vavilovВавиловский журнал генетики и селекцииVavilov Journal of Genetics and Breeding2500-3259Institute of Cytology and Genetics of Siberian Branch of the RAS10.18699/VJGB-22-26vavilov-3298Research ArticleАКТУАЛЬНЫЕ ТЕХНОЛОГИИMAINSTREAM TECHNOLOGIESMодельные системы вируса иммунодефицита человека (ВИЧ-1), используемые для оценки эффективности кандидатных вакцин и лекарственных препаратов против ВИЧ-1 in vitroModel systems of human immunodef iciency virus (HIV-1) for in vitro eff icacy assessment of candidate vaccines and drugs against HIV-1https://orcid.org/0000-0002-1684-9071РудометоваН. Б.RudometovaN. B.

р.п. Кольцово, Новосибирская область

Koltsovo, Novosibirsk Region

nadenkaand100@mail.ruhttps://orcid.org/0000-0001-8023-4453ЩербаковД. Н.ShcherbakovD. N.

р.п. Кольцово, Новосибирская область

Koltsovo, Novosibirsk Region

https://orcid.org/0000-0003-2808-4309РудометовА. П.RudometovA. P.

р.п. Кольцово, Новосибирская область

Koltsovo, Novosibirsk Region

https://orcid.org/0000-0001-5356-0843ИльичевА. А.IlyichevA. 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

Государственный научный центр вирусологии и биотехнологии «Вектор» РоспотребнадзораРоссияState Research Center of Virology and Biotechnology “Vector”Russian Federation202205042022262214221Copyright © Рудометова Н.Б., Щербаков Д.Н., Рудометов А.П., Ильичев А.А., Карпенко Л.И., 20222022Рудометова Н.Б., Щербаков Д.Н., Рудометов А.П., Ильичев А.А., Карпенко Л.И.Rudometova N.B., Shcherbakov D.N., Rudometov A.P., Ilyichev A.A., Karpenko L.I.This work is licensed under a Creative Commons Attribution 4.0 License.https://vavilov.elpub.ru/jour/article/view/3298

ВИЧ-инфекция по-прежнему остается одной из глобальных проблем здравоохранения во всем мире. Борьба с инфекцией ведется по нескольким направлениям. Во-первых, это профилактические мероприятия, которые включают просвещение населения по проблеме ВИЧ/СПИДа, пропаганду здорового образа жизни, защищенные половые контакты, доконтактную профилактику уязвимых групп населения. Во-вторых, прохождение своевременного тестирования на ВИЧ и применение антиретровирусной терапии в случае его обнаружения. В-третьих, это научные исследования, связанные как с поиском новых лекарственных агентов, так и с разработкой вакцины против ВИЧ-1. Ключевой момент при определении эффективности вакцин и химиотерапевтических препаратов – выбор инструмента, позволяющего быстро и точно оценить их эффективность in vitro. Классическим методом вирусологии, позволяющим оценить нейтрализующую активность сывороток животных, иммунизированных экспериментальными вакцинами, и эффективность химиотерапевтических агентов, является метод нейтрализации с использованием вирусных изолятов, а также инфекционных молекулярных клонов, которые представляют собой инфекционные вирусные частицы, полученные путем трансфекции клеток плазмидным вектором, содержащим полноразмерный геном ВИЧ-1, кодирующий структурные, регуляторные и вспомогательные белки вируса, необходимые для образования репликационно-компетентных вирусных частиц в культуре клеток. При этом метод нейтрализации с использованием вирусных изолятов и инфекционных молекулярных клонов отличается трудоемкостью, продолжительностью и требует повышенных мер биобезопасности. Альтернативным решением, устраняющим указанные недостатки и позволяющим проводить быстрый скрининг, является использование для анализа нейтрализующей активности псевдовирусов, которые представляют собой рекомбинантные вирусные частицы. В отличие от инфекционных вирусов, работа с псевдовирусами безопасна, поскольку геном псевдовирусов нарушен для того, чтобы их инфекция ограничивалась лишь одним циклом. Данный обзор посвящен описанию модельных вирусных систем, используемых для оценки эффективности вакцин и лекарственных препаратов против ВИЧ-1 in vitro: первичных изолятов ВИЧ-1 и лабораторно-адаптированных штаммов, инфекционных молекулярных клонов и env-псевдовирусов. Кратко представлена их сравнительная характеристика. Более подробно описана технология env-псевдовирусов ВИЧ-1.

HIV infection still remains a major challenge for healthcare systems of the world. There are several aspects on counteracting the HIV/AIDS epidemic. The f irst aspect covers preventive measures including educational campaigns on HIV/AIDS and promotion of a healthy lifestyle, protected sex, and pre-exposure prophylaxis of vulnerable groups. The second aspect is timely HIV testing and the use of antiretroviral therapy when test results come back positive. The third aspect is the scientif ic research associated with discovering new pharmaceutical agents and developing HIV-1 vaccines. Selecting an adequate tool for quick and accurate in vitro eff icacy assessment is the key aspect for eff icacy assessment of vaccines and chemotherapy drugs. The classical method of virology, which makes it possible to evaluate the neutralizing activity of the sera of animals immunized with experimental vaccines and the eff icacy of chemotherapy agents is the method of neutralization using viral isolates and infectious molecular clones, i. e. infectious viral particles obtained via cell transfection with a plasmid vector including the full-length HIV-1 genome coding structural, regulatory, and accessory proteins of the virus required for the cultivation of replication-competent viral particles in cell culture. However, neutralization assessment using viral isolates and infectious molecular clones is demanding in terms of time, effort, and biosafety measures. An alternative eliminating these disadvantages and allowing for rapid screening is the use of pseudoviruses, which are recombinant viral particles, for the analysis of neutralizing activity. Pseudotyped viruses have defective genomes restricting their replication to a single cycle, which renders them harmless compared to infectious viruses. The present review focuses on describing viral model systems for in vitro eff icacy assessment of vaccines and drugs against HIV-1, which include primary HIV-1 isolates, laboratoryadapted strains, infectious molecular clones, and env-pseudoviruses. A brief comparison of the listed models is presented. The HIV-1 env-pseudoviruses approach is described in more detail.

