Antibody dependent cellular cytotoxicity (ADCC) is an effector function performed by natural killer (NK) cells to target and clear viral infections and cancer. ADCC is a critical feature of several antibody and cellular therapeutics as well as vaccination strategies. Using microscopy to understand the details of molecular events driving ADCC is essential to improving such therapeutics but has been limited by technologies that cannot practically provide the spatial resolution necessary to study protein function at the single molecule level in cells. In this chapter, we describe a model system using MINFLUX nanoscopy to study the molecular distribution of human FcγRIIIa (CD16a), the IgG receptor, in the NK cell immunological synapse during ADCC. The technique described here will enable further exploration of how CD16a drives NK cell ADCC and can also be applied to the study of other important protein receptors for which nanometer localization precision is needed.

Evaluation of novel vaccine antigens and adjuvants traditionally relies on measuring antibody titers, which require time to develop following immunization. Quantification of antibody-secreting cells (ASCs) after primary and booster immunizations provides an earlier, mechanistically informative measure of vaccine-induced humoral immunity and can facilitate the optimization of vaccine formulations and immunization strategies. Here, a protocol for enumerating ASCs from peripheral blood mononuclear cells (PBMCs) and bone marrow (BM) cells of rhesus macaques is described; rhesus macaques are an important preclinical model for HIV vaccine research. This enzyme-linked immunospot (ELISpot) assay enables the quantification of both antigen-specific and total IgG, IgM, and IgA ASCs by measuring their absolute and relative frequencies. In peripheral blood, the assay captures the peak plasmablast response following immunization, whereas in bone marrow it detects the accumulation of antigen-specific plasma cells of different isotypes, thereby assessing the durability of long-lived ASCs. This protocol offers a robust and reproducible approach for evaluating immunogen and adjuvant combinations in preclinical vaccine studies and provides valuable data to support the translation of promising vaccine candidates into clinical development.

Antibody-dependent cellular cytotoxicity (ADCC) by natural killer (NK) cells is mediated by the activating IgG receptor CD16a (FcγRIIIa), yet the molecular mechanisms governing receptor activation remain poorly understood. We demonstrate that the membrane-distal domain 1 (D1) of CD16a functions as an allosteric checkpoint that controls ADCC independently of IgG-Fc binding. A nanobody, C28, that binds an electronegative patch in D1 dose-dependently blocks NK cell ADCC against multiple therapeutic antibodies without affecting direct cytotoxicity. A second nanobody, C21, binding an adjacent D1 epitope has no such effect. Cryo-EM structures of the CD16a-IgG-nanobody complex reveal that C28 allosterically competes with core-fucosylated IgG and stabilizes a closed D1 conformation resembling unliganded receptor, even when Fc is bound. Molecular dynamics simulations show that occupation of the D1 epitope rigidifies the IgG-binding site, stabilizing CD16a overall in contrast with IgG binding alone. The nanobody C28 restricts CD3ζ phosphorylation in both resting and ADCC-activated NK cells, revealing tonic inhibitory control upstream of the signaling cascade. Using MINFLUX nanoscopy, we also show that CD16a forms dimers of ~9 nm spacing on the NK cell surface, a geometry unaltered by the ADCC-enhancing L48H polymorphism. Drawing on structural parallels with the IgE receptor FcεRI, which is held inactive as a cholesterol-stabilized dimer, we propose that CD16a dimerization through D1 contacts represents a conserved autoinhibitory mechanism among Fc receptors. Consistent with this model, structure-guided disruption of the C28 epitope in NK-92 cells enhances ADCC potency and killing kinetics, providing a blueprint for engineering improved cellular immunotherapeutics.

