Ontology highlight
ABSTRACT:
PROVIDER: PRJNA656582 | ENA |
REPOSITORIES: ENA
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Project description:Germinal centers (GCs) are the engines of antibody evolution. Using HIV Env protein immunogen priming in rhesus monkeys (RM) followed by a long period without further immunization, we demonstrate GC B cells (BGC) lasted at least 6 months. A 186-fold BGC cell increase was present by week 10 compared to a conventional immunization. Single cell transcriptional profiling revealed both light and dark zone GC states were sustained. Antibody somatic hypermutation (SHM) of BGC cells continued to accumulate throughout the 29-week priming period, with evidence of selective pressure. Env-binding BGC cells were still 49-fold above baseline at 29 weeks, suggesting they could be active for even longer periods of time. High HIV neutralizing antibody titers were generated after a single booster immunization. Fully glycosylated HIV trimer protein is a complex antigen, posing significant immunodominance challenges for B cells. Memory B cells (BMem) generated under these long priming conditions had higher levels of SHM, and both BMem cells and antibodies were more likely to recognize non-immunodominant epitopes. Numerous BGC cell lineage phylogenies spanning the >6-month GC period were identified, demonstrating continuous GC activity and selection for at least 191 days with no additional antigen exposure. A long prime, slow delivery (12-day) immunization approach holds promise for difficult vaccine targets, and suggests that patience can have great value for tuning GCs to maximize antibody responses.
2022-09-15 | GSE209904 | GEO
Project description:HIV-1 infection begins with binding of the viral envelope glycoprotein Env to the host receptor CD4, triggering a series of conformational changes that lead to fusion of the virus and cell membranes. Env, a trimer of gp120 and gp41 subunits, occupies a ‘closed’ conformation with contacts between gp120 subunits at the apex, and transitions through an ‘open’ conformation with the gp120 subunits spread apart following CD4 binding. Using deep mutational scanning, sequence-fitness landscapes were mapped for full-length Env from the clade B BaL strain interacting with CD4, and broadly neutralizing antibodies VRC01 and PG16, which preferentially bind closed Env. Contacting residues are conserved for CD4 binding, and glycosylation at N262 is critical for accessing the high-affinity CD4-bound state. By comparison, VRC01 binding is resistant to most single amino acid substitutions, an ideal quality in a broadly neutralizing antibody. Also in contrast to CD4 interaction, Env interfacial residues are under tight selection for PG16 binding to maintain a closed conformation. Screening for mutations that enhanced PG16 binding, we identified several important sites, in particular neutralization of the electropositive apical cavity that we hypothesize promotes trimer opening by electrostatic repulsion. Mutations were combined to generate Quaternary Epitope Stabilized (QES) mutants with enhanced presentation of the PG16 epitope, and the mutations were partially transferable to other HIV-1 strains. These mutational analyses offer insight into Env conformational stabilization that may assist immunogen design.
2019-02-03 | GSE102276 | GEO
Project description:The HIV-1 genome gains access to the inside of a cell via the mechanism of the viral spike protein Env, which undergoes a series of major conformational rearrangements after binding target receptors that ultimately drive virus-cell membrane fusion. Env is expressed as a heterogenous ensemble of conformations, which can inappropriately misdirect the host immune response towards the production of non-protective, strain-specific antibodies. Potent, broadly neutralizing antibodies (bnAbs) frequently recognize a ‘closed’ Env conformation, and therefore Env has undergone significant engineering to stabilize the closed state for vaccine incorporation. Previously, we used deep mutational scanning of Env from a prototypical tier 1 clade B strain (BaL) to characterize the sequence-activity landscape for binding to PG16, a bnAb that preferentially binds the closed state. Mutations were identified that increased expression of closed Env and reduced conformational heterogeneity, but these mutations were only partially transferable to Env sequences from other strains. To generate an expanded set of mutations that may be broadly applicable to diverse HIV-1 strains, we present here the deep mutational scanning of Env from the tier 2 clade C strain DU422 for interactions with CD4 and PG16. Residues across the trimerization domain and trimer interface have low mutational tolerance for maintaining PG16 recognition. New mutations are identified that enhance presentation of the closed Env conformation, and these are applied to Env sequences spanning multiple clades and tiers.
2019-02-01 | GSE117328 | GEO
Project description:Generation of Tier 2 HIV neutralizing antibody (nAb) responses by immunization remains a challenging problem, and the immunological barriers to induction of such responses by Env immunogens remain unclear. We explored these barriers by combining a suite of innovative techniques, including longitudinal lymph node fine needle aspirates, germinal center (GC) B cell lineage tracking, and a new method for detecting and quantifying GC T follicular helper (GC Tfh) cells, in non-human primates immunized with a native-like HIV-1 Env trimer protein (BG505 SOSIP.v5.2). A majority of immunized animals (9/12) developed Tier 2 neutralizing antibodies (nAb). Tier 2 nAb development best correlated with GC B cell magnitude in response to later booster immunizations and the quality of the Tfh help. Notably, these immunological factors distinguished between qualitatively successful and unsuccessful vaccine Ab responses, as they correlated with nAb development but did not correlate with simple Env Ab binding titers. Therefore, direct probing of germinal centers in future vaccine trials is key, as this suite of technically robust approaches provides quantitation of the proximal immune correlates of neutralizing antibody development and could allow redesign of optimal multi-stage vaccination schedules.