ВИЧ-1первичные изолятыинфекционные молекулярные клоныenv-псевдовирусыанализ нейтрализации вирусаHIV-1primary isolatesinfectious molecular clonesenv-pseudovirusesvirus neutralization assayThe reported study was funded by the grant of the President of the Russian Federation MK-583.2020.4 and RFBR and Novosibirsk region, according to the research project No. 19-44-543013, and the state assignment of FBSI SRC VB “Vector”, Rospotrebnadzor.ReferencesArts E.J., Hazuda D.J. HIV-1 antiretroviral drug therapy. Cold Spring Harb. Perspect. Med. 2012;2(4):1-23. DOI 10.1101/cshperspect.a007161.Arts E.J., Hazuda D.J. HIV-1 antiretroviral drug therapy. Cold Spring Harb. Perspect. Med. 2012;2(4):1-23. DOI 10.1101/cshperspect.a007161.Baalwa J., Wang S., Parrish N.F., Decker J.M., Keele B.F., Learn G.H., Yue L., Ruzagira E., Ssemwanga D., Kamali A. Molecular identification, cloning and characterization of transmitted/founder HIV- 1 subtype A, D and A/D infectious molecular clones. Virology. 2013; 436(1):33-48. DOI 10.1016/j.virol.2012.10.009.Baalwa J., Wang S., Parrish N.F., Decker J.M., Keele B.F., Learn G.H., Yue L., Ruzagira E., Ssemwanga D., Kamali A. Molecular identification, cloning and characterization of transmitted/founder HIV- 1 subtype A, D and A/D infectious molecular clones. Virology. 2013; 436(1):33-48. DOI 10.1016/j.virol.2012.10.009.Chenine A.L., Merbah M., Wieczorek L., Molnar S., Mann B., Lee J., O’Sullivan A.M., Bose M., Sanders-Buell E., Kijak G.H., Herrera C., McLinden R., O’Connell R., Michael N.L., Robb M.L., Kim J.H., Polonis V.R., Tovanabutra S. Neutralization sensitivity of a novel HIV-1 CRF01_AE panel of infectious molecular clones. J. Acquir. Immune Def ic. Syndrom. 2018;78(3):348-355. DOI 10.1097/QAI.0000000000001675.Chenine A.L., Merbah M., Wieczorek L., Molnar S., Mann B., Lee J., O’Sullivan A.M., Bose M., Sanders-Buell E., Kijak G.H., Herrera C., McLinden R., O’Connell R., Michael N.L., Robb M.L., Kim J.H., Polonis V.R., Tovanabutra S. Neutralization sensitivity of a novel HIV-1 CRF01_AE panel of infectious molecular clones. J. Acquir. Immune Def ic. Syndrom. 2018;78(3):348-355. DOI 10.1097/QAI.0000000000001675.Cheresiz S.V., Grigoryev I.V., Semenova E.A., Pustylnyak V.O., Vlasov V.V., Pokrovsky A.G. A pseudovirus system for the testing of antiviral activity of compounds in different cell lines. Doklady Biochemistry and Biophysics. 2010;435:295-298. DOI 10.1134/S1607672910060049.Cheresiz S.V., Grigoryev I.V., Semenova E.A., Pustylnyak V.O., Vlasov V.V., Pokrovsky A.G. A pseudovirus system for the testing of antiviral activity of compounds in different cell lines. Doklady Biochemistry and Biophysics. 2010;435:295-298. DOI 10.1134/S1607672910060049.Dorr P., Westby M., Dobbs S., Griffin P., Irvine B., Macartney M., Mori J., Rickett G., Smith-Burchnell C., Napier C., Webster R., Armour D., Price D., Stammen B., Wood A., Perros M. Maraviroc (UK-427,857), a potent, orally bioavailable, and selective small-molecule inhibitor of chemokine receptor CCR5 with broadspectrum anti-human immunodeficiency virus type 1 activity. Antimicrob. Agents Chemother. 2005;49(11):4721-4732. DOI 10.1128/AAC.49.11.4721-4732.2005.Dorr P., Westby M., Dobbs S., Griffin P., Irvine B., Macartney M., Mori J., Rickett G., Smith-Burchnell C., Napier C., Webster R., Armour D., Price D., Stammen B., Wood A., Perros M. Maraviroc (UK-427,857), a potent, orally bioavailable, and selective small-molecule inhibitor of chemokine receptor CCR5 with broadspectrum anti-human immunodeficiency virus type 1 activity. Antimicrob. Agents Chemother. 2005;49(11):4721-4732. DOI 10.1128/AAC.49.11.4721-4732.2005.Edmonds T.G., Ding H., Yuan X., Wei Q., Smith K.S., Conway J.A., Wieczorek L., Brown B., Polonis V., West J.T., Montefiori D.C., Kappes J.C., Ochsenbauer Ch. Replication competent molecular clones of HIV-1 expressing Renilla luciferase facilitate the analysis of antibody inhibition in PBMC. Virology. 2010;408(1):1-13. DOI 10.1016/j.virol.2010.08.028.Edmonds T.G., Ding H., Yuan X., Wei Q., Smith K.S., Conway J.A., Wieczorek L., Brown B., Polonis V., West J.T., Montefiori D.C., Kappes J.C., Ochsenbauer Ch. Replication competent molecular clones of HIV-1 expressing Renilla luciferase facilitate the analysis of antibody inhibition in PBMC. Virology. 2010;408(1):1-13. DOI 10.1016/j.virol.2010.08.028.González N., Pérez-Olmeda M., Mateos E., Cascajero A., Alvarez A., Spijkers S., García-Pérez J., Sánchez-Palomino S., Ruiz-Mateos E., Leal M., Alcami J. A sensitive phenotypic assay for the determination of human immunodeficiency virus type 1 tropism. J. Antimicrob. Chemother. 2010;65(12):2493-2501. DOI 10.1093/jac/dkq379.González N., Pérez-Olmeda M., Mateos E., Cascajero A., Alvarez A., Spijkers S., García-Pérez J., Sánchez-Palomino S., Ruiz-Mateos E., Leal M., Alcami J. A sensitive phenotypic assay for the determination of human immunodeficiency virus type 1 tropism. J. Antimicrob. Chemother. 