In:
PLOS Pathogens, Public Library of Science (PLoS), Vol. 17, No. 10 ( 2021-10-22), p. e1009807-
Abstract:
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.
Type of Medium:
Online Resource
ISSN:
1553-7374
DOI:
10.1371/journal.ppat.1009807
DOI:
10.1371/journal.ppat.1009807.g001
DOI:
10.1371/journal.ppat.1009807.g002
DOI:
10.1371/journal.ppat.1009807.g003
DOI:
10.1371/journal.ppat.1009807.g004
DOI:
10.1371/journal.ppat.1009807.g005
DOI:
10.1371/journal.ppat.1009807.g006
DOI:
10.1371/journal.ppat.1009807.g007
DOI:
10.1371/journal.ppat.1009807.g008
DOI:
10.1371/journal.ppat.1009807.g009
DOI:
10.1371/journal.ppat.1009807.g010
DOI:
10.1371/journal.ppat.1009807.g011
DOI:
10.1371/journal.ppat.1009807.s001
DOI:
10.1371/journal.ppat.1009807.s002
DOI:
10.1371/journal.ppat.1009807.s003
DOI:
10.1371/journal.ppat.1009807.s004
DOI:
10.1371/journal.ppat.1009807.s005
DOI:
10.1371/journal.ppat.1009807.s006
DOI:
10.1371/journal.ppat.1009807.s007
DOI:
10.1371/journal.ppat.1009807.s008
DOI:
10.1371/journal.ppat.1009807.s009
DOI:
10.1371/journal.ppat.1009807.s010
DOI:
10.1371/journal.ppat.1009807.s011
DOI:
10.1371/journal.ppat.1009807.s012
DOI:
10.1371/journal.ppat.1009807.s013
DOI:
10.1371/journal.ppat.1009807.s014
DOI:
10.1371/journal.ppat.1009807.s015
DOI:
10.1371/journal.ppat.1009807.s016
DOI:
10.1371/journal.ppat.1009807.s017
DOI:
10.1371/journal.ppat.1009807.s018
DOI:
10.1371/journal.ppat.1009807.s019
DOI:
10.1371/journal.ppat.1009807.s020
Language:
English
Publisher:
Public Library of Science (PLoS)
Publication Date:
2021
detail.hit.zdb_id:
2205412-1
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