2016-12-31 | GSE89148 | GEO
Project description:We report deep mutational scanning data for the Env protein's LLP-2 domain in the NL4-3 strain HIV-1 Env. Processed Data repersents counts for each amino acid pre and post spread
2021-07-15 | GSE179046 | GEO
Project description:CD4 T follicular helper cells (Tfh) are essential for establishing serological memory and have distinct helper attributes that impact both the quantity and quality of the antibody response. Therefore, gaining insights into Tfh subsets promoting antibody persistence and functional capacity is crucial for vaccine design. We investigated the potential of inducing a mixed Tfh1/17 response to enhance HIV-1 Envelope (Env) antibodies to a DNA-prime/protein boost platform, utilizing rhesus macaques as our experimental model. Following immunization with Clade C gp140 protein formulated with cationic liposome-based formulation (CAF01), we successfully generated germinal center (GC) Tfh1/17 cells, in contrast to the predominance of GC Tfh1 cells induced with gp140 formulated in monophosphoryl lipid A+QS-21 (MPLA). Analysis of lymph nodes, employing proteomic and transcriptional approaches, demonstrated robust induction of GC responses across vaccine platforms with distinct qualitative and quantitative effects elicited by MPLA versus CAF01. While the induction of GC Tfh1 cells with MPLA and GC Tfh1/17 cells with CAF01 resulted in comparable peak HIV-Env antibody levels, there was a notable difference in antibody persistence. The MPLA group demonstrated significantly greater antibody persistence at week 8 and up to 30 weeks after final immunization compared to CAF01. Inducing GC Tfh1 cells with MPLA furthermore enhanced tier 1 neutralization titers, antibody functions, and Env-specific IgG in the rectal mucosa. Notably, IFNγ+ Env-specific Tfh responses, both in blood and lymph nodes, were higher with MPLA and correlated with Env antibody persistence. These findings suggest that vaccine platforms maximizing GC Tfh1 induction may confer protective immunity against HIV.
2023-06-16 | GSE234813 | GEO
Project description:Impact of Common Modifications on the Antigenic Profile and Glycosylation of Membrane-Expressed HIV-1 Envelope Glycoprotein
Recent HIV-1 vaccine development has centered on near native soluble envelope glycoprotein (Env) trimers. These trimers are artificially stabilized laterally (between protomers) and apically (between gp120 and gp41). These same stabilizing mutations have been leveraged for use in membrane-expressed Env mRNA vaccines, although their precise effects in this context are unclear. To address this question, we investigated the effects of Env mutations expressed on virus-like particle (VLP) in 293T cells. Uncleaved (UNC) trimers were laterally unstable upon gentle lysis from membranes. However, gp120/gp41 processing improved lateral stability. Due to inefficient gp120/gp41 processing, UNC is incorporated into VLPs. A linker between gp120 and gp41 (NFL) neither improved trimer stability nor its antigenic profile. An artificially introduced enterokinase cleavage site allowed processing post-expression, resulting in increased trimer stability. Gp41 N-helix mutations I559P and NT1-5 both imparted lateral trimer stability, but concomitantly reduced gp120/gp41 processing and/or impacted V2 apex and interface NAb binding. I559P consistently reduced recognition by HIV+ donor plasmas, further supporting antigenic differences. Mutations in the gp120 bridging sheet failed to stabilize membrane trimers in a pre-fusion conformation, reduced gp120/gp41 processing and exposed non-neutralizing epitopes. Reduced glycan maturation and increased sequon skipping were common effect of mutations. In some cases, this may be due to increased rigidity which limits access to glycan processing enzymes. In contrast, viral gp120 did not show glycan skipping. We observed a minor species of high mannose glycan only gp160 in particle preparations. This was unaffected by any mutations and instead bypasses normal folding and glycan maturation processes. Including the full gp41 cytoplasmic tail led to markedly reduced gp120/gp41 processing and increased the proportion of high mannose gp160. Remarkably, NAbs were unable to bind to full-length Env trimers. Overall, our findings suggest caution in leveraging mutations to ensure they impart valuable membrane trimer phenotypes for vaccine use.
The files enclosed are labelled according to the enzyme used in the digest. T = Trypsin, C = Chymotrypsin, A = Alpha-lytic protease
2023-07-12 | MSV000092416 | MassIVE