2010;65(12):2493-2501. DOI 10.1093/jac/dkq379.Gray G.E., Bekker L.G., Laher F., Malahleha M., Allen M., Moodie Z., Grunenberg N., Huang Yu., Grove D., Prigmore B. Vaccine efficacy of ALVAC-HIV and bivalent subtype C gp120–MF59 in adults. New Eng. J. Med. 2021;384(12):1089-1100. DOI 10.1056/NEJMoa2031499.Gray G.E., Bekker L.G., Laher F., Malahleha M., Allen M., Moodie Z., Grunenberg N., Huang Yu., Grove D., Prigmore B. Vaccine efficacy of ALVAC-HIV and bivalent subtype C gp120–MF59 in adults. New Eng. J. Med. 2021;384(12):1089-1100. DOI 10.1056/NEJMoa2031499.Hsu D.C., O’Connell R.J. Progress in HIV vaccine development. Hum. Vaccines Immunother. 2017;13(5):1018-1030. DOI 10.1080/21645515.2016.1276138.Hsu D.C., O’Connell R.J. Progress in HIV vaccine development. Hum. Vaccines Immunother. 2017;13(5):1018-1030. DOI 10.1080/21645515.2016.1276138.Hu J., Gao Q., He C., Huang A., Tang N., Wang K. Development of cell-based pseudovirus entry assay to identify potential viral entry inhibitors and neutralizing antibodies against SARS-CoV-2. Genes Dis. 2020;7(4):551-557. DOI 10.1016/j.gendis.2020.07.006.Hu J., Gao Q., He C., Huang A., Tang N., Wang K. Development of cell-based pseudovirus entry assay to identify potential viral entry inhibitors and neutralizing antibodies against SARS-CoV-2. Genes Dis. 2020;7(4):551-557. DOI 10.1016/j.gendis.2020.07.006.Huang J., Ofek G., Laub L., Louder M.K., Doria-Rose N.A., Longo N.S., Imamichi H., Bailer R.T., Chakrabarti B., Sharma S.K., Munir Alam S., Wang T., Yang Y., Zhang B., Migueles S.A., Wyatt R., Haynes B.F., Kwong P.D., Mascola J.R., Connors M. Broad and potent neutralization of HIV-1 by a gp41-specific human antibody. Nature. 2012;491(7424):406-412. DOI 10.1038/nature11544.Huang J., Ofek G., Laub L., Louder M.K., Doria-Rose N.A., Longo N.S., Imamichi H., Bailer R.T., Chakrabarti B., Sharma S.K., Munir Alam S., Wang T., Yang Y., Zhang B., Migueles S.A., Wyatt R., Haynes B.F., Kwong P.D., Mascola J.R., Connors M. Broad and potent neutralization of HIV-1 by a gp41-specific human antibody. Nature. 2012;491(7424):406-412. DOI 10.1038/nature11544.Hyseni I., Molesti E., Benincasa L., Piu P., Casa E., Temperton N.J., Manenti A., Montomoli E. Characterisation of SARS-CoV-2 lentiviral pseudotypes and correlation between pseudotype-based neutralisation assays and live virus-based micro neutralisation assays. Viruses. 2020;12(9):1-18. DOI 10.3390/v12091011.Hyseni I., Molesti E., Benincasa L., Piu P., Casa E., Temperton N.J., Manenti A., Montomoli E. Characterisation of SARS-CoV-2 lentiviral pseudotypes and correlation between pseudotype-based neutralisation assays and live virus-based micro neutralisation assays. Viruses. 2020;12(9):1-18. DOI 10.3390/v12091011.Jackson J.B., Coombs R.W., Sannerud K., Rhame F.S., Balfour H.H., Jr. Rapid and sensitive viral culture method for human immunodeficiency virus type 1. J. Clin. Microbiol. 1988;26(7):1416-1418. PMID 3165981.Jackson J.B., Coombs R.W., Sannerud K., Rhame F.S., Balfour H.H., Jr. Rapid and sensitive viral culture method for human immunodeficiency virus type 1. J. Clin. Microbiol. 1988;26(7):1416-1418. PMID 3165981.Johnson M.C., Lyddon T.D., Suarez R., Salcedo B., LePique M., Graham M., Ricana C.L., Robinson C.A., Ritter D.G. Optimized pseudotyping conditions for the SARS-COV-2 spike glycoprotein. J. Virol. 2020;94(21):1-10. DOI 10.1128/JVI.01062-20.Johnson M.C., Lyddon T.D., Suarez R., Salcedo B., LePique M., Graham M., Ricana C.L., Robinson C.A., Ritter D.G. Optimized pseudotyping conditions for the SARS-COV-2 spike glycoprotein. J. Virol. 2020;94(21):1-10. DOI 10.1128/JVI.01062-20.Johnston E., Dupnik K.M., Gonzales M.J., Winters M.A., Rhee S.Y., Imamichi T., Shafer R.W. Panel of prototypical infectious molecular HIV-1 clones containing multiple nucleoside reverse transcriptase inhibitor resistance mutations. AIDS. 2005;19(7):731-733. DOI 10.1097/01.aids.0000166098.54564.0c.Johnston E., Dupnik K.M., Gonzales M.J., Winters M.A., Rhee S.Y., Imamichi T., Shafer R.W. Panel of prototypical infectious molecular HIV-1 clones containing multiple nucleoside reverse transcriptase inhibitor resistance mutations. AIDS. 2005;19(7):731-733. DOI 10.1097/01.aids.0000166098.54564.0c.Jones L.D., Moody M.A., Thompson A.B. Innovations in HIV-1 vaccine design. Clin. Ther. 2020;42(3):499-514. DOI 10.1016/j.clinthera.2020.01.009.Jones L.D., Moody M.A., Thompson A.B. Innovations in HIV-1 vaccine design. Clin. Ther. 2020;42(3):499-514. DOI 10.1016/j.clinthera.2020.01.009.Kim J.H., Excler J.L., Michael N.L. Lessons from the RV144 Thai phase III HIV-1 vaccine trial and the search for correlates of protection. Annu. Rev. Med. 