Vaccination induces broadly neutralizing antibody precursors to HIV gp41.

A key barrier to the development of vaccines that induce broadly neutralizing antibodies (bnAbs) against human immunodeficiency virus (HIV) and other viruses of high antigenic diversity is the design of priming immunogens that induce rare bnAb-precursor B cells. The high neutralization breadth of the HIV bnAb 10E8 makes elicitation of 10E8-class bnAbs desirable; however, the recessed epitope within gp41 makes envelope trimers poor priming immunogens and requires that 10E8-class bnAbs possess a long heavy chain complementarity determining region 3 (HCDR3) with a specific binding motif. We developed germline-targeting epitope scaffolds with affinity for 10E8-class precursors and engineered nanoparticles for multivalent display. Scaffolds exhibited epitope structural mimicry and bound bnAb-precursor human naive B cells in ex vivo screens, protein nanoparticles induced bnAb-precursor responses in stringent mouse models and rhesus macaques, and mRNA-encoded nanoparticles triggered similar responses in mice. Thus, germline-targeting epitope scaffold nanoparticles can elicit rare bnAb-precursor B cells with predefined binding specificities and HCDR3 features.

Repeat modules and N-linked glycans define structure and antigenicity of a critical enterotoxigenic E. coli adhesin.

Enterotoxigenic Escherichia coli (ETEC) cause hundreds of millions of cases of infectious diarrhea annually, predominantly in children from low-middle income regions. Notably, in children, as well as human volunteers challenged with ETEC, diarrheal severity is significantly increased severity in blood group A (bgA) individuals. EtpA, is a secreted glycoprotein adhesin that functions as a blood group A lectin to promote critical interactions between ETEC and blood group A glycans on intestinal epithelia for effective bacterial adhesion and toxin delivery. EtpA is highly immunogenic resulting in robust antibody responses following natural infection and experimental challenge of human volunteers with ETEC. To understand how EtpA directs ETEC-blood group A interactions and stimulates adaptive immunity, we mutated EtpA, mapped its glycosylation by mass-spectrometry (MS), isolated polyclonal (pAbs) and monoclonal antibodies (mAbs) from vaccinated mice and ETEC-infected human volunteers, and determined structures of antibody-EtpA complexes by cryo-electron microscopy. Both bgA and mAbs that inhibited EtpA-bgA interactions and ETEC adhesion, bound to the C-terminal repeat domain highlighting this region as crucial for ETEC pathogen-host interaction. MS analysis uncovered extensive and heterogeneous N-linked glycosylation of EtpA and cryo-EM structures revealed that mAbs directly engage these unique glycan containing epitopes. Finally, electron microscopy-based polyclonal epitope mapping revealed antibodies targeting numerous distinct epitopes on N and C-terminal domains, suggesting that EtpA vaccination generates responses against neutralizing and decoy regions of the molecule. Collectively, we anticipate that these data will inform our general understanding of pathogen-host glycan interactions and adaptive immunity relevant to rational vaccine subunit design.

Heterologous prime-boost vaccination drives early maturation of HIV broadly neutralizing antibody precursors in humanized mice.

A protective HIV vaccine will likely need to induce broadly neutralizing antibodies (bnAbs). Vaccination with the germline-targeting immunogen eOD-GT8 60mer adjuvanted with AS01B was found to induce VRC01-class bnAb precursors in 97% of vaccine recipients in the IAVI G001 phase 1 clinical trial; however, heterologous boost immunizations with antigens more similar to the native glycoprotein will be required to induce bnAbs. Therefore, we designed core-g28v2 60mer, a nanoparticle immunogen to be used as a first boost after eOD-GT8 60mer priming. We found, using a humanized mouse model approximating human conditions of VRC01-class precursor B cell diversity, affinity, and frequency, that both protein- and mRNA-based heterologous prime-boost regimens induced VRC01-class antibodies that gained key mutations and bound to near-native HIV envelope trimers lacking the N276 glycan. We further showed that VRC01-class antibodies induced by mRNA-based regimens could neutralize pseudoviruses lacking the N276 glycan. These results demonstrated that heterologous boosting can drive maturation toward VRC01-class bnAb development and supported the initiation of the IAVI G002 phase 1 trial testing mRNA-encoded nanoparticle prime-boost regimens.

Vaccine priming of rare HIV broadly neutralizing antibody precursors in nonhuman primates.