The envelope surface glycoprotein (Env) on HIV-1 drives cell entry and genome delivery through its receptor binding and membrane fusion activities. Incorporation of Env onto assembling virions is governed by its cytoplasmic tail (Env-CT) and the matrix (MA) domain of the PR55Gag (Gag) polyprotein. To better understand how Gag recruits Env onto virions while reducing its antigenic profile by restricting Env copy number to low levels, we used cryo-electron tomography (cryo-ET) and subtomogram averaging combined with molecular dynamics simulations to investigate Env-MA association in intact viral particles. Full-length Env-CT was resolved directly over individual MA trimers in regions of MA lattice discontinuity. We observed that the conserved but enigmatic Kennedy-sequence motif in Env-CT forms a key linkage between Env and MA. Mutational analysis confirmed the impact of specific CT and MA interactions on Env incorporation. Gag maturation released MA lattice restraints on Env, facilitating its clustering and promoting fusion activity.

NK cells utilize effector functions, including antibody-dependent cellular cytotoxicity (ADCC), for the clearance of viral infection and cellular malignancies. While antibody-induced clustering of FcγRIIIa (CD16a) is thought to drive ADCC, the molecular basis for this activity has not been fully described. We used MINFLUX nanoscopy to map the spatial distribution of CD16a within the NK-cell ADCC immune synapse. In both resting and NK cells activated on supported lipid bilayers by Trastuzumab, we detected pairs of CD16a molecules approximately 18 nm apart that could be homodimers. NK-cell activation results in a modest increase of clusters of 4 or more CD16a localizations without a change in cluster characteristics, while CD16a pair distances do not significantly change, suggesting that subtle structural changes underpin ADCC-based activation. Our results provide the highest spatial resolution yet described for CD16a imaging, offering insight into how CD16a organization within the immune synapse could influence ADCC activity.

Natural Killer (NK) cells utilize effector functions, including antibody-dependent cellular cytotoxicity (ADCC), for the clearance of viral infection and cellular malignancies. NK cell ADCC is mediated by FcγRIIIa (CD16a) binding to the fragment crystallizable (Fc) region of immunoglobulin G (IgG) within immune complexes on a target cell surface. While antibody-induced clustering of CD16a is thought to drive ADCC, the molecular basis for this activity has not been fully described. Here we use MINFLUX nanoscopy to map the spatial distribution of stoichiometrically labeled CD16a across the NK cell membrane, revealing the presence of pairs of CD16a molecules with intra-doublet distance of approximately 17 nm. NK cells activated on supported lipid bilayers by Trastuzumab results in an increase of synaptic regions with greater CD16a density. Our results provide the highest spatial resolution yet described for CD16a imaging, offering new insight into how CD16a organization within the immune synapse could influence ADCC activity. MINFLUX holds great promise to further unravel the molecular details driving CD16a-based activation of NK cells.

Background High sequence identity between segmental duplications (SDs) can facilitate copy number variants (CNVs) via non-allelic homologous recombination (NAHR). These CNVs are one of the fundamental causes of genomic disorders such as the 3q29 deletion syndrome (del3q29S). There are 21 protein-coding genes lost or gained as a result of such recurrent 1.6-Mbp deletions or duplications, respectively, in the 3q29 locus. While NAHR plays a role in CNV occurrence, the factors that increase the risk of NAHR at this particular locus are not well understood. Methods We employed an optical genome mapping technique to characterize the 3q29 locus in 161 unaffected individuals, 16 probands with del3q29S and their parents, and 2 probands with the 3q29 duplication syndrome (dup3q29S). Long-read sequencing-based haplotype resolved de novo assemblies from 44 unaffected individuals, and 1 trio was used for orthogonal validation of haplotypes and deletion breakpoints. Results In total, we discovered 34 haplotypes, of which 19 were novel haplotypes. Among these 19 novel haplotypes, 18 were detected in unaffected individuals, while 1 novel haplotype was detected on the parent-of-origin chromosome of a proband with the del3q29S. Phased assemblies from 44 unaffected individuals enabled the orthogonal validation of 20 haplotypes. In 89% (16/18) of the probands, breakpoints were confined to paralogous copies of a 20-kbp segment within the 3q29 SDs. In one del3q29S proband, the breakpoint was confined to a 374-bp region using long-read sequencing. Furthermore, we categorized del3q29S cases into three classes and dup3q29S cases into two classes based on breakpoints. Finally, we found no evidence of inversions in parent-of-origin chromosomes. Conclusions We have generated the most comprehensive haplotype map for the 3q29 locus using unaffected individuals, probands with del3q29S or dup3q29S, and available parents, and also determined the deletion breakpoint to be within a 374-bp region in one proband with del3q29S. These results should provide a better understanding of the underlying genetic architecture that contributes to the etiology of del3q29S and dup3q29S.