2015;66:423-437. DOI 10.1146/annurevmed-052912-123749.Kim J.H., Excler J.L., Michael N.L. Lessons from the RV144 Thai phase III HIV-1 vaccine trial and the search for correlates of protection. Annu. Rev. Med. 2015;66:423-437. DOI 10.1146/annurevmed-052912-123749.Kong R., Xu K., Zhou T., Acharya P., Lemmin T., Liu K., Ozorowski G., Soto C., Taft J., Bailer R., Cale E.M., Chen L., Choi C.W., Chuang G., Doria-Rose N.A., Druz A., Georgiev I.S., Gorman J., Huang J., Gordon Joyce M., Louder M.K., Ma X., McKee K., O’Dell S., Pancera M., Yang Y., Blanchard S.C., Mothes W., Burton D.R., Koff W.C., Connors M., Ward A.B., Kwong P.D., Mascola J.R. Fusion peptide of HIV-1 as a site of vulnerability to neutralizing antibody. Science. 2016;352(6287):828-833. DOI 10.1126/science.aae0474.Kong R., Xu K., Zhou T., Acharya P., Lemmin T., Liu K., Ozorowski G., Soto C., Taft J., Bailer R., Cale E.M., Chen L., Choi C.W., Chuang G., Doria-Rose N.A., Druz A., Georgiev I.S., Gorman J., Huang J., Gordon Joyce M., Louder M.K., Ma X., McKee K., O’Dell S., Pancera M., Yang Y., Blanchard S.C., Mothes W., Burton D.R., Koff W.C., Connors M., Ward A.B., Kwong P.D., Mascola J.R. Fusion peptide of HIV-1 as a site of vulnerability to neutralizing antibody. Science. 2016;352(6287):828-833. DOI 10.1126/science.aae0474.Ladinsky M.S., Gnanapragasam P.N., Yang Z., West A.P., Kay M.S., Bjorkman P.J. Electron tomography visualization of HIV-1 fusion with target cells using fusion inhibitors to trap the pre-hairpin intermediate. eLife. 2020;9. DOI 10.7554/eLife.58411.Ladinsky M.S., Gnanapragasam P.N., Yang Z., West A.P., Kay M.S., Bjorkman P.J. Electron tomography visualization of HIV-1 fusion with target cells using fusion inhibitors to trap the pre-hairpin intermediate. eLife. 2020;9. DOI 10.7554/eLife.58411.Li M., Gao F., Mascola J.R., Stamatatos L., Polonis V.R., Koutsoukos M., Voss G., Goepfert P., Gilbert P., Greene K.M., Bilska M., Kothe D.L., Salazar-Gonzalez J.F., Wei X., Decker J.M., Hahn B.H., Montefiori D.C. Human immunodeficiency virus type 1 env clones from acute and early subtype B infections for standardized assessments of vaccine-elicited neutralizing antibodies. J. Virol. 2005;79(16):10108-10125. DOI 10.1128/JVI.79.16.10108-10125.2005.Li M., Gao F., Mascola J.R., Stamatatos L., Polonis V.R., Koutsoukos M., Voss G., Goepfert P., Gilbert P., Greene K.M., Bilska M., Kothe D.L., Salazar-Gonzalez J.F., Wei X., Decker J.M., Hahn B.H., Montefiori D.C. Human immunodeficiency virus type 1 env clones from acute and early subtype B infections for standardized assessments of vaccine-elicited neutralizing antibodies. J. Virol. 2005;79(16):10108-10125. DOI 10.1128/JVI.79.16.10108-10125.2005.Li Q., Liu Q., Huang W., Li X., Wang Y. Current status on the development of pseudoviruses for enveloped viruses. Rev. Med. Virol. 2018;28(1):1-10. DOI 10.1002/rmv.1963.Li Q., Liu Q., Huang W., Li X., Wang Y. Current status on the development of pseudoviruses for enveloped viruses. Rev. Med. Virol. 2018;28(1):1-10. DOI 10.1002/rmv.1963.Liu Y., Cao W., Sun M., Li T. Broadly neutralizing antibodies for HIV- 1: efficacies, challenges and opportunities. Emerg. Microbes Infect. 2020;9(1):194-206. DOI 10.1080/22221751.2020.1713707.Liu Y., Cao W., Sun M., Li T. Broadly neutralizing antibodies for HIV- 1: efficacies, challenges and opportunities. Emerg. Microbes Infect. 2020;9(1):194-206. DOI 10.1080/22221751.2020.1713707.Mascola J.R., Snyder S.W., Weislow O.S., Belay S.M., Belshe R.B., Schwartz D.H., Clements M.L., Dolin R., Graham B.S., Gorse G.J. Immunization with envelope subunit vaccine products elicits neutralizing antibodies against laboratory-adapted but not primary isolates of human immunodeficiency virus type 1. J. Infect. Dis. 1996; 173(2):340-348. DOI 10.1093/infdis/173.2.340.Mascola J.R., Snyder S.W., Weislow O.S., Belay S.M., Belshe R.B., Schwartz D.H., Clements M.L., Dolin R., Graham B.S., Gorse G.J. Immunization with envelope subunit vaccine products elicits neutralizing antibodies against laboratory-adapted but not primary isolates of human immunodeficiency virus type 1. J. Infect. Dis. 1996; 173(2):340-348. DOI 10.1093/infdis/173.2.340.Mascola J.R., D’Souza P., Gilbert P., Hahn B.H., Haigwood N.L., Morris L., Petropoulos C.J., Polonis V.R., Sarzotti M., Montefiori D.C. Recommendations for the design and use of standard virus panels to assess neutralizing antibody responses elicited by candidate human immunodeficiency virus type 1 vaccines. J. Virol. 2005;79(16): 10103-10107. DOI 10.1128/JVI.79.16.10103-10107.2005.Mascola J.R., D’Souza P., Gilbert P., Hahn B.H., Haigwood N.L., Morris L., Petropoulos C.J., Polonis V.R., Sarzotti M., Montefiori D.C. Recommendations for the design and use of standard virus panels to assess neutralizing antibody responses elicited by candidate human immunodeficiency virus type 1 vaccines. J. Virol. 