Germline-targeting immunogens hold promise for initiating the induction of broadly neutralizing antibodies (bnAbs) to HIV and other pathogens. However, antibody-antigen recognition is typically dominated by heavy chain complementarity determining region 3 (HCDR3) interactions, and vaccine priming of HCDR3-dominant bnAbs by germline-targeting immunogens has not been demonstrated in humans or outbred animals. In this work, immunization with N332-GT5, an HIV envelope trimer designed to target precursors of the HCDR3-dominant bnAb BG18, primed bnAb-precursor B cells in eight of eight rhesus macaques to substantial frequencies and with diverse lineages in germinal center and memory B cells. We confirmed bnAb-mimicking, HCDR3-dominant, trimer-binding interactions with cryo-electron microscopy. Our results demonstrate proof of principle for HCDR3-dominant bnAb-precursor priming in outbred animals and suggest that N332-GT5 holds promise for the induction of similar responses in humans.

mRNA-LNP HIV-1 trimer boosters elicit precursors to broad neutralizing antibodies.

Germline-targeting (GT) HIV vaccine strategies are predicated on deriving broadly neutralizing antibodies (bnAbs) through multiple boost immunogens. However, as the recruitment of memory B cells (MBCs) to germinal centers (GCs) is inefficient and may be derailed by serum antibody-induced epitope masking, driving further B cell receptor (BCR) modification in GC-experienced B cells after boosting poses a challenge. Using humanized immunoglobulin knockin mice, we found that GT protein trimer immunogen N332-GT5 could prime inferred-germline precursors to the V3-glycan-targeted bnAb BG18 and that B cells primed by N332-GT5 were effectively boosted by either of two novel protein immunogens designed to have minimum cross-reactivity with the off-target V1-binding responses. The delivery of the prime and boost immunogens as messenger RNA lipid nanoparticles (mRNA-LNPs) generated long-lasting GCs, somatic hypermutation, and affinity maturation and may be an effective tool in HIV vaccine development.

Comparative membrane proteomics reveals diverse cell regulators concentrated at the nuclear envelope.

The nuclear envelope (NE) is a subdomain of the ER with prominent roles in nuclear organization, which are largely mediated by its distinctive protein composition. We developed methods to reveal low-abundance transmembrane (TM) proteins concentrated at the NE relative to the peripheral ER. Using label-free proteomics that compared isolated NEs with cytoplasmic membranes, we first identified proteins with apparent NE enrichment. In subsequent authentication, ectopically expressed candidates were analyzed by immunofluorescence microscopy to quantify their targeting to the NE in cultured cells. Ten proteins from a validation set were found to associate preferentially with the NE, including oxidoreductases, enzymes for lipid biosynthesis, and regulators of cell growth and survival. We determined that one of the validated candidates, the palmitoyltransferase Zdhhc6, modifies the NE oxidoreductase Tmx4 and thereby modulates its NE levels. This provides a functional rationale for the NE concentration of Zdhhc6. Overall, our methodology has revealed a group of previously unrecognized proteins concentrated at the NE and additional candidates. Future analysis of these can potentially unveil new mechanistic pathways associated with the NE.

Evolving spike-protein N-glycosylation in SARS-CoV-2 variants.

Since >3 years, SARS-CoV-2 has plunged humans into a colossal pandemic. Henceforth, multiple waves of infection have swept through the human population, led by variants that were able to partially evade acquired immunity. The co-evolution of SARS-CoV-2 variants with human immunity provides an excellent opportunity to study the interaction between viral pathogens and their human hosts. The heavily N-glycosylated spike-protein of SARS-CoV-2 plays a pivotal role in initiating infection and is the target for host immune-response, both of which are impacted by host-installed N-glycans. Using highly-sensitive DeGlyPHER approach, we compared the N-glycan landscape on spikes of the SARS-CoV-2 Wuhan-Hu-1 strain to seven WHO-defined variants of concern/interest, using recombinantly expressed, soluble spike-protein trimers, sharing same stabilizing-mutations. We found that N-glycan processing is conserved at most sites. However, in multiple variants, processing of N-glycans from high mannose- to complex-type is reduced at sites N165, N343 and N616, implicated in spike-protein function.

DeGlyPHER: Highly sensitive site-specific analysis of N-linked glycans on proteins.

Traditional mass spectrometry-based glycoproteomic approaches have been widely used for site-specific N-glycoform analysis, but a large amount of starting material is needed to obtain sampling that is representative of the vast diversity of N-glycans on glycoproteins. These methods also often include a complicated workflow and very challenging data analysis. These limitations have prevented glycoproteomics from being adapted to high-throughput platforms, and the sensitivity of the analysis is currently inadequate for elucidating N-glycan heterogeneity in clinical samples. Heavily glycosylated spike proteins of enveloped viruses, recombinantly expressed as potential vaccines, are prime targets for glycoproteomic analysis. Since the immunogenicity of spike proteins may be impacted by their glycosylation patterns, site-specific analysis of N-glycoforms provides critical information for vaccine design. Using recombinantly expressed soluble HIV Env trimer, we describe DeGlyPHER, a modification of our previously reported sequential deglycosylation strategy to yield a "single-pot" process. DeGlyPHER is an ultrasensitive, simple, rapid, robust, and efficient approach for site-specific analysis of protein N-glycoforms, that we developed for analysis of limited quantities of glycoproteins.

