vavilovВавиловский журнал генетики и селекцииVavilov Journal of Genetics and Breeding2500-3259Institute of Cytology and Genetics of Siberian Branch of the RAS10.18699/VJ21.063vavilov-3114Research ArticleМОЛЕКУЛЯРНАЯ И КЛЕТОЧНАЯ БИОЛОГИЯMOLECULAR AND CELL BIOLOGYПрименение фагового дисплея для поиска ВИЧ-1-нейтрализующих антителPhage display as a tool for identifying HIV-1 broadly neutralizing antibodieshttps://orcid.org/0000-0001-5423-3457ЧикаевА. Н.ChikaevA. N.

Новосибирск

Novosibirsk

chikaev@mcb.nsc.ruhttps://orcid.org/0000-0003-2808-4309РудометовА. П.RudometovA. P.

р. п. Кольцово, Новосибирская область

Koltsovo, Novosibirsk region

https://orcid.org/0000-0002-6974-0686МеркульеваЮ. А.MerkulyevaYu. A.

р. п. Кольцово, Новосибирская область

Koltsovo, Novosibirsk region

https://orcid.org/0000-0003-4365-8809КарпенкоЛ. И.KarpenkoL. I.

р. п. Кольцово, Новосибирская область

Koltsovo, Novosibirsk region

Институт молекулярной и клеточной биологии Сибирского отделения Российской академии наукРоссияInstitute of Molecular and Cellular Biology of the Siberian Branch of the Russian Academy of SciencesRussian FederationГосударственный научный центр вирусологии и биотехнологии «Вектор» РоспотребнадзораРоссияState Research Center of Virology and Biotechnology “Vector”, RospotrebnadzorRussian Federation202110092021255562572Copyright © Чикаев А.Н., Рудометов А.П., Меркульева Ю.А., Карпенко Л.И., 20212021Чикаев А.Н., Рудометов А.П., Меркульева Ю.А., Карпенко Л.И.Chikaev A.N., Rudometov A.P., Merkulyeva Y.A., Karpenko L.I.This work is licensed under a Creative Commons Attribution 4.0 License.https://vavilov.elpub.ru/jour/article/view/3114

Комбинаторная белковая инженерия – востребованный инструмент для решения задач, связанных со скринингом большого разнообразия взаимодействующих молекул: разработки лекарств, средств диагностики, идентификации антител, поиска конформационных имитаторов антигенных детерминант для создания иммуногенов или компонентов вакцин. Среди всех подобных методик одна из наиболее популярных – технология фагового дисплея, появившаяся во второй половине 1980-х гг., однако в силу относительной простоты и универсальности по-прежнему активно применяющаяся для изучения белок-белковых, пептид-белковых и ДНК-белковых взаимодействий. Фаговый дисплей позволяет создавать высокопредставительные библиотеки пептидов, белков или их фрагментов, в которых каждая фаговая частица экспонирует на своей поверхности исследуемые пептиды или белки и одновременно несет в своем геноме последовательность ДНК, кодирующую экспонируемый пептид/белок. Процедура аффинной селекции позволяет находить специфические фаговые клоны практически к любой мишени, а за счет наличия физической связи между генотипом и фенотипом можно эффективно определить структуру отобранных молекул. Значительную роль технология фагового дисплея сыграла в исследованиях, направленных на изучение антигенной структуры вируса иммунодефицита человека (ВИЧ-1) и разработку средств борьбы с этим заболеванием. Серьезная проблема, из-за которой до сих пор не удается создать эффективную анти-ВИЧ-вакцину, – сильная антигенная изменчивость вируса. Согласно современным представлениям, для обеспечения защиты от инфицирования необходимо стимулировать индукцию в организме вируснейтрализующих антител, активных в отношении большого числа различных штаммов ВИЧ-1. Соответственно, идентификация подобных антител является важной исследовательской задачей. Тема настоящего обзора – применение фагового дисплея в качестве инструмента для поиска ВИЧ-1-нейтрализующих антител широкого спектра действия. Представлены ключевые характеристики технологии фагового дисплея, кратко описан процесс получения библиотек антител, проведения процедуры аффинной селекции, а также обсуждается феномен ВИЧ-1-нейтрализующих антител широкого спектра действия. Приводится обзор исследований, посвященных поиску кросс-нейтрализующих антител с использованием различных типов фаговых библиотек.

Combinatorial biology methods offer a good solution for targeting interactions of specific molecules by a high-throughput screening and are widely used for drug development, diagnostics, identification of novel monoclonal antibodies, search for linear peptide mimetics of discontinuous epitopes for the development of immunogens or vaccine components. Among all currently available techniques, phage display remains one of the most popular approaches. Despite being a fairly old method, phage display is still widely used for studying protein-protein, peptide-protein and DNA-protein interactions due to its relative simplicity and versatility. Phage display allows highly representative libraries of peptides, proteins or their fragments to be created. Each phage particle in a library displays peptides or proteins fused to its coat protein and simultaneously carries the DNA sequence encoding the displayed peptide/protein in its genome. The biopanning procedure allows isolation of specific clones for almost any target, and due to the physical link between the genotype and the phenotype of recombinant phage particles it is possible to determine the structure of selected molecules. Phage display technology continues to play an important role in HIV research. A major obstacle to the development of an effective HIV vaccine is an extensive genetic and antigenic variability of the virus. According to recent data, in order to provide protection against HIV infection, the so-called broadly neutralizing antibodies that are cross-reactive against multiple viral strains of HIV must be induced, which makes the identification of such antibodies a key area of HIV vaccinology. In this review, we discuss the use of phage display as a tool for identification of HIV-specific antibodies with broad neutralizing activity. We provide an outline of phage display technology, briefly describe the design of antibody phage libraries and the affinity selection procedure, and discuss the biology of HIV-1-specific broadly neutralizing antibodies. Finally, we summarize the studies aimed at identification of broadly neutralizing antibodies using various types of phage libraries.