2005;79(16): 10103-10107. DOI 10.1128/JVI.79.16.10103-10107.2005.Mavian C., Coman R.M., Zhang X., Pomeroy S., Ostrov D.A., Dunn B.M., Sleasman J.W., Goodenow M.M. Molecular dockingbased screening for novel inhibitors of the human immunodeficiency virus type 1 protease that effectively reduce the viral replication in human cells. bioRxiv. 2020;1-11. DOI 10.1101/2020.11.14.382895.Mavian C., Coman R.M., Zhang X., Pomeroy S., Ostrov D.A., Dunn B.M., Sleasman J.W., Goodenow M.M. Molecular dockingbased screening for novel inhibitors of the human immunodeficiency virus type 1 protease that effectively reduce the viral replication in human cells. bioRxiv. 2020;1-11. DOI 10.1101/2020.11.14.382895.Mohan G.S., Ye L., Li W., Monteiro A., Lin X., Sapkota B., Pollack B.P., Compans R.W., Yang C. Less is more: Ebola virus surface glycoprotein expression levels regulate virus production and infectivity. J. Virol. 2015;89(2):1205-1217. DOI 10.1128/JVI.01810-14.Mohan G.S., Ye L., Li W., Monteiro A., Lin X., Sapkota B., Pollack B.P., Compans R.W., Yang C. Less is more: Ebola virus surface glycoprotein expression levels regulate virus production and infectivity. J. Virol. 2015;89(2):1205-1217. DOI 10.1128/JVI.01810-14.Montefiori D.C., Roederer M., Morris L., Seaman M.S. Neutralization tiers of HIV-1. Curr. Opin. HIV AIDS. 2018;13(2):1-9. DOI 10.1097/COH.0000000000000442.Montefiori D.C., Roederer M., Morris L., Seaman M.S. Neutralization tiers of HIV-1. Curr. Opin. HIV AIDS. 2018;13(2):1-9. DOI 10.1097/COH.0000000000000442.Ng’uni T., Chasara C., Ndhlovu Z.M. Major scientific hurdles in HIV vaccine development: historical perspective and future directions. Front. Immun. 2020;11:1-17. DOI 10.3389/fimmu.2020.590780.Ng’uni T., Chasara C., Ndhlovu Z.M. Major scientific hurdles in HIV vaccine development: historical perspective and future directions. Front. Immun. 2020;11:1-17. DOI 10.3389/fimmu.2020.590780.Nie J., Liu L., Wang Q., Chen R., Ning T., Liu Q., Huang W., Wang Y. Nipah pseudovirus system enables evaluation of vaccines in vitro and in vivo using non-BSL-4 facilities. Emerg. Microbes Infect. 2019;8(1):272-281. DOI 10.1080/22221751.2019.1571871.Nie J., Liu L., Wang Q., Chen R., Ning T., Liu Q., Huang W., Wang Y. Nipah pseudovirus system enables evaluation of vaccines in vitro and in vivo using non-BSL-4 facilities. Emerg. Microbes Infect. 2019;8(1):272-281. DOI 10.1080/22221751.2019.1571871.Nie J., Wu X., Ma J., Cao S., Huang W., Liu Q., Li X., Li Y., Wang Y. Development of in vitro and in vivo rabies virus neutralization as says based on a high-titer pseudovirus system. Sci. Rep. 2017;7(1): 1-12. DOI 10.1038/srep42769.Nie J., Wu X., Ma J., Cao S., Huang W., Liu Q., Li X., Li Y., Wang Y. Development of in vitro and in vivo rabies virus neutralization as says based on a high-titer pseudovirus system. Sci. Rep. 2017;7(1): 1-12. DOI 10.1038/srep42769.Ochsenbauer C., Edmonds T.G., Ding H., Keele B.F., Decker J., Salazar M.G., Salazar-Gonzalez J.F., Shattock R., Haynes B.F., Shaw G.M., Hahn B.H., Kappes J.C. Generation of transmitted/founder HIV-1 infectious molecular clones and characterization of their replication capacity in CD4 T lymphocytes and monocyte-derived macrophages. J. Virol. 2012;86(5):2715-2728. DOI 10.1128/JVI.06157-11.Ochsenbauer C., Edmonds T.G., Ding H., Keele B.F., Decker J., Salazar M.G., Salazar-Gonzalez J.F., Shattock R., Haynes B.F., Shaw G.M., Hahn B.H., Kappes J.C. Generation of transmitted/founder HIV-1 infectious molecular clones and characterization of their replication capacity in CD4 T lymphocytes and monocyte-derived macrophages. J. Virol. 2012;86(5):2715-2728. DOI 10.1128/JVI.06157-11.Ou L., Kong W.P., Chuang G.Y., Ghosh M., Gulla K., O’Dell S., Varriale J., Barefoot N., Changela A., Chao C.W., Cheng Ch., Druz A., Kong R., McKee K., Rawi R., Sarfo E., Schön A., Shaddeau A., Tsybovsky Ya., Verardi R., Wang Sh. Preclinical development of a fusion peptide conjugate as an HIV vaccine immunogen. Sci. Rep. 2020;10(1):1-13. DOI 10.1038/s41598-020-59711-y.Ou L., Kong W.P., Chuang G.Y., Ghosh M., Gulla K., O’Dell S., Varriale J., Barefoot N., Changela A., Chao C.W., Cheng Ch., Druz A., Kong R., McKee K., Rawi R., Sarfo E., Schön A., Shaddeau A., Tsybovsky Ya., Verardi R., Wang Sh. Preclinical development of a fusion peptide conjugate as an HIV vaccine immunogen. Sci. Rep. 2020;10(1):1-13. DOI 10.1038/s41598-020-59711-y.Pace C.S., Fordyce M.W., Franco D., Kao C.Y., Seaman M.S., Ho D.D. Anti-CD4 monoclonal antibody ibalizumab exhibits breadth and potency against HIV-1, with natural resistance mediated by the loss of a V5 glycan in envelope. J. Acquir. Immune Defic. Syndr. 