Barriers to effective gene therapy for many diseases include the number of modified target cells required to achieve therapeutic outcomes and host immune responses to expressed therapeutic proteins. As long-lived cells specialized for protein secretion, antibody-secreting B cells are an attractive target for foreign protein expression in blood and tissue. To neutralize HIV-1, we developed a lentiviral vector (LV) gene therapy platform for delivery of the anti-HIV-1 immunoadhesin, eCD4-Ig, to B cells. The EμB29 enhancer/promoter in the LV limited gene expression in non-B cell lineages. By engineering a knob-in-hole-reversed (KiHR) modification in the CH3-Fc eCD4-Ig domain, we reduced interactions between eCD4-Ig and endogenous B cell immunoglobulin G proteins, which improved HIV-1 neutralization potency. Unlike previous approaches in non-lymphoid cells, eCD4-Ig-KiHR produced in B cells promoted HIV-1 neutralizing protection without requiring exogenous TPST2, a tyrosine sulfation enzyme required for eCD4-Ig-KiHR function. This finding indicated that B cell machinery is well suited to produce therapeutic proteins. Lastly, to overcome the inefficient transduction efficiency associated with VSV-G LV delivery to primary B cells, an optimized measles pseudotyped LV packaging methodology achieved up to 75% transduction efficiency. Overall, our findings support the utility of B cell gene therapy platforms for therapeutic protein delivery.

Human immunodeficiency virus 1 (HIV-1) infection is initiated by binding of the viral envelope glycoprotein (Env) to the cell-surface receptor CD41-4. Although high-resolution structures of Env in a complex with the soluble domains of CD4 have been determined, the binding process is less understood in native membranes5-13. Here we used cryo-electron tomography to monitor Env-CD4 interactions at the membrane-membrane interfaces formed between HIV-1 and CD4-presenting virus-like particles. Env-CD4 complexes organized into clusters and rings, bringing the opposing membranes closer together. Env-CD4 clustering was dependent on capsid maturation. Subtomogram averaging and classification revealed that Env bound to one, two and finally three CD4 molecules, after which Env adopted an open state. Our data indicate that asymmetric HIV-1 Env trimers bound to one and two CD4 molecules are detectable intermediates during virus binding to host cell membranes, which probably has consequences for antibody-mediated immune responses and vaccine immunogen design.

The 1.6 Mb 3q29 deletion is associated with developmental and psychiatric phenotypes, including a 40-fold increased risk for schizophrenia. Reduced birth weight and a high prevalence of feeding disorders in patients suggest underlying metabolic dysregulation. We investigated 3q29 deletion-induced metabolic changes using our previously generated heterozygous B6.Del16 +/ Bdh1-Tfrc mouse model. Animals were provided either standard chow (STD) or high-fat diet (HFD). Growth curves were performed on HFD mice to assess weight change ( n = 30–50/group). Indirect calorimetry and untargeted metabolomics were performed on STD and HFD mice to evaluate metabolic phenotypes ( n = 8–14/group). A behavioral battery was performed on STD and HFD mice to assess behavior change after the HFD challenge ( n = 5–13/group). We found that B6.Del16 +/ Bdh1-Tfrc animals preferentially use dietary lipids as an energy source. Untargeted metabolomics of liver tissue showed a strong sex-dependent effect of the 3q29 deletion on fat metabolism. A HFD partially rescued the 3q29 deletion-associated weight deficit in females, but not males. Untargeted metabolomics of liver tissue after HFD revealed persistent fat metabolism alterations in females. The HFD did not affect B6.Del16 +/ Bdh1-Tfrc behavioral phenotypes, suggesting that 3q29 deletion-associated metabolic and behavioral outcomes are uncoupled. Our data suggest that dietary interventions to improve weight phenotypes in 3q29 deletion syndrome patients are unlikely to exacerbate behavioral manifestations. Our study also highlights the importance of assessing sex in metabolic studies and suggests that mechanisms underlying 3q29 deletion-associated metabolic phenotypes are sex-specific.