фаговый дисплейбиблиотеки антителВИЧ-1нейтрализующие антитела широкого спектра действия (bnAbs)phage displayantibody librariesHIV-1broadly neutralizing antibodies (bnAbs)The reported study was funded by the Russian Foundation for Basic Research (project No. 20-04-00879).ReferencesAlfaleh M.A., Alsaab H.O., Mahmoud A.B., Alkayyal A.A., Jones M.L., Mahler S.M., Hashem A.M. Phage display derived monoclonal antibodies: from bench to bedside. Front. Immunol. 2020;11:1986. DOI 10.3389/fimmu.2020.01986.Alfaleh M.A., Alsaab H.O., Mahmoud A.B., Alkayyal A.A., Jones M.L., Mahler S.M., Hashem A.M. Phage display derived monoclonal antibodies: from bench to bedside. Front. Immunol. 2020;11:1986. DOI 10.3389/fimmu.2020.01986.Ashby M., Petkova A., Gani J., Mikut R., Hilpert K. Use of peptide libraries for identification and optimization of novel antimicrobial peptides. Curr. Top. Med. Chem. 2017;17(5):537-553. DOI 10.2174/1568026616666160713125555.Ashby M., Petkova A., Gani J., Mikut R., Hilpert K. Use of peptide libraries for identification and optimization of novel antimicrobial peptides. Curr. Top. Med. Chem. 2017;17(5):537-553. DOI 10.2174/1568026616666160713125555.Barbas C.F., 3rd, Crowe J.E., Jr., Cababa D., Jones T.M., Zebedee S.L., Murphy B.R., Chanock R.M., Burton D.R. Human monoclonal Fab fragments derived from a combinatorial library bind to respiratory syncytial virus F glycoprotein and neutralize infectivity. Proc. Natl. Acad. Sci. USA. 1992;89(21):10164-10168. DOI 10.1073/pnas.89.21.10164.Barbas C.F., 3rd, Crowe J.E., Jr., Cababa D., Jones T.M., Zebedee S.L., Murphy B.R., Chanock R.M., Burton D.R. Human monoclonal Fab fragments derived from a combinatorial library bind to respiratory syncytial virus F glycoprotein and neutralize infectivity. Proc. Natl. Acad. Sci. USA. 1992;89(21):10164-10168. DOI 10.1073/pnas.89.21.10164.Barbas C.F., 3rd, Kang A.S., Lerner R.A., Benkovic S.J. Assembly of combinatorial antibody libraries on phage surfaces: the gene III site. Proc. Natl. Acad. Sci. USA. 1991;88(18):7978-7982. DOI 10.1073/pnas.88.18.7978.Barbas C.F., 3rd, Kang A.S., Lerner R.A., Benkovic S.J. Assembly of combinatorial antibody libraries on phage surfaces: the gene III site. Proc. Natl. Acad. Sci. USA. 1991;88(18):7978-7982. DOI 10.1073/pnas.88.18.7978.Burton D.R., Barbas C.F., 3rd, Persson M.A., Koenig S., Chanock R.M., Lerner R.A. A large array of human monoclonal antibodies to type 1 human immunodeficiency virus from combinatorial libraries of asymptomatic seropositive individuals. Proc. Natl. Acad. Sci. USA. 1991;88(22):10134-10137. DOI 10.1073/pnas.88.22.10134.Burton D.R., Barbas C.F., 3rd, Persson M.A., Koenig S., Chanock R.M., Lerner R.A. A large array of human monoclonal antibodies to type 1 human immunodeficiency virus from combinatorial libraries of asymptomatic seropositive individuals. Proc. Natl. Acad. Sci. USA. 1991;88(22):10134-10137. DOI 10.1073/pnas.88.22.10134.Burton D.R., Hangartner L. Broadly neutralizing antibodies to HIV and their role in vaccine design. Annu. Rev. Immunol. 2016;34:635-659. DOI 10.1146/annurev-immunol-041015-055515.Burton D.R., Hangartner L. Broadly neutralizing antibodies to HIV and their role in vaccine design. Annu. Rev. Immunol. 2016;34:635-659. DOI 10.1146/annurev-immunol-041015-055515.Burton D.R., Pyati J., Koduri R., Sharp S.J., Thornton G.B., Parren P.W., Sawyer L.S., Hendry R.M., Dunlop N., Nara P.L. Efficient neutralization of primary isolates of HIV-1 by a recombinant human monoclonal antibody. Science. 1994;266(5187):1024-1027. DOI 10.1126/science.7973652.Burton D.R., Pyati J., Koduri R., Sharp S.J., Thornton G.B., Parren P.W., Sawyer L.S., Hendry R.M., Dunlop N., Nara P.L. Efficient neutralization of primary isolates of HIV-1 by a recombinant human monoclonal antibody. Science. 1994;266(5187):1024-1027. DOI 10.1126/science.7973652.Castel G., Chteoui M., Heyd B., Tordo N. Phage display of combinatorial peptide libraries: application to antiviral research. Molecules. 2011;16(5):3499-3518. DOI 10.3390/molecules16053499.Castel G., Chteoui M., Heyd B., Tordo N. Phage display of combinatorial peptide libraries: application to antiviral research. Molecules. 2011;16(5):3499-3518. DOI 10.3390/molecules16053499.Chames P., Baty D. Phage display and selections on biotinylated antigens. In: Kotermann R., Dübel S. (Eds.). Antibody Engineering. Humana Press, 2010;151-164. DOI 10.1007/978-3-642-01144-3_11.Chames P., Baty D. Phage display and selections on biotinylated antigens. In: Kotermann R., Dübel S. (Eds.). Antibody Engineering. Humana Press, 2010;151-164. DOI 10.1007/978-3-642-01144-3_11.Chan C.E., Lim A.P., Macary P.A., Hanson B.J. The role of phage display in therapeutic antibody discovery. Int. Immunol. 2014;26(12): 649-657. DOI 10.1093/intimm/dxu082.Chan C.E., Lim A.P., Macary P.A., Hanson B.J. The role of phage display in therapeutic antibody discovery. Int. Immunol. 2014;26(12): 649-657. DOI 10.1093/intimm/dxu082.Choudhry V., Zhang M.Y., Dimitrova D., Prabakaran P., Dimitrov A.S., Fouts T.R., Dimitrov D.S. Antibody-based inhibitors of HIV infection. Expert Opin. Biol. Ther. 2006;6(5):523-531. DOI 10.1517/14712598.6.5.523.Choudhry V., Zhang M.Y., Dimitrova D., Prabakaran P., Dimitrov A.S., Fouts T.R., Dimitrov D.S. Antibody-based inhibitors of HIV infection. Expert Opin. Biol. Ther. 2006;6(5):523-531. DOI 10.1517/14712598.6.5.523.Christensen D.J., Gottlin E.B., Benson R.E., Hamilton P.T. Phage display for target-based antibacterial drug discovery. Drug Discov. Today. 2001;6(14):721-727. DOI 10.1016/s1359-6446(01)01853-0.Christensen D.J., Gottlin E.B., Benson R.E., Hamilton P.T. Phage display for target-based antibacterial drug discovery. Drug Discov. Today. 2001;6(14):721-727. DOI 10.1016/s1359-6446(01)01853-0.Clackson T., Hoogenboom H.R., Griffiths A.D., Winter G. Making antibody fragments using phage display libraries. Nature. 1991; 352(6336):624-628. DOI 10.1038/352624a0.Clackson T., Hoogenboom H.R., Griffiths A.D., Winter G. Making antibody fragments using phage display libraries. Nature. 1991; 352(6336):624-628. DOI 10.1038/352624a0.Clark J.R., March J.B. Bacteriophage-mediated nucleic acid immunisation. FEMS Immunol. Med. Microbiol. 2004;40(1):21-26. DOI 10.1016/S0928-8244(03)00344-4.Clark J.R., March J.B. Bacteriophage-mediated nucleic acid immunisation. FEMS Immunol. Med. Microbiol. 2004;40(1):21-26. DOI 10.1016/S0928-8244(03)00344-4.Corti D., Langedijk J.P., Hinz A., Seaman M.S., Vanzetta F., FernandezRodriguez B.M., Silacci C., Pinna D., Jarrossay D., Balla-Jhagjhoorsingh S., Willems B., Zekveld M.J., Dreja H., O’sullivan E., Pade C., Orkin C., Jeffs S.A., Montefiori D.C., Davis D., Weissenhorn W., Mcknight A., Heeney J.L., Sallusto F., Sattentau Q.J., Weiss R.A., Lanzavecchia A. Analysis of memory B cell responses and isolation of novel monoclonal antibodies with neutralizing breadth from HIV-1-infected individuals. PLoS One. 2010;5(1):e8805. DOI 10.1371/journal.pone.0008805.Corti D., Langedijk J.P., Hinz A., Seaman M.S., Vanzetta F., FernandezRodriguez B.M., Silacci C., Pinna D., Jarrossay D., Balla-Jhagjhoorsingh S., Willems B., Zekveld M.J., Dreja H., O’sullivan E., Pade C., Orkin C., Jeffs S.A., Montefiori D.C., Davis D., Weissenhorn W., Mcknight A., Heeney J.L., Sallusto F., Sattentau Q.J., Weiss R.A., Lanzavecchia A. Analysis of memory B cell responses and isolation of novel monoclonal antibodies with neutralizing breadth from HIV-1-infected individuals. PLoS One. 2010;5(1):e8805. DOI 10.1371/journal.pone.0008805.Cuevas J.M., Geller R., Garijo R., Lopez-Aldeguer J., Sanjuan R. Extremely high mutation rate of HIV-1 in vivo. PLoS Biol. 2015;13(9): e1002251. DOI 10.1371/journal.pbio.1002251.Cuevas J.M., Geller R., Garijo R., Lopez-Aldeguer J., Sanjuan R. Extremely high mutation rate of HIV-1 in vivo. PLoS Biol. 2015;13(9): e1002251. DOI 10.1371/journal.pbio.1002251.Dashti A., Devico A.L., Lewis G.K., Sajadi M.M. Broadly neutralizing antibodies against HIV: back to blood. Trends Mol. Med. 2019; 25(3):228-240. DOI 10.1016/j.molmed.2019.01.007.Dashti A., Devico A.L., Lewis G.K., Sajadi M.M. Broadly neutralizing antibodies against HIV: back to blood. Trends Mol. Med. 2019; 25(3):228-240. DOI 10.1016/j.molmed.2019.01.007.Davies J., Riechmann L. Antibody VH domains as small recognition units. Biotechnology (NY ). 