2013;62(1):1-9. DOI 10.1097/QAI.0b013e3182732746.Pace C.S., Fordyce M.W., Franco D., Kao C.Y., Seaman M.S., Ho D.D. Anti-CD4 monoclonal antibody ibalizumab exhibits breadth and potency against HIV-1, with natural resistance mediated by the loss of a V5 glycan in envelope. J. Acquir. Immune Defic. Syndr. 2013;62(1):1-9. DOI 10.1097/QAI.0b013e3182732746.Peden K., Emerman M., Montagnier L. Changes in growth properties on passage in tissue culture of viruses derived from infectious molecular clones of HIV-1LAI, HIV-1MAL, and HIV-1ELI. Virology. 1991;185:661-672. DOI 10.1016/0042-6822(91)90537-L.Peden K., Emerman M., Montagnier L. Changes in growth properties on passage in tissue culture of viruses derived from infectious molecular clones of HIV-1LAI, HIV-1MAL, and HIV-1ELI. Virology. 1991;185:661-672. DOI 10.1016/0042-6822(91)90537-L.Phanuphak N., Gulick R.M. HIV treatment and prevention 2019: current standards of care. Curr. Opin. HIV AIDS. 2020;15(1):4-12. DOI 10.1097/COH.0000000000000588.Phanuphak N., Gulick R.M. HIV treatment and prevention 2019: current standards of care. Curr. Opin. HIV AIDS. 2020;15(1):4-12. DOI 10.1097/COH.0000000000000588.Platt E.J., Wehrly K., Kuhmann S.E., Chesebro B., Kabat D. Effects of CCR5 and CD4 cell surface concentrations on infections by macrophagetropic isolates of human immunodeficiency virus type 1. J. Virol. 1998;72:2855-2864. DOI 10.1128/JVI.72.4.2855-2864.1998.Platt E.J., Wehrly K., Kuhmann S.E., Chesebro B., Kabat D. Effects of CCR5 and CD4 cell surface concentrations on infections by macrophagetropic isolates of human immunodeficiency virus type 1. J. Virol. 1998;72:2855-2864. DOI 10.1128/JVI.72.4.2855-2864.1998.Polonis V.R., Brown B.K., Borges A.R., Zolla-Pazner S., Dimitrov D.S., Zhang M.Y., Barnett S.W., Ruprecht R.M., Scarlatti G., Fenyö E., Montefiori D.C., McCutchan F.E., Michael N.L. Recent advances in the characterization of HIV-1 neutralization assays for standardized evaluation of the antibody response to infection and vaccination. Virology. 2008;375(2):315-320. DOI 10.1016/j.virol.2008.02.007.Polonis V.R., Brown B.K., Borges A.R., Zolla-Pazner S., Dimitrov D.S., Zhang M.Y., Barnett S.W., Ruprecht R.M., Scarlatti G., Fenyö E., Montefiori D.C., McCutchan F.E., Michael N.L. Recent advances in the characterization of HIV-1 neutralization assays for standardized evaluation of the antibody response to infection and vaccination. Virology. 2008;375(2):315-320. DOI 10.1016/j.virol.2008.02.007.Princen K., Hatse S., Vermeire K., De Clercq E., Schols D. Establishment of a novel CCR5 and CXCR4 expressing CD4+ cell line which is highly sensitive to HIV and suitable for high-throughput evaluation of CCR5 and CXCR4 antagonists. Retrovirology. 2004;1(1): 1-13. DOI 10.1186/1742-4690-1-2.Princen K., Hatse S., Vermeire K., De Clercq E., Schols D. Establishment of a novel CCR5 and CXCR4 expressing CD4+ cell line which is highly sensitive to HIV and suitable for high-throughput evaluation of CCR5 and CXCR4 antagonists. Retrovirology. 2004;1(1): 1-13. DOI 10.1186/1742-4690-1-2.Pugach P., Marozsan A.J., Ketas T.J., Landes E.L., Moore J.P., Kuhmann S.E. HIV-1 clones resistant to a small molecule CCR5 inhibitor use the inhibitor-bound form of CCR5 for entry. Virology. 2007;361(1):212-228. DOI 10.1016/j.virol.2006.11.004.Pugach P., Marozsan A.J., Ketas T.J., Landes E.L., Moore J.P., Kuhmann S.E. HIV-1 clones resistant to a small molecule CCR5 inhibitor use the inhibitor-bound form of CCR5 for entry. Virology. 2007;361(1):212-228. DOI 10.1016/j.virol.2006.11.004.Rudometov A.P., Chikaev A.N., Rudometova N.B., Antonets D.V., Lomzov A.A., Kaplina O.N., Ilyichev A.A., Karpenko L.I. Artificial anti-HIV-1 immunogen comprising epitopes of broadly neutralizing antibodies 2F5, 10E8, and a peptide mimic of VRC01 discontinuous epitope. Vaccines. 2019a;7(3):1-18. DOI 10.3390/vaccines7030083.Rudometov A.P., Chikaev A.N., Rudometova N.B., Antonets D.V., Lomzov A.A., Kaplina O.N., Ilyichev A.A., Karpenko L.I. Artificial anti-HIV-1 immunogen comprising epitopes of broadly neutralizing antibodies 2F5, 10E8, and a peptide mimic of VRC01 discontinuous epitope. Vaccines. 2019a;7(3):1-18. DOI 10.3390/vaccines7030083.Rudometov A.P., Rudometova N.B., Shcherbakov D.N., Lomzov A.A., Kaplina O.N., Shcherbakova N.S., Ilyichev A.A., Bakulina A. Yu., Karpenko L.I. The structural and immunological properties of chimeric proteins containing HIV-1 MPER sites. Acta Naturae. 2019b; 11(3):56-65. DOI 10.32607/20758251-2019-11-3-56-65.Rudometov A.P., Rudometova N.B., Shcherbakov D.N., Lomzov A.A., Kaplina O.N., Shcherbakova N.S., Ilyichev A.A., Bakulina A. Yu., Karpenko L.I. The structural and immunological properties of chimeric proteins containing HIV-1 MPER sites. Acta Naturae. 