Human immunodeficiency virus 1 (HIV-1) infection is initiated by binding of the viral envelope glycoprotein (Env) to the cell-surface receptor CD4. Although high resolution structures of Env complexed with soluble domains of CD4 have been determined, the binding process is less understood on native membranes. Here, we apply cryo-electron tomography (cryo-ET) to monitor Env-CD4 interactions at membrane-membrane interfaces formed between HIV-1 and CD4-presenting virus-like particles. Env-CD4 complexes organized into clusters and rings, bringing opposing membranes closer together. Additionally, Env-CD4 clustering was dependent on capsid maturation. Subtomogram averaging and classification revealed that Env bound one, two, and finally three CD4 molecules, upon which Env adopted a partially open state. Our data indicate that asymmetric HIV-1 Env trimers bound to one and two CD4 molecules are detectable intermediates during virus binding to host cell membranes, which likely has consequences for antibody-mediated immune responses and vaccine immunogen design.

HIV-1 Env mediates viral entry into host cells and is the sole target for neutralizing antibodies. However, Env structure and organization in its native virion context has eluded detailed characterization. Here, we used cryo-electron tomography to analyze Env in mature and immature HIV-1 particles. Immature particles showed distinct Env positioning relative to the underlying Gag lattice, providing insights into long-standing questions about Env incorporation. A 9.1-Å sub-tomogram-averaged reconstruction of virion-bound Env in conjunction with structural mass spectrometry revealed unexpected features, including a variable central core of the gp41 subunit, heterogeneous glycosylation between protomers, and a flexible stalk that allows Env tilting and variable exposure of neutralizing epitopes. Together, our results provide an integrative understanding of HIV assembly and structural variation in Env antigen presentation.

Identifying causative gene(s) within disease-associated large genomic regions of copy number variants (CNVs) is challenging. Here, by targeted sequencing of genes within schizophrenia (SZ)-associated CNVs in 1,779 SZ cases and 1,418 controls, we identified three rare putative loss-of-function (LoF) mutations in OTU deubiquitinase 7A (OTUD7A) within the 15q13.3 deletion in cases, but none in controls. To tie OTUD7A LoF with any SZ-relevant cellular phenotypes, we modeled the OTUD7A LoF mutation, rs757148409, in human induced pluripotent stem cell (hiPSC)-derived induced excitatory neurons (iNs) by CRISPR/Cas9 engineering. The mutant iNs showed a ∼50% decrease in OTUD7A expression without undergoing nonsense-mediated mRNA decay. The mutant iNs also exhibited marked reduction of dendritic complexity, density of synaptic proteins GluA1 and PSD-95, and neuronal network activity. Congruent with the neuronal phenotypes in mutant iNs, our transcriptomic analysis showed that the set of OTUD7A LoF-downregulated genes was enriched for those relating to synapse development and function, and was associated with SZ and other neuropsychiatric disorders. These results suggest that OTUD7A LoF impairs synapse development and neuronal function in human neurons, providing mechanistic insight into the possible role of OTUD7A in driving neuropsychiatric phenotypes associated with the 15q13.3 deletion.