1995;13(5):475-479. DOI 10.1038/nbt0595-475.Davies J., Riechmann L. Antibody VH domains as small recognition units. Biotechnology (NY ). 1995;13(5):475-479. DOI 10.1038/nbt0595-475.Del Moral-Sanchez I., Sliepen K. Strategies for inducing effective neutralizing antibody responses against HIV-1. Expert Rev. Vaccines. 2019;18(11):1127-1143. DOI 10.1080/14760584.2019.1690458.Del Moral-Sanchez I., Sliepen K. Strategies for inducing effective neutralizing antibody responses against HIV-1. Expert Rev. Vaccines. 2019;18(11):1127-1143. DOI 10.1080/14760584.2019.1690458.Dhillon A.K., Donners H., Pantophlet R., Johnson W.E., Decker J.M., Shaw G.M., Lee F.H., Richman D.D., Doms R.W., Vanham G., Burton D.R. Dissecting the neutralizing antibody specificities of broadly neutralizing sera from human immunodeficiency virus type 1-infected donors. J. Virol. 2007;81(12):6548-6562. DOI 10.1128/JVI.02749-06.Dhillon A.K., Donners H., Pantophlet R., Johnson W.E., Decker J.M., Shaw G.M., Lee F.H., Richman D.D., Doms R.W., Vanham G., Burton D.R. Dissecting the neutralizing antibody specificities of broadly neutralizing sera from human immunodeficiency virus type 1-infected donors. J. Virol. 2007;81(12):6548-6562. DOI 10.1128/JVI.02749-06.Doria-Rose N.A., Klein R.M., Manion M.M., O’dell S., Phogat A., Chakrabarti B., Hallahan C.W., Migueles S.A., Wrammert J., Ahmed R., Nason M., Wyatt R.T., Mascola J.R., Connors M. Frequency and phenotype of human immunodeficiency virus envelope-specific B cells from patients with broadly cross-neutralizing antibodies. J. Virol. 2009;83(1):188-199. DOI 10.1128/JVI.01583-08.Doria-Rose N.A., Klein R.M., Manion M.M., O’dell S., Phogat A., Chakrabarti B., Hallahan C.W., Migueles S.A., Wrammert J., Ahmed R., Nason M., Wyatt R.T., Mascola J.R., Connors M. Frequency and phenotype of human immunodeficiency virus envelope-specific B cells from patients with broadly cross-neutralizing antibodies. J. Virol. 2009;83(1):188-199. DOI 10.1128/JVI.01583-08.Duan H., Chen X., Boyington J.C., Cheng C., Zhang Y., Jafari A.J., Stephens T., Tsybovsky Y., Kalyuzhniy O., Zhao P., Menis S., Nason M.C., Normandin E., Mukhamedova M., Dekosky B.J., Wells L., Schief W.R., Tian M., Alt F.W., Kwong P.D., Mascola J.R. Glycan masking focuses immune responses to the HIV-1 CD4-binding site and enhances elicitation of VRC01-class precursor antibodies. Immunity. 2018;49(2):301-311 e305. DOI 10.1016/j.immuni.2018.07.005.Duan H., Chen X., Boyington J.C., Cheng C., Zhang Y., Jafari A.J., Stephens T., Tsybovsky Y., Kalyuzhniy O., Zhao P., Menis S., Nason M.C., Normandin E., Mukhamedova M., Dekosky B.J., Wells L., Schief W.R., Tian M., Alt F.W., Kwong P.D., Mascola J.R. Glycan masking focuses immune responses to the HIV-1 CD4-binding site and enhances elicitation of VRC01-class precursor antibodies. Immunity. 2018;49(2):301-311 e305. DOI 10.1016/j.immuni.2018.07.005.Felici F., Castagnoli L., Musacchio A., Jappelli R., Cesareni G. Selection of antibody ligands from a large library of oligopeptides expressed on a multivalent exposition vector. J. Mol. Biol. 1991;222(2):301-310. DOI 10.1016/0022-2836(91)90213-p.Felici F., Castagnoli L., Musacchio A., Jappelli R., Cesareni G. Selection of antibody ligands from a large library of oligopeptides expressed on a multivalent exposition vector. J. Mol. Biol. 1991;222(2):301-310. DOI 10.1016/0022-2836(91)90213-p.Forsman A., Beirnaert E., Aasa-Chapman M.M., Hoorelbeke B., Hijazi K., Koh W., Tack V., Szynol A., Kelly C., Mcknight A., Verrips T., De Haard H., Weiss R.A. Llama antibody fragments with crosssubtype human immunodeficiency virus type 1 (HIV-1)-neutralizing properties and high affinity for HIV-1 gp120. J. Virol. 2008;82(24): 12069-12081. DOI 10.1128/JVI.01379-08.Forsman A., Beirnaert E., Aasa-Chapman M.M., Hoorelbeke B., Hijazi K., Koh W., Tack V., Szynol A., Kelly C., Mcknight A., Verrips T., De Haard H., Weiss R.A. Llama antibody fragments with crosssubtype human immunodeficiency virus type 1 (HIV-1)-neutralizing properties and high affinity for HIV-1 gp120. J. Virol. 2008;82(24): 12069-12081. DOI 10.1128/JVI.01379-08.Gach J.S., Quendler H., Tong T., Narayan K.M., Du S.X., Whalen R.G., Binley J.M., Forthal D.N., Poignard P., Zwick M.B. A human antibody to the CD4 binding site of gp120 capable of highly potent but sporadic cross clade neutralization of primary HIV-1. PLoS One. 2013;8(8):e72054. DOI 10.1371/journal.pone.0072054.Gach J.S., Quendler H., Tong T., Narayan K.M., Du S.X., Whalen R.G., Binley J.M., Forthal D.N., Poignard P., Zwick M.B. A human antibody to the CD4 binding site of gp120 capable of highly potent but sporadic cross clade neutralization of primary HIV-1. PLoS One. 2013;8(8):e72054. DOI 10.1371/journal.pone.0072054.Greenberg A.S., Avila D., Hughes M., Hughes A., McKinney E.C., Flajnik M.F. A new antigen receptor gene family that undergoes rearrangement and extensive somatic diversification in sharks. Nature. 1995;374(6518):168-173. DOI 10.1038/374168a0.Greenberg A.S., Avila D., Hughes M., Hughes A., McKinney E.C., Flajnik M.F. A new antigen receptor gene family that undergoes rearrangement and extensive somatic diversification in sharks. Nature. 1995;374(6518):168-173. DOI 10.1038/374168a0.Griffiths A.D., Duncan A.R. Strategies for selection of antibodies by phage display. Curr. Opin. Biotechnol. 1998;9(1):102-108. DOI 10.1016/s0958-1669(98)80092-x.Griffiths A.D., Duncan A.R. Strategies for selection of antibodies by phage display. Curr. Opin. Biotechnol. 1998;9(1):102-108. DOI 10.1016/s0958-1669(98)80092-x.Habeshaw J.A., Dalgleish A.G., Bountiff L., Newell A.L., Wilks D., Walker L.C., Manca F. AIDS pathogenesis: HIV envelope and its interaction with cell proteins. Immunol. Today. 1990;11(11):418-425. DOI 10.1016/0167-5699(90)90162-3.Habeshaw J.A., Dalgleish A.G., Bountiff L., Newell A.L., Wilks D., Walker L.C., Manca F. AIDS pathogenesis: HIV envelope and its interaction with cell proteins. Immunol. Today. 1990;11(11):418-425. DOI 10.1016/0167-5699(90)90162-3.Hammers C.M., Stanley J.R. Antibody phage display: technique and applications. J. Invest. Dermatol. 2014;134(2):1-5. DOI 10.1038/jid.2013.521.Hammers C.M., Stanley J.R. Antibody phage display: technique and applications. J. Invest. Dermatol. 2014;134(2):1-5. DOI 10.1038/jid.2013.521.Hess K.L., Jewell C.M. Phage display as a tool for vaccine and immunotherapy development. Bioeng. Transl. Med. 2020;5(1):e10142. DOI 10.1002/btm2.10142.Hess K.L., Jewell C.M. Phage display as a tool for vaccine and immunotherapy development. Bioeng. Transl. Med. 2020;5(1):e10142. DOI 10.1002/btm2.10142.Hessell A.J., Rakasz E.G., Poignard P., Hangartner L., Landucci G., Forthal D.N., Koff W.C., Watkins D.I., Burton D.R. Broadly neutralizing human anti-HIV antibody 2G12 is effective in protection against mucosal SHIV challenge even at low serum neutralizing titers. PLoS Pathog. 