun (2018) 9:1251. doi: 10.1038/s41467-018-03632-y PubMed DOI PMC

Pejchal R, Doores KJ, Walker LM, Khayat R, Huang PS, Wang SK, et al. A potent and broad neutralizing antibody recognizes and penetrates the HIV glycan shield. Science (2011) 334:1097–103. doi: 10.1126/science.1213256 PubMed DOI PMC

Sok D, Doores KJ, Briney B, Le KM, Saye-Francisco KL, Ramos A, et al. Promiscuous glycan site recognition by antibodies to the high-mannose patch of gp120 broadens neutralization of HIV. Sci Trans Med (2014) 6(236):236ra63 doi: 10.1126/scitranslmed.3008104 PubMed DOI PMC

Moore PL, Gray ES, Wibmer CK, Bhiman JN, Nonyane M, Sheward DJ, et al. Evolution of an HIV glycan-dependent broadly neutralizing antibody epitope through immune escape. Nat Med (2012) 18:1688. doi: 10.1038/nm.2985 PubMed DOI PMC

Krumm SA, Mohammed H, Le KM, Crispin M, Wrin T, Poignard P, et al. Mechanisms of escape from the PGT128 family of anti-HIV broadly neutralizing antibodies. Retrovirology (2016) 13:8. doi: 10.1186/s12977-016-0241-5 PubMed DOI PMC

Doores KJ, Kong L, Krumm SA, Le KM, Sok D, Laserson U, et al. Two classes of broadly neutralizing antibodies within a single lineage directed to the high-mannose patch of HIV envelope. J Virol (2015) 89:6525–5. doi: 10.1128/JVI.00593-15 PubMed DOI PMC

Cai H, Zhang RS, Orwenyo J, Giddens J, Yang Q, Labranche CC, et al. Synthetic HIV V3 glycopeptide immunogen carrying a N334 n-glycan induces glycan-dependent antibodies with promiscuous site recognition. J Med Chem (2018) 61:10116–25. doi: 10.1021/acs.jmedchem.8b01290 PubMed DOI PMC

Lyumkis D, Julien JP, De Val N, Cupo A, Potter CS, Klasse PJ, et al. Cryo-EM structure of a fully glycosylated soluble cleaved HIV-1 envelope trimer. Science (2013) 342:1484–90. doi: 10.1126/science.1245627 PubMed DOI PMC

Li Y, Cleveland B, Klots I, Travis B, Richardson BA, Anderson D, et al. Removal of a single n-linked glycan in human immunodeficiency virus type 1 gp120 results in an enhanced ability to induce neutralizing antibody responses. J Virol (2008) 82:638–51. doi: 10.1128/JVI.01691-07 PubMed DOI PMC

Zolla-Pazner S, Cohen SS, Boyd D, Kong XP, Seaman M, Nussenzweig M, et al. Structure/Function studies involving the V3 region of the HIV-1 envelope delineate multiple factors that affect neutralization sensitivity. J Virol (2016) 90:636–49. doi: 10.1128/JVI.01645-15 PubMed DOI PMC

Moyo T, Ferreira RC, Davids R, Sonday Z, Moore PL, Travers SA, et al. Chinks in the armor of the HIV-1 envelope glycan shield: Implications for immune escape from anti-glycan broadly neutralizing antibodies. Virology (2017) 501:12–24. doi: 10.1016/j.virol.2016.10.026 PubMed DOI

Goo L, Jalalian-Lechak Z, Richardson BA, Overbaugh J. A combination of broadly neutralizing HIV-1 monoclonal antibodies targeting distinct epitopes effectively neutralizes variants found in early infection. J Virol (2012) 86:10857–61. doi: 10.1128/JVI.01414-12 PubMed DOI PMC

Bricault CA, Yusim K, Seaman MS, Yoon H, Theiler J, Giorgi EE, et al. HIV-1 neutralizing antibody signatures and application to epitope-targeted vaccine design. Cell Host Microbe (2019) 25:59–72.e58. doi: 10.1016/j.chom.2018.12.001 PubMed DOI PMC

van Regenmortel MHV. What does it mean to develop an HIV vaccine by rational design? Arch Virol (2021) 166:27–33. doi: 10.1007/s00705-020-04884-0 PubMed DOI

Wagh K, Bhattacharya T, Williamson C, Robles A, Bayne M, Garrity J, et al. Optimal combinations of broadly neutralizing antibodies for prevention and treatment of HIV-1 clade c infection. PloS Pathog (2016) 12:e1005520. doi: 10.1371/journal.ppat.1005520 PubMed DOI PMC

Gautam R, Nishimura Y, Pegu A, Nason MC, Klein F, Gazumyan A, et al. A single injection of anti-HIV-1 antibodies protects against repeated SHIV challenges. Nature (2016) 533:105. doi: 10.1038/nature17677 PubMed DOI PMC

Klein F, Halper-Stromberg A, Horwitz JA, Gruell H, Scheid JF, Bournazos S, et al. HIV Therapy by a combination of broadly neutralizing antibodies in humanized mice. Nature (2012) 492:118. doi: 10.1038/nature11604 PubMed DOI PMC

Klein F, Nogueira L, Nishimura Y, Phad G, West AP, Halper-Stromberg A, et al. Enhanced HIV-1 immunotherapy by commonly arising antibodies that target virus escape variants. J Exp Med (2014) 211:2361–72. doi: 10.1084/jem.20141050 PubMed DOI PMC

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