Dataset Information

Human Immunoglobulin Repertoire-Guided Immunogen Design Targeting HIV V2-Apex Broadly Neutralizing Antibody Precursors

ABSTRACT: Broadly neutralizing antibodies (bnAbs) to the HIV envelope (Env) V2-apex region are important leads for HIV vaccine design. Most V2-apex bnAbs engage Env with an uncommonly long heavy chain complementarity-determining region 3 (HCDR3), suggesting that rarity of bnAb precursors poses a challenge for vaccine priming. We created precursor sequence definitions for V2-apex HCDR3-dependent bnAbs and searched for related precursors in human antibody heavy chain ultradeep sequencing data from 14 HIV-unexposed donors. We found potential precursors in a majority of donors for only two long-HCDR3 V2-apex bnAbs, PCT64 and PG9, identifying these bnAbs as priority vaccine targets. We then engineered ApexGT Env trimers that bind inferred germlines for PCT64 and PG9 and have higher affinities for bnAbs; determined cryo-EM structures of ApexGT trimers bound to inferred germline and bnAb forms of PCT64 and PG9; and developed an mRNA-encoded cell-surface trimer for our lead ApexGT candidate. The methods and immunogens developed here have promise to assist the development of an HIV vaccine.

INSTRUMENT(S): Q Exactive HF-X

ORGANISM(S): Homo Sapiens (human) Human Immunodeficiency Virus

TISSUE(S): Epithelial Cell

SUBMITTER: Sabyasachi Baboo

LAB HEAD: John R. Yates III

PROVIDER: PXD033766 | Pride | 2023-03-11

REPOSITORIES: Pride

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			Action	DRS
	20220310_Apex_GT2-1_1.ms1	Other
	20220310_Apex_GT2-1_1.ms2	Other
	20220310_Apex_GT2-1_1.mzXML	Mzxml
	20220310_Apex_GT2-1_1.raw	Raw
	20220310_Apex_GT2-1_2.ms1	Other

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Human immunoglobulin repertoire analysis guides design of vaccine priming immunogens targeting HIV V2-apex broadly neutralizing antibody precursors.

Willis Jordan R JR Berndsen Zachary T ZT Ma Krystal M KM Steichen Jon M JM Schiffner Torben T Landais Elise E Liguori Alessia A Kalyuzhniy Oleksandr O Allen Joel D JD Baboo Sabyasachi S Omorodion Oluwarotimi O Diedrich Jolene K JK Hu Xiaozhen X Georgeson Erik E Phelps Nicole N Eskandarzadeh Saman S Groschel Bettina B Kubitz Michael M Adachi Yumiko Y Mullin Tina-Marie TM Alavi Nushin B NB Falcone Samantha S Himansu Sunny S Carfi Andrea A Wilson Ian A IA Yates John R JR Paulson James C JC Crispin Max M Ward Andrew B AB Schief William R WR

Immunity 20220929 11

Broadly neutralizing antibodies (bnAbs) to the HIV envelope (Env) V2-apex region are important leads for HIV vaccine design. Most V2-apex bnAbs engage Env with an uncommonly long heavy-chain complementarity-determining region 3 (HCDR3), suggesting that the rarity of bnAb precursors poses a challenge for vaccine priming. We created precursor sequence definitions for V2-apex HCDR3-dependent bnAbs and searched for related precursors in human antibody heavy-chain ultradeep sequencing data from 14 HI ...[more]

PMID: 36179689

Similar Datasets

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

Project description:The HIV-1 envelope glycoprotein (Env) is the sole neutralizing determinant on the surface of the virus. The Env gp120 and gp41 subunits mediate receptor binding and membrane fusion and are generated from the gp160 precursor by cellular furins. This cleavage event is required for viral entry. One approach to generate HIV-1 neutralizing antibodies following immunization is to express membrane-bound Env anchored on the cell-surface by genetic means using the natural HIV gp41 transmembrane (TM) spanning domain. To simplify the process of Env trimer membrane expression we sought to remove the need for Env precursor cleavage while maintaining native-like conformation following genetic expression. To accomplish these objectives, we selected our previously developed 'native flexibly linked' (NFL) stabilized soluble trimers that are both near native in conformation and cleavage-independent. We genetically fused the NFL construct to the HIV TM domain by using a short linker or by restoring the native membrane external proximal region, absent in soluble trimers, to express the full HIV Env ectodomain on the plasma membrane. Both forms of cell-surface NFL trimers, without and with the MPER, displayed favorable antigenic profiles by flow cytometry when expressed from plasmid DNA or mRNA. These results were consistent with the presence of well-ordered cell surface native-like trimeric Env, a necessary requirement to generate neutralizing antibodies by vaccination. Inoculation of rabbits with mRNA lipid nanoparticles (LNP) expressing membrane-bound stabilized HIV Env NFL trimers generated tier 2 neutralizing antibody serum titers in immunized animals. Multiple inoculations of mRNA LNPs generated similar neutralizing antibody titers compared to immunizations of matched NFL soluble proteins in adjuvant. Given the recent success of mRNA vaccines to prevent severe COVID, these are important developments for genetic expression of native-like HIV Env trimers in animals and potentially in humans.

Project description:Despite the critical role antigen-specific T cells play in containing viral infections, their aggregate frequencies in peripheral blood have not correlated with clinical protection during HIV infection. However, a subset of HIV-specific T cells, which are capable of simultaneously producing multiple effector cytokines, termed polyfunctional T cells, have correlated with delayed disease progression. HIV-specific polyfunctional T cells have been enumerated and characterized using the intracellular cytokine staining (ICS) assay, which allows for simultaneous multiplexed analysis of cytokine expression at the single-cell level. In addition, using an unbiased computational analysis of ICS data generated through the RV144 HIV vaccine case-control study, we identified vaccine-induced HIV envelope (Env)-specific polyfunctional CD4 T cells as a novel correlate of reduced risk of HIV infection. However, very little is known about these cells and what differentiates them from other vaccine-elicited T cells. In addition, the use of sample fixation in the ICS assay prevents those cells from being further interrogated for transcriptional differences. Thus, we developed a novel live-cell multiplexed cytokine capture assay which allows for identification, sorting and transcriptional profiling of individual antigen-specific polyfunctional T cells by single-cell RNA-sequencing (scRNA-seq). We applied these methods to peripheral blood samples from participants who had received the RV144 vaccine regimen as part of the HVTN 097 clinical trial. Using this approach, we discovered transcriptional signatures that specifically identify the single-cell Env-specific CD4 T-cell functional profiles which had correlated with reduced risk of HIV infection in RV144. Interestingly, this signature is particularly characterized by upregulation of Th2 cytokines, supporting the hypothesis that the vaccine-induced Env-specific CD4 T cells likely contributed to the reduced risk of infection by providing T cell help to the Env-specific antibody responses. By interrogating the gene signatures that correlated with polyfunctionality, we have identified potential mechanisms of how vaccine-elicited polyfunctional T cells may contribute to reduced risk of HIV infection.