2009;5(5):e1000433. DOI 10.1371/journal.ppat.1000433.Hessell A.J., Rakasz E.G., Poignard P., Hangartner L., Landucci G., Forthal D.N., Koff W.C., Watkins D.I., Burton D.R. Broadly neutralizing human anti-HIV antibody 2G12 is effective in protection against mucosal SHIV challenge even at low serum neutralizing titers. PLoS Pathog. 2009;5(5):e1000433. DOI 10.1371/journal.ppat.1000433.Hraber P., Korber B.T., Lapedes A.S., Bailer R.T., Seaman M.S., Gao H., Greene K.M., Mccutchan F., Williamson C., Kim J.H., Tovanabutra S., Hahn B.H., Swanstrom R., Thomson M.M., Gao F., Harris L., Giorgi E., Hengartner N., Bhattacharya T., Mascola J.R., Montefiori D.C. Impact of clade geography and age of the epidemic on HIV-1 neutralization by antibodies. J. Virol. 2014;88(21):12623-12643. DOI 10.1128/JVI.01705-14.Hraber P., Korber B.T., Lapedes A.S., Bailer R.T., Seaman M.S., Gao H., Greene K.M., Mccutchan F., Williamson C., Kim J.H., Tovanabutra S., Hahn B.H., Swanstrom R., Thomson M.M., Gao F., Harris L., Giorgi E., Hengartner N., Bhattacharya T., Mascola J.R., Montefiori D.C. Impact of clade geography and age of the epidemic on HIV-1 neutralization by antibodies. J. Virol. 2014;88(21):12623-12643. DOI 10.1128/JVI.01705-14.Huang J.X., Bishop-Hurley S.L., Cooper M.A. Development of antiinfectives using phage display: biological agents against bacteria viruses and parasites. Antimicrob. Agents Chemother. 2012;56(9): 4569-4582. DOI 10.1128/AAC.00567-12.Huang J.X., Bishop-Hurley S.L., Cooper M.A. Development of antiinfectives using phage display: biological agents against bacteria viruses and parasites. Antimicrob. Agents Chemother. 2012;56(9): 4569-4582. DOI 10.1128/AAC.00567-12.Ilyichev A.A., Minenkova O.O., Kishchenko G.P., Tat’kov S.I., Karpishev N.N., Eroshkin A.M., Ofitzerov V.I., Akimenko Z.A., Petrenko V.A., Sandakhchiev L.S. Inserting foreign peptides into the major coat protein of bacteriophage M13. FEBS Lett. 1992;301(3):322-324. DOI 10.1016/0014-5793(92)80267-k.Ilyichev A.A., Minenkova O.O., Kishchenko G.P., Tat’kov S.I., Karpishev N.N., Eroshkin A.M., Ofitzerov V.I., Akimenko Z.A., Petrenko V.A., Sandakhchiev L.S. Inserting foreign peptides into the major coat protein of bacteriophage M13. FEBS Lett. 1992;301(3):322-324. DOI 10.1016/0014-5793(92)80267-k.Jardine J., Julien J.P., Menis S., Ota T., Kalyuzhniy O., Mcguire A., Sok D., Huang P.S., Macpherson S., Jones M., Nieusma T., Mathison J., Baker D., Ward A.B., Burton D.R., Stamatatos L., Nemazee D., Wilson I.A., Schief W.R. Rational HIV immunogen design to target specific germline B cell receptors. Science. 2013;340(6133):711-716. DOI 10.1126/science.1234150.Jardine J., Julien J.P., Menis S., Ota T., Kalyuzhniy O., Mcguire A., Sok D., Huang P.S., Macpherson S., Jones M., Nieusma T., Mathison J., Baker D., Ward A.B., Burton D.R., Stamatatos L., Nemazee D., Wilson I.A., Schief W.R. Rational HIV immunogen design to target specific germline B cell receptors. Science. 2013;340(6133):711-716. DOI 10.1126/science.1234150.Julg B., Barouch D.H. Neutralizing antibodies for HIV-1 prevention. Curr. Opin. HIV AIDS. 2019;14(4):318-324. DOI 10.1097/COH.0000000000000556.Julg B., Barouch D.H. Neutralizing antibodies for HIV-1 prevention. Curr. Opin. HIV AIDS. 2019;14(4):318-324. DOI 10.1097/COH.0000000000000556.Karuna S.T., Corey L. Broadly neutralizing antibodies for HIV prevention. Annu. Rev. Med. 2020;71(1):329-346. DOI 10.1146/annurev-med-110118-045506.Karuna S.T., Corey L. Broadly neutralizing antibodies for HIV prevention. Annu. Rev. Med. 2020;71(1):329-346. DOI 10.1146/annurev-med-110118-045506.Kay B.K., Adey N.B., He Y.S., Manfredi J.P., Mataragnon A.H., Fowlkes D.M. An M13 phage library displaying random 38-amino-acid peptides as a source of novel sequences with affinity to selected targets. Gene. 1993;128(1):59-65. DOI 10.1016/0378-1119(93)90153-t.Kay B.K., Adey N.B., He Y.S., Manfredi J.P., Mataragnon A.H., Fowlkes D.M. An M13 phage library displaying random 38-amino-acid peptides as a source of novel sequences with affinity to selected targets. Gene. 1993;128(1):59-65. DOI 10.1016/0378-1119(93)90153-t.Kennedy P.J., Oliveira C., Granja P.L., Sarmento B. Monoclonal antibodies: technologies for early discovery and engineering. Crit. Rev. Biotechnol. 2018;38(3):394-408. DOI 10.1080/07388551.2017.1357002.Kennedy P.J., Oliveira C., Granja P.L., Sarmento B. Monoclonal antibodies: technologies for early discovery and engineering. Crit. Rev. Biotechnol. 2018;38(3):394-408. DOI 10.1080/07388551.2017.1357002.Kishchenko G., Batliwala H., Makowski L. Structure of a foreign peptide displayed on the surface of bacteriophage M13. J. Mol. Biol. 1994;241(2):208-213. DOI 10.1006/jmbi.1994.1489.Kishchenko G., Batliwala H., Makowski L. Structure of a foreign peptide displayed on the surface of bacteriophage M13. J. Mol. Biol. 1994;241(2):208-213. DOI 10.1006/jmbi.1994.1489.Koch K., Kalusche S., Torres J.L., Stanfield R.L., Danquah W., Khazanehdari K., Von Briesen H., Geertsma E.R., Wilson I.A., Wernery U., Koch-Nolte F., Ward A.B., Dietrich U. Selection of nanobodies with broad neutralizing potential against primary HIV-1 strains using soluble subtype C gp140 envelope trimers. Sci. Rep. 2017;7(1):8390. DOI 10.1038/s41598-017-08273-7.Koch K., Kalusche S., Torres J.L., Stanfield R.L., Danquah W., Khazanehdari K., Von Briesen H., Geertsma E.R., Wilson I.A., Wernery U., Koch-Nolte F., Ward A.B., Dietrich U. Selection of nanobodies with broad neutralizing potential against primary HIV-1 strains using soluble subtype C gp140 envelope trimers. Sci. Rep. 2017;7(1):8390. DOI 10.1038/s41598-017-08273-7.Kowalski M., Potz J., Basiripour L., Dorfman T., Goh W.C., Terwilliger E., Dayton A., Rosen C., Haseltine W., Sodroski J. Functional regions of the envelope glycoprotein of human immunodeficiency virus type 1. Science. 1987;237(4820):1351-1355. DOI 10.1126/science.3629244.Kowalski M., Potz J., Basiripour L., Dorfman T., Goh W.C., Terwilliger E., Dayton A., Rosen C., Haseltine W., Sodroski J. Functional regions of the envelope glycoprotein of human immunodeficiency virus type 1. Science. 1987;237(4820):1351-1355. DOI 10.1126/science.3629244.Kwong P.D., Mascola J.R. HIV-1 vaccines based on antibody identification B cell ontogeny and epitope structure. Immunity. 2018;48(5): 855-871. DOI 10.1016/j.immuni.2018.04.029.Kwong P.D., Mascola J.R. HIV-1 vaccines based on antibody identification B cell ontogeny and epitope structure. Immunity. 2018;48(5): 855-871. DOI 10.1016/j.immuni.2018.04.029.Labrijn A.F., Poignard P., Raja A., Zwick M.B., Delgado K., Franti M., Binley J., Vivona V., Grundner C., Huang C.C., Venturi M., Petropoulos C.J., Wrin T., Dimitrov D.S., Robinson J., Kwong P.D., Wyatt R.T., Sodroski J., Burton D.R. Access of antibody molecules to the conserved coreceptor binding site on glycoprotein gp120 is sterically restricted on primary human immunodeficiency virus type 1. J. Virol. 2003;77(19):10557-10565. DOI 10.1128/jvi.77.19.10557-10565.2003.Labrijn A.F., Poignard P., Raja A., Zwick M.B., Delgado K., Franti M., Binley J., Vivona V., Grundner C., Huang C.C., Venturi M., Petropoulos C.J., Wrin T., Dimitrov D.S., Robinson J., Kwong P.D., Wyatt R.T., Sodroski J., Burton D.R. Access of antibody molecules to the conserved coreceptor binding site on glycoprotein gp120 is sterically restricted on primary human immunodeficiency virus type 1. J. Virol. 2003;77(19):10557-10565. DOI 10.1128/jvi.77.19.10557-10565.2003.Ledsgaard L., Kilstrup M., Karatt-Vellatt A., McCafferty J., Laustsen A.H. Basics of antibody phage display technology. Toxins (Basel ). 2018;10(6). DOI 10.3390/toxins10060236.Ledsgaard L., Kilstrup M., Karatt-Vellatt A., McCafferty J., Laustsen A.H. Basics of antibody phage display technology. Toxins (Basel ). 