2019b; 11(3):56-65. DOI 10.32607/20758251-2019-11-3-56-65.Sarzotti-Kelsoe M., Bailer R.T., Turk E., Lin C.L., Bilska M., Greene K.M., Gao H., Todd C.A., Ozaki D., Seaman M.S., Mascola J.R., Montefiori D.C. Optimization and validation of the TZM-bl assay for standardized assessments of neutralizing antibodies against HIV-1. J. Immunol. Methods. 2014;409:131-146. DOI 10.1016/j.jim.2013.11.022.Sarzotti-Kelsoe M., Bailer R.T., Turk E., Lin C.L., Bilska M., Greene K.M., Gao H., Todd C.A., Ozaki D., Seaman M.S., Mascola J.R., Montefiori D.C. Optimization and validation of the TZM-bl assay for standardized assessments of neutralizing antibodies against HIV-1. J. Immunol. Methods. 2014;409:131-146. DOI 10.1016/j.jim.2013.11.022.Seaman M.S., Janes H., Hawkins N., Grandpre L.E., Devoy C., Giri A., Coffey R.T., Harris L., Wood B., Daniels M.G. Tiered categorization of a diverse panel of HIV-1 Env pseudoviruses for assessment of neutralizing antibodies. J. Virol. 2010;84(3):1439-1452. DOI 10.1128/JVI.02108-09.Seaman M.S., Janes H., Hawkins N., Grandpre L.E., Devoy C., Giri A., Coffey R.T., Harris L., Wood B., Daniels M.G. Tiered categorization of a diverse panel of HIV-1 Env pseudoviruses for assessment of neutralizing antibodies. J. Virol. 2010;84(3):1439-1452. DOI 10.1128/JVI.02108-09.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. PMID 26798488.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. PMID 26798488.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.Su S., Rasquinha G., Du L., Wang Q., Xu W., Li W., Lu L., Jiang S. A peptide-based HIV-1 fusion inhibitor with two tail-anchors and palmitic acid exhibits substantially improved in vitro and ex vivo anti-HIV-1 activity and prolonged in vivo half-life. Molecules. 2019;24(6):1-13. DOI 10.3390/molecules24061134.Su S., Rasquinha G., Du L., Wang Q., Xu W., Li W., Lu L., Jiang S. A peptide-based HIV-1 fusion inhibitor with two tail-anchors and palmitic acid exhibits substantially improved in vitro and ex vivo anti-HIV-1 activity and prolonged in vivo half-life. Molecules. 2019;24(6):1-13. DOI 10.3390/molecules24061134.Van’t Wout A.B., Schuitemaker H., Kootstra N.A. Isolation and propagation of HIV-1 on peripheral blood mononuclear cells. Nat. Protoc. 2008;3(3):363-370. DOI 10.1038/nprot.2008.3.Van’t Wout A.B., Schuitemaker H., Kootstra N.A. Isolation and propagation of HIV-1 on peripheral blood mononuclear cells. Nat. Protoc. 2008;3(3):363-370. DOI 10.1038/nprot.2008.3.Varghese V., Mitsuya Y., Fessel W.J., Liu T.F., Melikian G.L., Katzenstein D.A., Schiffer C.A., Holmes S.P., Shafer R.W. Prototypical recombinant multi-protease-inhibitor-resistant infectious molecular clones of human immunodeficiency virus type 1. Antimicrob. Agents Chemother. 2013;57(9):4290-4299. DOI 10.1128/AAC.00614-13.Varghese V., Mitsuya Y., Fessel W.J., Liu T.F., Melikian G.L., Katzenstein D.A., Schiffer C.A., Holmes S.P., Shafer R.W. Prototypical recombinant multi-protease-inhibitor-resistant infectious molecular clones of human immunodeficiency virus type 1. Antimicrob. Agents Chemother. 2013;57(9):4290-4299. DOI 10.1128/AAC.00614-13.Voronin Y., Chohan B., Emerman M., Overbaugh J. Primary isolates of human immunodeficiency virus type 1 are usually dominated by the major variants found in blood. J. Virol. 2007;81(19):10232-10241. DOI 10.1128/JVI.01035-07.Voronin Y., Chohan B., Emerman M., Overbaugh J. Primary isolates of human immunodeficiency virus type 1 are usually dominated by the major variants found in blood. J. Virol. 2007;81(19):10232-10241. DOI 10.1128/JVI.01035-07.Wagstaff K.M., Headey S., Telwatte S., Tyssen D., Hearps A.C., Thomas D.R., Tachedjian G., Jans D.A. Molecular dissection of an inhibitor targeting the HIV integrase dependent preintegration complex nuclear import. Cell. Microbiol. 2019;21(1):1-13. DOI 10.1111/cmi.12953.Wagstaff K.M., Headey S., Telwatte S., Tyssen D., Hearps A.C., Thomas D.R., Tachedjian G., Jans D.A. Molecular dissection of an inhibitor targeting the HIV integrase dependent preintegration complex nuclear import. Cell. Microbiol. 2019;21(1):1-13. DOI 10.1111/cmi.12953.Wang Z., Hong K., Zhang J., Zhang L., Li D., Ren L., Liang H., Shao Y. Construction and characterization of highly infectious fulllength molecular clones of a HIV-1 CRF07_BC isolate from Xinjiang, China. PLoS One. 2013;8(11):1-9. DOI 10.1371/journal.pone.0079177.Wang Z., Hong K., Zhang J., Zhang L., Li D., Ren L., Liang H., Shao Y. Construction and characterization of highly infectious fulllength molecular clones of a HIV-1 CRF07_BC isolate from Xinjiang, China. PLoS One. 2013;8(11):1-9. DOI 10.1371/journal.pone.0079177.Wei X., Decker J.M., Liu H., Zhang Z., Arani R.B., Kilby J.M., Saag M.S., Wu X., Shaw G.M., Kappes J.C. Emergence of resistant human immunodeficiency virus type 1 in patients receiving fusion inhibitor (T-20) monotherapy. Antimicrob. Agents Chemother. 