Comparative membrane proteomics reveals diverse cell regulators concentrated at the nuclear envelope.

The nuclear envelope (NE) is a subdomain of the ER with prominent roles in nuclear organization, largely mediated by its distinctive protein composition. We developed methods to reveal novel, low abundance transmembrane (TM) proteins concentrated at the NE relative to the peripheral ER. Using label-free proteomics that compared isolated NEs to cytoplasmic membranes, we first identified proteins with apparent NE enrichment. In subsequent authentication, ectopically expressed candidates were analyzed by immunofluorescence microscopy to quantify their targeting to the NE in cultured cells. Ten proteins from a validation set were found to associate preferentially with the NE, including oxidoreductases, enzymes for lipid biosynthesis and regulators of cell growth and survival. We determined that one of the validated candidates, the palmitoyltransferase Zdhhc6, modifies the NE oxidoreductase Tmx4 and thereby modulates its NE levels. This provides a functional rationale for the NE concentration of Zdhhc6. Overall, our methodology has revealed a group of previously unrecognized proteins concentrated at the NE and additional candidates. Future analysis of these can potentially unveil new mechanistic pathways associated with the NE.

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

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 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 bound inferred germlines for PCT64 and PG9 and had higher affinities for bnAbs, determined cryo-EM structures of ApexGT trimers complexed with inferred-germline and bnAb forms of PCT64 and PG9, and developed an mRNA-encoded cell-surface ApexGT trimer. These methods and immunogens have promise to assist HIV vaccine development.

Shared and Distinctive Neighborhoods of Emerin and Lamin B Receptor Revealed by Proximity Labeling and Quantitative Proteomics.

Emerin and lamin B receptor (LBR) are abundant transmembrane proteins of the nuclear envelope that are concentrated at the inner nuclear membrane (INM). Although both proteins interact with chromatin and nuclear lamins, they have distinctive biochemical and functional properties. Here, we have deployed proximity labeling using the engineered biotin ligase TurboID (TbID) and quantitative proteomics to compare the neighborhoods of emerin and LBR in cultured mouse embryonic fibroblasts. Our analysis revealed 232 high confidence proximity partners that interact selectively with emerin and/or LBR, 49 of which are shared by both. These included previously characterized NE-concentrated proteins, as well as a host of additional proteins not previously linked to emerin or LBR functions. Many of these are TM proteins of the ER, including two E3 ubiquitin ligases. Supporting these results, we found that 11/12 representative proximity relationships identified by TbID also were detected at the NE with the proximity ligation assay. Overall, this work presents methodology that may be used for large-scale mapping of the landscape of the INM and reveals a group of new proteins with potential functional connections to emerin and LBR.

Intercepting IRE1 kinase-FMRP signaling prevents atherosclerosis progression.

Fragile X Mental Retardation protein (FMRP), widely known for its role in hereditary intellectual disability, is an RNA-binding protein (RBP) that controls translation of select mRNAs. We discovered that endoplasmic reticulum (ER) stress induces phosphorylation of FMRP on a site that is known to enhance translation inhibition of FMRP-bound mRNAs. We show ER stress-induced activation of Inositol requiring enzyme-1 (IRE1), an ER-resident stress-sensing kinase/endoribonuclease, leads to FMRP phosphorylation and to suppression of macrophage cholesterol efflux and apoptotic cell clearance (efferocytosis). Conversely, FMRP deficiency and pharmacological inhibition of IRE1 kinase activity enhances cholesterol efflux and efferocytosis, reducing atherosclerosis in mice. Our results provide mechanistic insights into how ER stress-induced IRE1 kinase activity contributes to macrophage cholesterol homeostasis and suggests IRE1 inhibition as a promising new way to counteract atherosclerosis.

DeGlyPHER: An Ultrasensitive Method for the Analysis of Viral Spike N-Glycoforms.

Viruses can evade the host immune system by displaying numerous glycans on their surface "spike-proteins" that cover immune epitopes. We have developed an ultrasensitive "single-pot" method to assess glycan occupancy and the extent of glycan processing from high-mannose to complex forms at each N-glycosylation site. Though aimed at characterizing glycosylation of viral spike-proteins as potential vaccines, this method is applicable for the analysis of site-specific glycosylation of any glycoprotein.