2018;10(6). DOI 10.3390/toxins10060236.Lutje Hulsik D., Liu Y.Y., Strokappe N.M., Battella S., El Khattabi M., Mccoy L.E., Sabin C., Hinz A., Hock M., Macheboeuf P., Bonvin A.M., Langedijk J.P., Davis D., Forsman Quigley A., AasaChapman M.M., Seaman M.S., Ramos A., Poignard P., Favier A., Simorre J.P., Weiss R.A., Verrips C.T., Weissenhorn W., Rutten L. A gp41 MPER-specific llama VHH requires a hydrophobic CDR3 for neutralization but not for antigen recognition. PLoS Pathog. 2013;9(3):e1003202. DOI 10.1371/journal.ppat.1003202.Lutje Hulsik D., Liu Y.Y., Strokappe N.M., Battella S., El Khattabi M., Mccoy L.E., Sabin C., Hinz A., Hock M., Macheboeuf P., Bonvin A.M., Langedijk J.P., Davis D., Forsman Quigley A., AasaChapman M.M., Seaman M.S., Ramos A., Poignard P., Favier A., Simorre J.P., Weiss R.A., Verrips C.T., Weissenhorn W., Rutten L. A gp41 MPER-specific llama VHH requires a hydrophobic CDR3 for neutralization but not for antigen recognition. PLoS Pathog. 2013;9(3):e1003202. DOI 10.1371/journal.ppat.1003202.Lynch R.M., Boritz E., Coates E.E., Dezure A., Madden P., Costner P., Enama M.E., Plummer S., Holman L., Hendel C.S., Gordon I., Casazza J., Conan-Cibotti M., Migueles S.A., Tressler R., Bailer R.T., Mcdermott A., Narpala S., O’dell S., Wolf G., Lifson J.D., Freemire B.A., Gorelick R.J., Pandey J.P., Mohan S., Chomont N., Fromentin R., Chun T.W., Fauci A.S., Schwartz R.M., Koup R.A., Douek D.C., Hu Z., Capparelli E., Graham B.S., Mascola J.R., Ledgerwood J.E., VRC 601 Study Team. Virologic effects of broadly neutralizing antibody VRC01 administration during chronic HIV-1 infection. Sci. Transl. Med. 2015;7(319):319ra206. DOI 10.1126/scitranslmed.aad5752.Lynch R.M., Boritz E., Coates E.E., Dezure A., Madden P., Costner P., Enama M.E., Plummer S., Holman L., Hendel C.S., Gordon I., Casazza J., Conan-Cibotti M., Migueles S.A., Tressler R., Bailer R.T., Mcdermott A., Narpala S., O’dell S., Wolf G., Lifson J.D., Freemire B.A., Gorelick R.J., Pandey J.P., Mohan S., Chomont N., Fromentin R., Chun T.W., Fauci A.S., Schwartz R.M., Koup R.A., Douek D.C., Hu Z., Capparelli E., Graham B.S., Mascola J.R., Ledgerwood J.E., VRC 601 Study Team. Virologic effects of broadly neutralizing antibody VRC01 administration during chronic HIV-1 infection. Sci. Transl. Med. 2015;7(319):319ra206. DOI 10.1126/scitranslmed.aad5752.Mahomed S., Garrett N., Baxter C., Abdool Karim Q., Abdool Karim S.S. Clinical trials of broadly neutralizing monoclonal antibodies for Human Immunodeficiency Virus prevention: a review. J. Infect. Dis. 2021;223(3):370-380. DOI 10.1093/infdis/jiaa377.Mahomed S., Garrett N., Baxter C., Abdool Karim Q., Abdool Karim S.S. Clinical trials of broadly neutralizing monoclonal antibodies for Human Immunodeficiency Virus prevention: a review. J. Infect. Dis. 2021;223(3):370-380. DOI 10.1093/infdis/jiaa377.Mahomed S., Garrett N., Karim Q.A., Zuma N.Y., Capparelli E., Baxter C., Gengiah T., Archary D., Samsunder N., Rose N.D., Moore P., Williamson C., Barouch D.H., Fast P.E., Pozzetto B., Hankins C., Carlton K., Ledgerwood J., Morris L., Mascola J., Abdool Karim S. Assessing the safety and pharmacokinetics of the anti-HIV monoclonal antibody CAP256V2LS alone and in combination with VRC07-523LS and PGT121 in South African women: study protocol for the first-in-human CAPRISA 012B phase I clinical trial. BMJ Open. 2020;10(11):e042247. DOI 10.1136/bmjopen-2020-042247.Mahomed S., Garrett N., Karim Q.A., Zuma N.Y., Capparelli E., Baxter C., Gengiah T., Archary D., Samsunder N., Rose N.D., Moore P., Williamson C., Barouch D.H., Fast P.E., Pozzetto B., Hankins C., Carlton K., Ledgerwood J., Morris L., Mascola J., Abdool Karim S. Assessing the safety and pharmacokinetics of the anti-HIV monoclonal antibody CAP256V2LS alone and in combination with VRC07-523LS and PGT121 in South African women: study protocol for the first-in-human CAPRISA 012B phase I clinical trial. BMJ Open. 2020;10(11):e042247. DOI 10.1136/bmjopen-2020-042247.Mascola J.R., Haynes B.F. HIV-1 neutralizing antibodies: understanding nature’s pathways. Immunol. Rev. 2013;254(1):225-244. DOI 10.1111/imr.12075.Mascola J.R., Haynes B.F. HIV-1 neutralizing antibodies: understanding nature’s pathways. Immunol. Rev. 2013;254(1):225-244. DOI 10.1111/imr.12075.McCafferty J., Griffiths A.D., Winter G., Chiswell D.J. Phage antibodies: filamentous phage displaying antibody variable domains. Nature. 1990;348(6301):552-554. DOI 10.1038/348552a0.McCafferty J., Griffiths A.D., Winter G., Chiswell D.J. Phage antibodies: filamentous phage displaying antibody variable domains. Nature. 1990;348(6301):552-554. DOI 10.1038/348552a0.Mccoy L.E., Burton D.R. Identification and specificity of broadly neutralizing antibodies against HIV. Immunol. Rev. 2017;275(1):11-20. DOI 10.1111/imr.12484.Mccoy L.E., Burton D.R. Identification and specificity of broadly neutralizing antibodies against HIV. Immunol. Rev. 2017;275(1):11-20. DOI 10.1111/imr.12484.Mccoy L.E., Quigley A.F., Strokappe N.M., Bulmer-Thomas B., Seaman M.S., Mortier D., Rutten L., Chander N., Edwards C.J., Ketteler R., Davis D., Verrips T., Weiss R.A. Potent and broad neutralization of HIV-1 by a llama antibody elicited by immunization. J. Exp. Med. 2012;209(6):1091-1103. DOI 10.1084/jem.20112655.Mccoy L.E., Quigley A.F., Strokappe N.M., Bulmer-Thomas B., Seaman M.S., Mortier D., Rutten L., Chander N., Edwards C.J., Ketteler R., Davis D., Verrips T., Weiss R.A. Potent and broad neutralization of HIV-1 by a llama antibody elicited by immunization. J. Exp. Med. 2012;209(6):1091-1103. DOI 10.1084/jem.20112655.Mccoy L.E., Rutten L., Frampton D., Anderson I., Granger L., Bashford-Rogers R., Dekkers G., Strokappe N.M., Seaman M.S., Koh W., Grippo V., Kliche A., Verrips T., Kellam P., Fassati A., Weiss R.A. Molecular evolution of broadly neutralizing llama antibodies to the CD4-binding site of HIV-1. PLoS Pathog. 2014;10(12):e1004552. DOI 10.1371/journal.ppat.1004552.Mccoy L.E., Rutten L., Frampton D., Anderson I., Granger L., Bashford-Rogers R., Dekkers G., Strokappe N.M., Seaman M.S., Koh W., Grippo V., Kliche A., Verrips T., Kellam P., Fassati A., Weiss R.A. Molecular evolution of broadly neutralizing llama antibodies to the CD4-binding site of HIV-1. PLoS Pathog. 2014;10(12):e1004552. DOI 10.1371/journal.ppat.1004552.Medina-Ramirez M., Garces F., Escolano A., Skog P., De Taeye S.W., Del Moral-Sanchez I., Mcguire A.T., Yasmeen A., Behrens A.J., Ozorowski G., Van Den Kerkhof T., Freund N.T., Dosenovic P., Hua Y., Gitlin A.D., Cupo A., Van Der Woude P., Golabek M., Sliepen K., Blane T., Kootstra N., Van Breemen M.J., Pritchard L.K., Stanfield R.L., Crispin M., Ward A.B., Stamatatos L., Klasse P.J., Moore J.P., Nemazee D., Nussenzweig M.C., Wilson I.A., Sanders R.W. Design and crystal structure of a native-like HIV-1 envelope trimer that engages multiple broadly neutralizing antibody precursors in vivo. J. Exp. Med. 2017;214(9):2573-2590. DOI 10.1084/jem.20161160.Medina-Ramirez M., Garces F., Escolano A., Skog P., De Taeye S.W., Del Moral-Sanchez I., Mcguire A.T., Yasmeen A., Behrens A.J., Ozorowski G., Van Den Kerkhof T., Freund N.T., Dosenovic P., Hua Y., Gitlin A.D., Cupo A., Van Der Woude P., Golabek M., Sliepen K., Blane T., Kootstra N., Van Breemen M.J., Pritchard L.K., Stanfield R.L., Crispin M., Ward A.B., Stamatatos L., Klasse P.J., Moore J.P., Nemazee D., Nussenzweig M.C., Wilson I.A., Sanders R.W. Design and crystal structure of a native-like HIV-1 envelope trimer that engages multiple broadly neutralizing antibody precursors in vivo. J. Exp. Med. 2017;214(9):2573-2590. DOI 10.1084/jem.20161160.Miller M.D., Geleziunas R., Bianchi E., Lennard S., Hrin R., Zhang H., Lu M., An Z., Ingallinella P., Finotto M., Mattu