Engineering well-expressed, V2-immunofocusing HIV-1 envelope glycoprotein membrane trimers for use in heterologous prime-boost vaccine regimens

HIV-1 vaccine immunofocusing strategies may be able to induce broadly-reactive neutralizing antibodies (NAbs). Here, we engineered a panel of diverse, membrane-resident native HIV-1 trimers vulnerable to two broad targets—the V2 apex and fusion peptide (FP). Selection criteria included i) high expre...

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Autores principales:	Emma T. Crooks, Francisco Almanza, Alessio D’Addabbo, Erika Duggan, Jinsong Zhang, Kshitij Wagh, Huihui Mou, Joel D. Allen, Alyssa Thomas, Keiko Osawa, Bette T. Korber, Yaroslav Tsybovsky, Evan Cale, John Nolan, Max Crispin, Laurent K. Verkoczy, James M. Binley
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2021
Materias:	Immunologic diseases. Allergy RC581-607 Biology (General) QH301-705.5
Acceso en línea:	https://doaj.org/article/30ea4c2a548142c8b204822f276498bc
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spelling	oai:doaj.org-article:30ea4c2a548142c8b204822f276498bc2021-11-11T06:02:01ZEngineering well-expressed, V2-immunofocusing HIV-1 envelope glycoprotein membrane trimers for use in heterologous prime-boost vaccine regimens1553-73661553-7374https://doaj.org/article/30ea4c2a548142c8b204822f276498bc2021-10-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC8565784/?tool=EBIhttps://doaj.org/toc/1553-7366https://doaj.org/toc/1553-7374HIV-1 vaccine immunofocusing strategies may be able to induce broadly-reactive neutralizing antibodies (NAbs). Here, we engineered a panel of diverse, membrane-resident native HIV-1 trimers vulnerable to two broad targets—the V2 apex and fusion peptide (FP). Selection criteria included i) high expression and ii) infectious function, so that trimer neutralization sensitivity can be profiled in pseudovirus (PV) assays. Initially, we boosted the expression of 17 candidate trimers by truncating gp41 and introducing a gp120-gp41 SOS disulfide to prevent gp120 shedding. "Repairs" were made to fill glycan holes and eliminate other strain-specific aberrations. A new neutralization assay allowed PV infection when our standard assay was insufficient. Trimers with exposed V3 loops, a target of non-NAbs, were discarded. To try to increase V2-sensitivity, we removed clashing glycans and modified the C-strand. Notably, a D167N mutation improved V2-sensitivity in several cases. Glycopeptide analysis of JR-FL trimers revealed near complete sequon occupation and that filling the N197 glycan hole was well-tolerated. In contrast, sequon optimization and inserting/removing glycans at other positions frequently had global "ripple" effects on glycan maturation and sequon occupation throughout the gp120 outer domain and gp41. V2 MAb CH01 selectively bound to trimers with small high mannose glycans near the base of the V1 loop, thereby avoiding clashes. Knocking in a rare N49 glycan was found to perturb gp41 glycans, increasing FP NAb sensitivity—and sometimes improving expression. Finally, a biophysical analysis of VLPs revealed that i) ~25% of particles bear Env spikes, ii) spontaneous particle budding is high and only increases 4-fold upon Gag transfection, and iii) Env+ particles express ~30–40 spikes. Taken together, we identified 7 diverse trimers with a range of sensitivities to two targets to allow rigorous testing of immunofocusing vaccine concepts. Author summary Despite almost 40 years of innovation, a vaccine to induce antibodies that block HIV infection remains elusive. Challenges include the unparalleled sequence diversity of HIV’s surface spikes and its dense sugar coat that limits antibody access. A growing number of monoclonal antibodies from HIV infected donors provide vaccine blueprints, but have been difficult to induce by vaccination, due to their unusual features. However, two targets, one at the viral spike apex and another at the side of the spikes are more forgiving in their ’demands’ for unusual antibodies. Here, we made a diverse panel of HIV spikes vulnerable at these two sites to be used as vaccines to try to focus antibodies on these targets. Our selection criteria for these spikes were: i) that when expressed on particles, they are infectious, allowing us to evaluate immunogens and vaccine sera using particles made with the same trimers, ii) that spikes are easy to produce by cells in quantities sufficient for vaccine use. Ultimately, we selected 7 trimers that will allow us to explore concepts that could bring us closer to an HIV vaccine.Emma T. CrooksFrancisco AlmanzaAlessio D’AddabboErika DugganJinsong ZhangKshitij WaghHuihui MouJoel D. AllenAlyssa ThomasKeiko OsawaBette T. KorberYaroslav TsybovskyEvan CaleJohn NolanMax CrispinLaurent K. VerkoczyJames M. BinleyPublic Library of Science (PLoS)articleImmunologic diseases. AllergyRC581-607Biology (General)QH301-705.5ENPLoS Pathogens, Vol 17, Iss 10 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Immunologic diseases. Allergy RC581-607 Biology (General) QH301-705.5