Identification of new transmembrane proteins concentrated at the nuclear envelope using organellar proteomics of mesenchymal cells.

The double membrane nuclear envelope (NE), which is contiguous with the ER, contains nuclear pore complexes (NPCs) - the channels for nucleocytoplasmic transport, and the nuclear lamina (NL) - a scaffold for NE and chromatin organization. Since numerous human diseases linked to NE proteins occur in mesenchyme-derived cells, we used proteomics to characterize NE and other subcellular fractions isolated from mesenchymal stem cells and from adipocytes and myocytes. Based on spectral abundance, we calculated enrichment scores for proteins in the NE fractions. We demonstrated by quantitative immunofluorescence microscopy that five little-characterized proteins with high enrichment scores are substantially concentrated at the NE, with Itprip exposed at the outer nuclear membrane, Smpd4 enriched at the NPC, and Mfsd10, Tmx4, and Arl6ip6 likely residing in the inner nuclear membrane. These proteins provide new focal points for studying the functions of the NE. Moreover, our datasets provide a resource for evaluating additional potential NE proteins.

"Dark matter" worlds of unstable RNA and protein.

Astrophysicists use the term "dark matter" to describe the majority of the matter and/or energy in the universe that is hidden from view, and biologists now apply it to the new families of RNA they are uncovering. We review evidence for an analogous hidden world containing peptides. The critical experiments involved pulse-labeling human cells with tagged amino acids for periods as short as five seconds. Results are extraordinary in two respects: both nucleus and cytoplasm become labeled, and most signals disappear with a half-life of less than one minute. Just as the synthesis of each mature mRNA is regulated by the abortive production of hundreds of shorter transcripts that are quickly degraded, it seems that the synthesis of each full-length protein in the stable proteome is regulated by an apparently wasteful production and degradation of shorter peptides. Some of the nuclear synthesis is probably a byproduct of nuclear ribosomes proofreading newly-made RNA for inappropriately-placed termination codons (a process that triggers "nonsense-mediated decay"). We speculate that some "dark-matter" peptides will play other important roles in the cell.

Most human proteins made in both nucleus and cytoplasm turn over within minutes.

In bacteria, protein synthesis can be coupled to transcription, but in eukaryotes it is believed to occur solely in the cytoplasm. Using pulses as short as 5 s, we find that three analogues--L-azidohomoalanine, puromycin (detected after attaching fluors using 'click' chemistry or immuno-labeling), and amino acids tagged with 'heavy' 15N and 13C (detected using secondary ion mass spectrometry)--are incorporated into the nucleus and cytoplasm in a process sensitive to translational inhibitors. The nuclear incorporation represents a significant fraction of the total, and labels in both compartments have half-lives of less than a minute; results are consistent with most newly-made peptides being destroyed soon after they are made. As nascent RNA bearing a premature termination codon (detected by fluorescence in situ hybridization) is also eliminated by a mechanism sensitive to a translational inhibitor, the nuclear turnover of peptides is probably a by-product of proof-reading the RNA for stop codons (a process known as nonsense-mediated decay). We speculate that the apparently-wasteful turnover of this previously-hidden ('dark-matter') world of peptide is involved in regulating protein production.

TNFα signals through specialized factories where responsive coding and miRNA genes are transcribed.

Tumour necrosis factor alpha (TNFα) is a potent cytokine that signals through nuclear factor kappa B (NFκB) to activate a subset of human genes. It is usually assumed that this involves RNA polymerases transcribing responsive genes wherever they might be in the nucleus. Using primary human endothelial cells, variants of chromosome conformation capture (including 4C and chromatin interaction analysis with paired-end tag sequencing), and fluorescence in situ hybridization to detect single nascent transcripts, we show that TNFα induces responsive genes to congregate in discrete 'NFκB factories'. Some factories further specialize in transcribing responsive genes encoding micro-RNAs that target downregulated mRNAs. We expect all signalling pathways to contain this extra leg, where responding genes are transcribed in analogous specialized factories.

The proteomes of transcription factories containing RNA polymerases I, II or III.

Human nuclei contain three RNA polymerases (I, II and III) that transcribe different groups of genes; the active forms of all three are difficult to isolate because they are bound to the substructure. Here we describe a purification approach for isolating active RNA polymerase complexes from mammalian cells. After isolation, we analyzed their protein content by mass spectrometry. Each complex represents part of the core of a transcription factory. For example, the RNA polymerase II complex contains subunits unique to RNA polymerase II plus various transcription factors but shares a number of ribonucleoproteins with the other polymerase complexes; it is also rich in polymerase II transcripts. We also describe a native chromosome conformation capture method to confirm that the complexes remain attached to the same pairs of DNA templates found in vivo.