M., Finnefrock A.C., Bramhill D., Cook J., Eckert D.M., Hampton R., Patel M., Jarantow S., Joyce J., Ciliberto G., Cortese R., Lu P., Strohl W., Schleif W., Mcelhaugh M., Lane S., Lloyd C., Lowe D., Osbourn J., Vaughan T., Emini E., Barbato G., Kim P.S., Hazuda D.J., Shiver J.W., Pessi A. A human monoclonal antibody neutralizes diverse HIV-1 isolates by binding a critical gp41 epitope. Proc. Natl. Acad. Sci. USA. 2005; 102(41):14759-14764. DOI 10.1073/pnas.0506927102.Miller M.D., Geleziunas R., Bianchi E., Lennard S., Hrin R., Zhang H., Lu M., An Z., Ingallinella P., Finotto M., Mattu M., Finnefrock A.C., Bramhill D., Cook J., Eckert D.M., Hampton R., Patel M., Jarantow S., Joyce J., Ciliberto G., Cortese R., Lu P., Strohl W., Schleif W., Mcelhaugh M., Lane S., Lloyd C., Lowe D., Osbourn J., Vaughan T., Emini E., Barbato G., Kim P.S., Hazuda D.J., Shiver J.W., Pessi A. A human monoclonal antibody neutralizes diverse HIV-1 isolates by binding a critical gp41 epitope. Proc. Natl. Acad. Sci. USA. 2005; 102(41):14759-14764. DOI 10.1073/pnas.0506927102.Minenkova O.O., Ilyichev A.A., Kishchenko G.P., Petrenko V.A. Design of specific immunogens using filamentous phage as the carrier. Gene. 1993;128(1):85-88. DOI 10.1016/0378-1119(93)90157-x.Minenkova O.O., Ilyichev A.A., Kishchenko G.P., Petrenko V.A. Design of specific immunogens using filamentous phage as the carrier. Gene. 1993;128(1):85-88. DOI 10.1016/0378-1119(93)90157-x.Moldt B., Rakasz E.G., Schultz N., Chan-Hui P.Y., Swiderek K., Weisgrau K.L., Piaskowski S.M., Bergman Z., Watkins D.I., Poignard P., Burton D.R. Highly potent HIV-specific antibody neutralization in vitro translates into effective protection against mucosal SHIV challenge in vivo. Proc. Natl. Acad. Sci. USA. 2012;109(46):18921-18925. DOI 10.1073/pnas.1214785109.Moldt B., Rakasz E.G., Schultz N., Chan-Hui P.Y., Swiderek K., Weisgrau K.L., Piaskowski S.M., Bergman Z., Watkins D.I., Poignard P., Burton D.R. Highly potent HIV-specific antibody neutralization in vitro translates into effective protection against mucosal SHIV challenge in vivo. Proc. Natl. Acad. Sci. USA. 2012;109(46):18921-18925. DOI 10.1073/pnas.1214785109.Montefiori D.C., Baba T.W., Li A., Bilska M., Ruprecht R.M. Neutralizing and infection-enhancing antibody responses do not correlate with the differential pathogenicity of SIVmac239delta3 in adult and infant rhesus monkeys. J. Immunol. 1996;157(12):5528-5535.Montefiori D.C., Baba T.W., Li A., Bilska M., Ruprecht R.M. Neutralizing and infection-enhancing antibody responses do not correlate with the differential pathogenicity of SIVmac239delta3 in adult and infant rhesus monkeys. J. Immunol. 1996;157(12):5528-5535.Moon J.S., Choi E.J., Jeong N.N., Sohn J.R., Han D.W., Oh J.W. Research progress of M13 bacteriophage-based biosensors. Nanomaterials (Basel ). 2019;9(10). DOI 10.3390/nano9101448.Moon J.S., Choi E.J., Jeong N.N., Sohn J.R., Han D.W., Oh J.W. Research progress of M13 bacteriophage-based biosensors. Nanomaterials (Basel ). 2019;9(10). DOI 10.3390/nano9101448.Moulard M., Phogat S.K., Shu Y., Labrijn A.F., Xiao X., Binley J.M., Zhang M.Y., Sidorov I.A., Broder C.C., Robinson J., Parren P.W., Burton D.R., Dimitrov D.S. Broadly cross-reactive HIV-1-neutralizing human monoclonal Fab selected for binding to gp120-CD4CCR5 complexes. Proc. Natl. Acad. Sci. USA. 2002;99(10):6913-6918. DOI 10.1073/pnas.102562599.Moulard M., Phogat S.K., Shu Y., Labrijn A.F., Xiao X., Binley J.M., Zhang M.Y., Sidorov I.A., Broder C.C., Robinson J., Parren P.W., Burton D.R., Dimitrov D.S. Broadly cross-reactive HIV-1-neutralizing human monoclonal Fab selected for binding to gp120-CD4CCR5 complexes. Proc. Natl. Acad. Sci. USA. 2002;99(10):6913-6918. DOI 10.1073/pnas.102562599.Mouquet H., Nussenzweig M.C. HIV: Roadmaps to a vaccine. Nature. 2013;496(7446):441-442. DOI 10.1038/nature12091.Mouquet H., Nussenzweig M.C. HIV: Roadmaps to a vaccine. Nature. 2013;496(7446):441-442. DOI 10.1038/nature12091.Mullen L.M., Nair S.P., Ward J.M., Rycroft A.N., Henderson B. Phage display in the study of infectious diseases. Trends Microbiol. 2006; 14(3):141-147. DOI 10.1016/j.tim.2006.01.006.Mullen L.M., Nair S.P., Ward J.M., Rycroft A.N., Henderson B. Phage display in the study of infectious diseases. Trends Microbiol. 2006; 14(3):141-147. DOI 10.1016/j.tim.2006.01.006.Muyldermans S. A guide to: generation and design of nanobodies. FEBS J. 2021;288(7):2084-2102. DOI 10.1111/febs.15515.Muyldermans S. A guide to: generation and design of nanobodies. FEBS J. 2021;288(7):2084-2102. DOI 10.1111/febs.15515.Nelson J.D., Brunel F.M., Jensen R., Crooks E.T., Cardoso R.M., Wang M., Hessell A., Wilson I.A., Binley J.M., Dawson P.E., Burton D.R., Zwick M.B. An affinity-enhanced neutralizing antibody against the membrane-proximal external region of human immunodeficiency virus type 1 gp41 recognizes an epitope between those of 2F5 and 4E10. J. Virol. 2007;81(8):4033-4043. DOI 10.1128/JVI.02588-06.Nelson J.D., Brunel F.M., Jensen R., Crooks E.T., Cardoso R.M., Wang M., Hessell A., Wilson I.A., Binley J.M., Dawson P.E., Burton D.R., Zwick M.B. An affinity-enhanced neutralizing antibody against the membrane-proximal external region of human immunodeficiency virus type 1 gp41 recognizes an epitope between those of 2F5 and 4E10. J. Virol. 2007;81(8):4033-4043. DOI 10.1128/JVI.02588-06.Nemudraya A.A., Richter V.A., Kuligina E.V. Phage peptide libraries as a source of targeted ligands. Acta Naturae. 2016;8(1):48-57. DOI 10.32607/20758251-2016-8-1-48-57.Nemudraya A.A., Richter V.A., Kuligina E.V. Phage peptide libraries as a source of targeted ligands. Acta Naturae. 2016;8(1):48-57. DOI 10.32607/20758251-2016-8-1-48-57.Nixon A.E., Sexton D.J., Ladner R.C. Drugs derived from phage display: from candidate identification to clinical practice. MAbs. 2014; 6(1):73-85. DOI 10.4161/mabs.27240.Nixon A.E., Sexton D.J., Ladner R.C. Drugs derived from phage display: from candidate identification to clinical practice. MAbs. 2014; 6(1):73-85. DOI 10.4161/mabs.27240.Petrenko V.A., Jayanna P.K. Phage protein-targeted cancer nanomedicines. FEBS Lett. 2014;588(2):341-349. DOI 10.1016/j.febslet.2013.11.011.Petrenko V.A., Jayanna P.K. Phage protein-targeted cancer nanomedicines. FEBS Lett. 2014;588(2):341-349. DOI 10.1016/j.febslet.2013.11.011.Putney S. How antibodies block HIV infection: paths to an AIDS vaccine. Trends Biochem. Sci. 1992;17(5):191-196. DOI 10.1016/0968-0004(92)90265-b.Putney S. How antibodies block HIV infection: paths to an AIDS vaccine. Trends Biochem. Sci. 1992;17(5):191-196. DOI 10.1016/0968-0004(92)90265-b.Roben P., Moore J.P., Thali M., Sodroski J., Barbas C.F., 3rd, Burton D.R. Recognition properties of a panel of human recombinant Fab fragments to the CD4 binding site of gp120 that show differing abilities to neutralize human immunodeficiency virus type 1. J. Virol. 1994;68(8):4821-4828. DOI 10.1128/JVI.68.8.4821-4828.1994.Roben P., Moore J.P., Thali M., Sodroski J., Barbas C.F., 3rd, Burton D.R. Recognition properties of a panel of human recombinant Fab fragments to the CD4 binding site of gp120 that show differing abilities to neutralize human immunodeficiency virus type 1. J. Virol. 