2002; 46:1896-1905. DOI 10.1128/aac.46.6.1896-1905.2002.Wei X., Decker J.M., Liu H., Zhang Z., Arani R.B., Kilby J.M., Saag M.S., Wu X., Shaw G.M., Kappes J.C. Emergence of resistant human immunodeficiency virus type 1 in patients receiving fusion inhibitor (T-20) monotherapy. Antimicrob. Agents Chemother. 2002; 46:1896-1905. DOI 10.1128/aac.46.6.1896-1905.2002.Wu J., Zhao C., Liu Q., Huang W., Wang Y. Development and application of a bioluminescent imaging mouse model for Chikungunya virus based on pseudovirus system. Vaccine. 2017;35(47):6387-6394. DOI 10.1016/j.vaccine.2017.10.007.Wu J., Zhao C., Liu Q., Huang W., Wang Y. Development and application of a bioluminescent imaging mouse model for Chikungunya virus based on pseudovirus system. Vaccine. 2017;35(47):6387-6394. DOI 10.1016/j.vaccine.2017.10.007.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. 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. Rational design of envelope identifies broadly neutralizing human monoclonal antibodies to HIV-1. Science. 2010;329(5993):856-861. DOI 10.1126/science.1187659.Xu K., Acharya P., Kong R., Cheng C., Chuang G.Y., Liu K., Louder M.K., O’Dell S., Rawi R., Sastry M. Epitope-based vaccine design yields fusion peptide-directed antibodies that neutralize diverse strains of HIV-1. Nat. Med. 2018;24(6):857-867. DOI 10.1038/s41591-018-0042-6.Xu K., Acharya P., Kong R., Cheng C., Chuang G.Y., Liu K., Louder M.K., O’Dell S., Rawi R., Sastry M. Epitope-based vaccine design yields fusion peptide-directed antibodies that neutralize diverse strains of HIV-1. Nat. Med. 2018;24(6):857-867. DOI 10.1038/s41591-018-0042-6.Zaitsev B.N., Taranov O.S., Rudometova N.B., Shcherbakova N.S., Ilyichev A.A., Karpenko L.I. An optimized method for counting viral particles using electron microscopy. Vavilovskii Zhurnal Genetiki i Selektsii = Vavilov Journal of Genetics and Breeding. 2019;23: 337-342. DOI 10.18699/VJ19.498.Zaitsev B.N., Taranov O.S., Rudometova N.B., Shcherbakova N.S., Ilyichev A.A., Karpenko L.I. An optimized method for counting viral particles using electron microscopy. Vavilovskii Zhurnal Genetiki i Selektsii = Vavilov Journal of Genetics and Breeding. 2019;23: 337-342. DOI 10.18699/VJ19.498.Zhang L., Lei S., Xie H., Li Q., Liu S., Liu Q., Huang W., Xiao X., Wang Y. Screening and identification of Marburg virus entry inhibitors using approved drugs. Virol. Sin. 2020;35:235-239. DOI 10.1007/s12250-019-00184-3.Zhang L., Lei S., Xie H., Li Q., Liu S., Liu Q., Huang W., Xiao X., Wang Y. Screening and identification of Marburg virus entry inhibitors using approved drugs. Virol. Sin. 2020;35:235-239. DOI 10.1007/s12250-019-00184-3.Zhang X., Yan F., Tang K., Chen Q., Guo J., Zhu W., He S., Banadyga L., Qiu X., Guo Y. Identification of a clinical compound losmapimod that blocks Lassa virus entry. Antiviral Res. 2019;167:68-77. DOI 10.1016/j.antiviral.2019.03.014.Zhang X., Yan F., Tang K., Chen Q., Guo J., Zhu W., He S., Banadyga L., Qiu X., Guo Y. Identification of a clinical compound losmapimod that blocks Lassa virus entry. Antiviral Res. 2019;167:68-77. DOI 10.1016/j.antiviral.2019.03.014.Zhao G., Du L., Ma C., Li Y., Li L., Poon V.K., Wang L., Yu F., Zheng B.J., Jiang S., Zhou Y. A safe and convenient pseudovirusbased inhibition assay to detect neutralizing antibodies and screen for viral entry inhibitors against the novel human coronavirus MERSCoV. Virol. J. 2013;10(1):1-8. DOI 10.1186/1743-422X-10-266.Zhao G., Du L., Ma C., Li Y., Li L., Poon V.K., Wang L., Yu F., Zheng B.J., Jiang S., Zhou Y. A safe and convenient pseudovirusbased inhibition assay to detect neutralizing antibodies and screen for viral entry inhibitors against the novel human coronavirus MERSCoV. Virol. J. 2013;10(1):1-8. DOI 10.1186/1743-422X-10-266.Zyryanova D.P., Bogacheva N.V., Totmenin A.V., Gashnikova N.M. HIV-1 CRF63_02A6 models as a tool for evaluating efficacy of developing antiretroviral drugs. Infektsiya i Immunitet = Russian Journal of Infection and Immunity. 2020;10(4):769-774. DOI 10.15789/2220-7619-MHC-1261. (in Russian)Zyryanova D.P., Bogacheva N.V., Totmenin A.V., Gashnikova N.M. HIV-1 CRF63_02A6 models as a tool for evaluating efficacy of developing antiretroviral drugs. Infektsiya i Immunitet = Russian Journal of Infection and Immunity. 2020;10(4):769-774. DOI 10.15789/2220-7619-MHC-1261. (in Russian)Zyryanova D.P., Totmenin A.V., Bogacheva N.V., Gashnikova N.M. Construction and characterization of infectious molecular clones of HIV-1 CRF63_02A6. AIDS Res. Hum. Retrovir. 2020;36(3):227-233. DOI 10.1089/aid.2019.0177.Zyryanova D.P., Totmenin A.V., Bogacheva N.V., Gashnikova N.M. Construction and characterization of infectious molecular clones of HIV-1 CRF63_02A6. AIDS Res. Hum. Retrovir. 2020;36(3):227-233. DOI 10.1089/aid.2019.0177.

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