spellingShingle	Immunologic diseases. Allergy RC581-607 Biology (General) QH301-705.5 Emma T. Crooks Francisco Almanza Alessio D’Addabbo Erika Duggan Jinsong Zhang Kshitij Wagh Huihui Mou Joel D. Allen Alyssa Thomas Keiko Osawa Bette T. Korber Yaroslav Tsybovsky Evan Cale John Nolan Max Crispin Laurent K. Verkoczy James M. Binley Engineering well-expressed, V2-immunofocusing HIV-1 envelope glycoprotein membrane trimers for use in heterologous prime-boost vaccine regimens
description	HIV-1 vaccine immunofocusing strategies may be able to induce broadly-reactive neutralizing antibodies (NAbs). Here, we engineered a panel of diverse, membrane-resident native HIV-1 trimers vulnerable to two broad targets—the V2 apex and fusion peptide (FP). Selection criteria included i) high expression and ii) infectious function, so that trimer neutralization sensitivity can be profiled in pseudovirus (PV) assays. Initially, we boosted the expression of 17 candidate trimers by truncating gp41 and introducing a gp120-gp41 SOS disulfide to prevent gp120 shedding. "Repairs" were made to fill glycan holes and eliminate other strain-specific aberrations. A new neutralization assay allowed PV infection when our standard assay was insufficient. Trimers with exposed V3 loops, a target of non-NAbs, were discarded. To try to increase V2-sensitivity, we removed clashing glycans and modified the C-strand. Notably, a D167N mutation improved V2-sensitivity in several cases. Glycopeptide analysis of JR-FL trimers revealed near complete sequon occupation and that filling the N197 glycan hole was well-tolerated. In contrast, sequon optimization and inserting/removing glycans at other positions frequently had global "ripple" effects on glycan maturation and sequon occupation throughout the gp120 outer domain and gp41. V2 MAb CH01 selectively bound to trimers with small high mannose glycans near the base of the V1 loop, thereby avoiding clashes. Knocking in a rare N49 glycan was found to perturb gp41 glycans, increasing FP NAb sensitivity—and sometimes improving expression. Finally, a biophysical analysis of VLPs revealed that i) ~25% of particles bear Env spikes, ii) spontaneous particle budding is high and only increases 4-fold upon Gag transfection, and iii) Env+ particles express ~30–40 spikes. Taken together, we identified 7 diverse trimers with a range of sensitivities to two targets to allow rigorous testing of immunofocusing vaccine concepts. Author summary Despite almost 40 years of innovation, a vaccine to induce antibodies that block HIV infection remains elusive. Challenges include the unparalleled sequence diversity of HIV’s surface spikes and its dense sugar coat that limits antibody access. A growing number of monoclonal antibodies from HIV infected donors provide vaccine blueprints, but have been difficult to induce by vaccination, due to their unusual features. However, two targets, one at the viral spike apex and another at the side of the spikes are more forgiving in their ’demands’ for unusual antibodies. Here, we made a diverse panel of HIV spikes vulnerable at these two sites to be used as vaccines to try to focus antibodies on these targets. Our selection criteria for these spikes were: i) that when expressed on particles, they are infectious, allowing us to evaluate immunogens and vaccine sera using particles made with the same trimers, ii) that spikes are easy to produce by cells in quantities sufficient for vaccine use. Ultimately, we selected 7 trimers that will allow us to explore concepts that could bring us closer to an HIV vaccine.
format	article
author	Emma T. Crooks Francisco Almanza Alessio D’Addabbo Erika Duggan Jinsong Zhang Kshitij Wagh Huihui Mou Joel D. Allen Alyssa Thomas Keiko Osawa Bette T. Korber Yaroslav Tsybovsky Evan Cale John Nolan Max Crispin Laurent K. Verkoczy James M. Binley
author_facet	Emma T. Crooks Francisco Almanza Alessio D’Addabbo Erika Duggan Jinsong Zhang Kshitij Wagh Huihui Mou Joel D. Allen Alyssa Thomas Keiko Osawa Bette T. Korber Yaroslav Tsybovsky Evan Cale John Nolan Max Crispin Laurent K. Verkoczy James M. Binley
author_sort	Emma T. Crooks
title	Engineering well-expressed, V2-immunofocusing HIV-1 envelope glycoprotein membrane trimers for use in heterologous prime-boost vaccine regimens
title_short	Engineering well-expressed, V2-immunofocusing HIV-1 envelope glycoprotein membrane trimers for use in heterologous prime-boost vaccine regimens
title_full	Engineering well-expressed, V2-immunofocusing HIV-1 envelope glycoprotein membrane trimers for use in heterologous prime-boost vaccine regimens
title_fullStr	Engineering well-expressed, V2-immunofocusing HIV-1 envelope glycoprotein membrane trimers for use in heterologous prime-boost vaccine regimens
title_full_unstemmed	Engineering well-expressed, V2-immunofocusing HIV-1 envelope glycoprotein membrane trimers for use in heterologous prime-boost vaccine regimens
title_sort	engineering well-expressed, v2-immunofocusing hiv-1 envelope glycoprotein membrane trimers for use in heterologous prime-boost vaccine regimens
publisher	Public Library of Science (PLoS)
publishDate	2021
url	https://doaj.org/article/30ea4c2a548142c8b204822f276498bc
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Engineering well-expressed, V2-immunofocusing HIV-1 envelope glycoprotein membrane trimers for use in heterologous prime-boost vaccine regimens

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