1994;68(8):4821-4828. DOI 10.1128/JVI.68.8.4821-4828.1994.Rusert P., Kouyos R.D., Kadelka C., Ebner H., Schanz M., Huber M., Braun D.L., Hoze N., Scherrer A., Magnus C., Weber J., Uhr T., Cippa V., Thorball C.W., Kuster H., Cavassini M., Bernasconi E., Hoffmann M., Calmy A., Battegay M., Rauch A., Yerly S., Aubert V., Klimkait T., Boni J., Fellay J., Regoes R.R., Gunthard H.F., Trkola A., Swiss Hivcs T. Determinants of HIV-1 broadly neutralizing antibody induction. Nat. Med. 2016;22(11):1260-1267. DOI 10.1038/nm.4187.Rusert P., Kouyos R.D., Kadelka C., Ebner H., Schanz M., Huber M., Braun D.L., Hoze N., Scherrer A., Magnus C., Weber J., Uhr T., Cippa V., Thorball C.W., Kuster H., Cavassini M., Bernasconi E., Hoffmann M., Calmy A., Battegay M., Rauch A., Yerly S., Aubert V., Klimkait T., Boni J., Fellay J., Regoes R.R., Gunthard H.F., Trkola A., Swiss Hivcs T. Determinants of HIV-1 broadly neutralizing antibody induction. Nat. Med. 2016;22(11):1260-1267. DOI 10.1038/nm.4187.Scheid J.F., Horwitz J.A., Bar-On Y., Kreider E.F., Lu C.L., Lorenzi J.C., Feldmann A., Braunschweig M., Nogueira L., Oliveira T., Shimeliovich I., Patel R., Burke L., Cohen Y.Z., Hadrigan S., Settler A., Witmer-Pack M., West A.P., Jr., Juelg B., Keler T., Hawthorne T., Zingman B., Gulick R.M., Pfeifer N., Learn G.H., Seaman M.S., Bjorkman P.J., Klein F., Schlesinger S.J., Walker B.D., Hahn B.H., Nussenzweig M.C., Caskey M. HIV-1 antibody 3BNC117 suppresses viral rebound in humans during treatment interruption. Nature. 2016;535(7613):556-560. DOI 10.1038/nature18929.Scheid J.F., Horwitz J.A., Bar-On Y., Kreider E.F., Lu C.L., Lorenzi J.C., Feldmann A., Braunschweig M., Nogueira L., Oliveira T., Shimeliovich I., Patel R., Burke L., Cohen Y.Z., Hadrigan S., Settler A., Witmer-Pack M., West A.P., Jr., Juelg B., Keler T., Hawthorne T., Zingman B., Gulick R.M., Pfeifer N., Learn G.H., Seaman M.S., Bjorkman P.J., Klein F., Schlesinger S.J., Walker B.D., Hahn B.H., Nussenzweig M.C., Caskey M. HIV-1 antibody 3BNC117 suppresses viral rebound in humans during treatment interruption. Nature. 2016;535(7613):556-560. DOI 10.1038/nature18929.Schoofs T., Klein F., Braunschweig M., Kreider E.F., Feldmann A., Nogueira L., Oliveira T., Lorenzi J.C., Parrish E.H., Learn G.H., West A.P., Jr., Bjorkman P.J., Schlesinger S.J., Seaman M.S., Czartoski J., Mcelrath M.J., Pfeifer N., Hahn B.H., Caskey M., Nussenzweig M.C. HIV-1 therapy with monoclonal antibody 3BNC117 elicits host immune responses against HIV-1. Science. 2016; 352(6288):997-1001. DOI 10.1126/science.aaf0972.Schoofs T., Klein F., Braunschweig M., Kreider E.F., Feldmann A., Nogueira L., Oliveira T., Lorenzi J.C., Parrish E.H., Learn G.H., West A.P., Jr., Bjorkman P.J., Schlesinger S.J., Seaman M.S., Czartoski J., Mcelrath M.J., Pfeifer N., Hahn B.H., Caskey M., Nussenzweig M.C. HIV-1 therapy with monoclonal antibody 3BNC117 elicits host immune responses against HIV-1. Science. 2016; 352(6288):997-1001. DOI 10.1126/science.aaf0972.Scott J.K., Smith G.P. Searching for peptide ligands with an epitope library. Science. 1990;249(4967):386-390. DOI 10.1126/science.1696028.Scott J.K., Smith G.P. Searching for peptide ligands with an epitope library. Science. 1990;249(4967):386-390. DOI 10.1126/science.1696028.Shcherbakov D.N., Bakulina A.Y., Karpenko L.I., Ilyichev A.A. Broadly neutralizing antibodies against HIV-1 as a novel aspect of the immune response. Acta Naturae. 2015;7(4):11-21.Shcherbakov D.N., Bakulina A.Y., Karpenko L.I., Ilyichev A.A. Broadly neutralizing antibodies against HIV-1 as a novel aspect of the immune response. Acta Naturae. 2015;7(4):11-21.Shingai M., Donau O.K., Plishka R.J., Buckler-White A., Mascola J.R., Nabel G.J., Nason M.C., Montefiori D., Moldt B., Poignard P., Diskin R., Bjorkman P.J., Eckhaus M.A., Klein F., Mouquet H., Cetrulo Lorenzi J.C., Gazumyan A., Burton D.R., Nussenzweig M.C., Martin M.A., Nishimura Y. Passive transfer of modest titers of potent and broadly neutralizing anti-HIV monoclonal antibodies block SHIV infection in macaques. J. Exp. Med. 2014;211(10):2061-2074. DOI 10.1084/jem.20132494.Shingai M., Donau O.K., Plishka R.J., Buckler-White A., Mascola J.R., Nabel G.J., Nason M.C., Montefiori D., Moldt B., Poignard P., Diskin R., Bjorkman P.J., Eckhaus M.A., Klein F., Mouquet H., Cetrulo Lorenzi J.C., Gazumyan A., Burton D.R., Nussenzweig M.C., Martin M.A., Nishimura Y. Passive transfer of modest titers of potent and broadly neutralizing anti-HIV monoclonal antibodies block SHIV infection in macaques. J. Exp. Med. 2014;211(10):2061-2074. DOI 10.1084/jem.20132494.Skerra A., Pluckthun A. Assembly of a functional immunoglobulin Fv fragment in Escherichia coli. Science. 1988;240(4855):1038-1041. DOI 10.1126/science.3285470.Skerra A., Pluckthun A. Assembly of a functional immunoglobulin Fv fragment in Escherichia coli. Science. 1988;240(4855):1038-1041. DOI 10.1126/science.3285470.Smith G.P. Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface. Science. 1985; 228(4705):1315-1317. DOI 10.1126/science.4001944.Smith G.P. Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface. Science. 1985; 228(4705):1315-1317. DOI 10.1126/science.4001944.Smith G.P., Petrenko V.A. Phage display. Chem. Rev. 1997;97(2):391-410. DOI 10.1021/cr960065d.Smith G.P., Petrenko V.A. Phage display. Chem. Rev. 1997;97(2):391-410. DOI 10.1021/cr960065d.Sok D., Burton D.R. Recent progress in broadly neutralizing antibodies to HIV. Nat. Immunol. 2018;19(11):1179-1188. DOI 10.1038/s41590-018-0235-7.Sok D., Burton D.R. Recent progress in broadly neutralizing antibodies to HIV. Nat. Immunol. 2018;19(11):1179-1188. DOI 10.1038/s41590-018-0235-7.Sozhamannan S., Hofmann E.R. The state of the art in biodefense related bacterial pathogen detection using bacteriophages: how it started and how it’s going. Viruses. 2020;12(12). DOI 10.3390/v12121393.Sozhamannan S., Hofmann E.R. The state of the art in biodefense related bacterial pathogen detection using bacteriophages: how it started and how it’s going. Viruses. 2020;12(12). DOI 10.3390/v12121393.Stamatatos L., Pancera M., Mcguire A.T. Germline-targeting immunogens. Immunol. Rev. 2017;275(1):203-216. DOI 10.1111/imr.12483.Stamatatos L., Pancera M., Mcguire A.T. Germline-targeting immunogens. Immunol. Rev. 2017;275(1):203-216. DOI 10.1111/imr.12483.Stephenson K.E., Wagh K., Korber B., Barouch D.H. Vaccines and broadly neutralizing antibodies for HIV-1 prevention. Annu. Rev. Immunol. 2020;38(673-703). DOI 10.1146/annurev-immunol-080219-023629.Stephenson K.E., Wagh K., Korber B., Barouch D.H. Vaccines and broadly neutralizing antibodies for HIV-1 prevention. Annu. Rev. Immunol. 2020;38(673-703). DOI 10.1146/annurev-immunol-080219-023629.Strokappe N.M., Hock M., Rutten L., Mccoy L.E., Back J.W., Caillat C., Haffke M., Weiss R.A., Weissenhorn W., Verrips T. Super potent bispecific llama VHH antibodies neutralize HIV via a combination of gp41 and gp120 epitopes. Antibodies (Basel ). 2019;8(2). DOI 10.3390/antib8020038.Strokappe N.M., Hock M., Rutten L., Mccoy L.E., Back J.W., Caillat C., Haffke M., Weiss R.A., Weissenhorn W., Verrips T. Super potent bispecific llama VHH antibodies neutralize HIV via a combination of gp41 and gp120 epitopes. Antibodies (Basel ). 2019;8(2). DOI 10.3390/antib8020038.Strokappe N., Szynol A., Aasa-Chapman M., Gorlani A., Forsman Quigley A., Hulsik D.L., Chen L., Weiss R., De Haard H., Verrips T. Llama antibody fragments recognizing various epitopes of the CD4bs neutralize a broad range of HIV-1 subtypes A, B and C. PLoS One. 2012;7(3):e33298. DOI 10.1371/journal.pone.0033298.Strokappe N., Szynol A., Aasa-Chapman M., Gorlani A., Forsman Quigley A., Hulsik D.L., Chen L., Weiss R., De Haard H., Verrips T. Llama antibody fragments recognizing various epitopes of the CD4bs neutralize a broad range of HIV-1 subtypes A, B and C. PLoS One. 2012;7(3):e33298. DOI 10.1371/journal.pone.0033298.Tikunova N.V., Morozova V.V. Phage display on the base of filamentous bacteriophages: application for recombinant antibodies selection. Acta Naturae. 2009;1(3):20-28.Tikunova N.V., Morozova V.V. Phage display on the base of filamentous bacteriophages: application for recombinant antibodies selection. Acta Naturae. 2009;1(3):20-28.Walker L.M., Phogat S.K., Chan-Hui P.Y., Wagner D., Phung P., Goss J.L., Wrin T., Simek M.D., Fling S., Mitcham J.L., Lehrman J.K., Priddy F.H., Olsen O.A., Frey S.M., Hammond P.W., Investigators P.G.P., Kaminsky S., Zamb T., Moyle M., Koff W.C., Poignard P., Burton D.R. Broad and potent neutralizing antibodies from an African donor reveal a new HIV-1 vaccine target. Science. 2009;326(5950):285-289. DOI 10.1126/science.1178746.Walker L.M., Phogat S.K., Chan-Hui P.Y., Wagner D., Phung P., Goss J.L., Wrin T., Simek M.D., Fling S., Mitcham J.L., Lehrman J.K., Priddy F.H., Olsen O.A., Frey S.M., Hammond P.W., Investigators P.G.P., Kaminsky S., Zamb T., Moyle M., Koff W.C., Poignard P., Burton D.R. Broad and potent neutralizing antibodies from an African donor reveal a new HIV-1 vaccine target. Science. 2009;326(5950):285-289. DOI 10.1126/science.1178746.Walker L.M., Simek M.D., Priddy F., Gach J.S., Wagner D., Zwick M.B., Phogat S.K., Poignard P., Burton D.R. A limited number of antibody specificities mediate broad and potent serum neutralization in selected HIV-1 infected individuals. PLoS Pathog. 2010;6(8):e1001028. DOI 10.1371/journal.ppat.1001028.Walker L.M., Simek M.D., Priddy F., Gach J.S., Wagner D., Zwick M.B., Phogat S.K., Poignard P., Burton D.R. A limited number of antibody specificities mediate broad and potent serum neutralization in selected HIV-1 infected individuals. PLoS Pathog. 2010;6(8):e1001028. DOI 10.1371/journal.ppat.1001028.Weiss R.A., Verrips C.T. Nanobodies that neutralize HIV. Vaccines (Basel ). 2019;7(3). DOI 10.3390/vaccines7030077.Weiss R.A., Verrips C.T. Nanobodies that neutralize HIV. Vaccines (Basel ). 2019;7(3). DOI 10.3390/vaccines7030077.Winter G., Griffiths A.D., Hawkins R.E., Hoogenboom H.R. Making antibodies by phage display technology. Annu. Rev. Immunol. 1994;12(433-455). DOI 10.1146/annurev.iy.12.040194.002245.Winter G., Griffiths A.D., Hawkins R.E., Hoogenboom H.R. Making antibodies by phage display technology. Annu. Rev. Immunol. 1994;12(433-455). DOI 10.1146/annurev.iy.12.040194.002245.Wu X., Yang Z.Y., Li Y., Hogerkorp C.M., Schief W.R., Seaman M.S., Zhou T., Schmidt S.D., Wu L., Xu L., Longo N.S., Mckee K., O’dell S., Louder M.K., Wycuff D.L., Feng Y., Nason M., DoriaRose N., Connors M., Kwong P.D., Roederer M., Wyatt R.T., Nabel G.J., Mascola J.R. Rational design of envelope identifies broadly neutralizing human monoclonal antibodies to HIV-1. Science. 2010; 329(5993):856-861. DOI 10.1126/science.1187659.Wu X., Yang Z.Y., Li Y., Hogerkorp C.M., Schief W.R., Seaman M.S., Zhou T., Schmidt S.D., Wu L., Xu L., Longo N.S., Mckee K., O’dell S., Louder M.K., Wycuff D.L., Feng Y., Nason M., DoriaRose N., Connors M., Kwong P.D., Roederer M., Wyatt R.T., Nabel G.J., Mascola J.R. Rational design of envelope identifies broadly neutralizing human monoclonal antibodies to HIV-1. Science. 2010; 329(5993):856-861. DOI 10.1126/science.1187659.Zhang M.Y., Choudhry V., Sidorov I.A., Tenev V., Vu B.K., Choudhary A., Lu H., Stiegler G.M., Katinger H.W., Jiang S., Broder C.C., Dimitrov D.S. Selection of a novel gp41-specific HIV-1 neutralizing human antibody by competitive antigen panning. J. Immunol. Methods. 2006;317(1-2):21-30. DOI 10.1016/j.jim.2006.09.016.Zhang M.Y., Choudhry V., Sidorov I.A., Tenev V., Vu B.K., Choudhary A., Lu H., Stiegler G.M., Katinger H.W., Jiang S., Broder C.C., Dimitrov D.S. Selection of a novel gp41-specific HIV-1 neutralizing human antibody by competitive antigen panning. J. Immunol. Methods. 2006;317(1-2):21-30. DOI 10.1016/j.jim.2006.09.016.Zhang M.Y., Shu Y., Phogat S., Xiao X., Cham F., Bouma P., Choudhary A., Feng Y.R., Sanz I., Rybak S., Broder C.C., Quinnan G.V., Evans T., Dimitrov D.S. Broadly cross-reactive HIV neutralizing human monoclonal antibody Fab selected by sequential antigen panning of a phage display library. J. Immunol. Methods. 2003; 283(1-2):17-25. http://dx.doi.org/10.1016/j.jim.2003.07.003.Zhang M.Y., Shu Y., Phogat S., Xiao X., Cham F., Bouma P., Choudhary A., Feng Y.R., Sanz I., Rybak S., Broder C.C., Quinnan G.V., Evans T., Dimitrov D.S. Broadly cross-reactive HIV neutralizing human monoclonal antibody Fab selected by sequential antigen panning of a phage display library. J. Immunol. Methods. 2003; 283(1-2):17-25. http://dx.doi.org/10.1016/j.jim.2003.07.003.Zhang M.Y., Shu Y., Rudolph D., Prabakaran P., Labrijn A.F., Zwick M.B., Lal R.B., Dimitrov D.S. Improved breadth and potency of an HIV-1-neutralizing human single-chain antibody by random mutagenesis and sequential antigen panning. J. Mol. Biol. 2004a; 335(1):209-219. DOI 10.1016/j.jmb.2003.09.055.Zhang M.Y., Shu Y., Rudolph D., Prabakaran P., Labrijn A.F., Zwick M.B., Lal R.B., Dimitrov D.S. Improved breadth and potency of an HIV-1-neutralizing human single-chain antibody by random mutagenesis and sequential antigen panning. J. Mol. Biol. 2004a; 335(1):209-219. DOI 10.1016/j.jmb.2003.09.055.Zhang M.Y., Xiao X., Sidorov I.A., Choudhry V., Cham F., Zhang P.F., Bouma P., Zwick M., Choudhary A., Montefiori D.C., Broder C.C., Burton D.R., Quinnan G.V., Jr., Dimitrov D.S. Identification and characterization of a new cross-reactive human immunodeficiency virus type 1-neutralizing human monoclonal antibody. J. Virol. 2004b;78(17):9233-9242. DOI 10.1128/JVI.78.17.9233-9242.2004.Zhang M.Y., Xiao X., Sidorov I.A., Choudhry V., Cham F., Zhang P.F., Bouma P., Zwick M., Choudhary A., Montefiori D.C., Broder C.C., Burton D.R., Quinnan G.V., Jr., Dimitrov D.S. Identification and characterization of a new cross-reactive human immunodeficiency virus type 1-neutralizing human monoclonal antibody. J. Virol. 2004b;78(17):9233-9242. DOI 10.1128/JVI.78.17.9233-9242.2004.Zhang M.Y., Yuan T., Li J., Rosa Borges A., Watkins J.D., Guenaga J., Yang Z., Wang Y., Wilson R., Li Y., Polonis V.R., Pincus S.H., Ruprecht R.M., Dimitrov D.S. Identification and characterization of a broadly cross-reactive HIV-1 human monoclonal antibody that binds to both gp120 and gp41. PLoS One. 2012;7(9):e44241. DOI 10.1371/journal.pone.0044241.Zhang M.Y., Yuan T., Li J., Rosa Borges A., Watkins J.D., Guenaga J., Yang Z., Wang Y., Wilson R., Li Y., Polonis V.R., Pincus S.H., Ruprecht R.M., Dimitrov D.S. Identification and characterization of a broadly cross-reactive HIV-1 human monoclonal antibody that binds to both gp120 and gp41. PLoS One. 2012;7(9):e44241. DOI 10.1371/journal.pone.0044241.Zhao A., Tohidkia M.R., Siegel D.L., Coukos G., Omidi Y. Phage antibody display libraries: a powerful antibody discovery platform for immunotherapy. Crit. Rev. Biotechnol. 2016;36(2):276-289. DOI 10.3109/07388551.2014.958978.Zhao A., Tohidkia M.R., Siegel D.L., Coukos G., Omidi Y. Phage antibody display libraries: a powerful antibody discovery platform for immunotherapy. Crit. Rev. Biotechnol. 2016;36(2):276-289. DOI 10.3109/07388551.2014.958978.Zwick M.B., Bonnycastle L.L., Menendez A., Irving M.B., Barbas C.F., 3rd, Parren P.W., Burton D.R., Scott J.K. Identification and characterization of a peptide that specifically binds the human broadly neutralizing anti-human immunodeficiency virus type 1 antibody b12. J. Virol. 2001;75(14):6692-6699. DOI 10.1128/JVI.75.14.6692-6699.2001.Zwick M.B., Bonnycastle L.L., Menendez A., Irving M.B., Barbas C.F., 3rd, Parren P.W., Burton D.R., Scott J.K. Identification and characterization of a peptide that specifically binds the human broadly neutralizing anti-human immunodeficiency virus type 1 antibody b12. J. Virol. 2001;75(14):6692-6699. DOI 10.1128/JVI.75.14.6692-6699.2001.

The authors declare that there are no conflicts of interest present.