A major goal of HIV vaccine design is to elicit broadly neutralizing antibodies (bNAbs). Such bNAbs target HIV's trimeric, membrane-embedded envelope glycoprotein spikes (mEnv). Soluble Env (sEnv) trimers have been used as vaccines, but engineering sEnvs for stability, multivalency, and desired antigenicity is problematic and deletes key neutralizing epitopes on glycoprotein 41 (gp41) while creating neoepitopes that elicit unwanted antibodies. Meanwhile, multivalent mEnv vaccines are challenging to develop due to trimer instability and low mEnv copy number amid other extraneous proteins on virus-like particles. Here, we describe a multivalent mEnv vaccine platform that does not require protein engineering or extraneous proteins. mEnv trimers were fixed, purified, and combined with naked liposomes in mild detergent. On removal of detergent, mEnv spikes were observed embedded in liposome particles (mean diameter, 133 nm) in correct orientation. These particles were recognized by HIV bNAbs and not non-NAbs and are designated mEnv liposomes (MELs). Following a sequential immunization scheme in rabbits, MELs elicited antibodies that neutralized tier 2 HIV isolates. Analysis of serum antibody specificities, including those to epitopes involving a missing conserved N-glycosylation site at position 197 near the CD4 binding site on two of the immunogens, provides clues on how NAb responses can be improved with modified immunogens. In sum, MELs are a biochemically defined platform that enables rational immunization strategies to elicit HIV bNAbs using multimerized mEnv. IMPORTANCE A vaccine that induced broadly neutralizing antibodies against HIV would likely end the AIDS pandemic. Such antibodies target membrane-embedded envelope glycoprotein spikes (mEnv) that HIV uses to enter cells. Due to HIV Env's low expression and instability, soluble stabilized Env trimers have been used as vaccine candidates, but these have an altered base that disrupts targets of HIV broadly neutralizing antibodies that bind near the membrane and are not available for all HIV isolates. Here, we describe membrane Env liposomes (MELs) that display a multivalent array of stable mEnvs on liposome particles. MELs showed the expected antibody recognition properties, including targeting parts of mEnv missing on soluble Envs. Immunization with MELs elicited antibodies that neutralized diverse HIV isolates. The MEL platform facilitates vaccine development with potentially any HIV Env at high valency, and a similar approach may be useful for eliciting antibodies to membrane-embedded targets of therapeutic interest.

Broadly neutralizing antibodies (bnAbs) against HIV-1 are frequently associated with the presence of autoreactivity/polyreactivity, a property that can limit their use as therapeutic agents. The bnAb 4E10, targeting the conserved Membrane proximal external region (MPER) of HIV-1, displays almost pan-neutralizing activity across globally circulating HIV-1 strains but exhibits nonspecific off-target interactions with lipid membranes. The hydrophobic apex of the third complementarity-determining region of the heavy chain (CDRH3) loop, which is essential for viral neutralization, critically contributes to this detrimental effect. Here, we have replaced the aromatic/hydrophobic residues from the apex of the CDRH3 of 4E10 with a single aromatic molecule through chemical modification to generate a variant that preserves the neutralization potency and breadth of 4E10 but with reduced autoreactivity. Collectively, our study suggests that the localized accumulation of aromaticity by chemical modification provides a pathway to ameliorate the adverse effects triggered by the CDRH3 of anti-HIV-1 MPER bnAbs.

BNAbs of V H 1-69 germline origin from a Chinese patient recognize the V3 glycan supersite via a disulfide-locked long HCDR3 loop.

The contribution of membrane interfacial interactions to recognition of membrane-embedded antigens by antibodies is currently unclear. This report demonstrates the optimization of this type of antibodies via chemical modification of regions near the membrane but not directly involved in the recognition of the epitope. Using the HIV-1 antibody 10E8 as a model, linear and polycyclic synthetic aromatic compounds are introduced at selected sites. Molecular dynamics simulations predict the favorable interactions of these synthetic compounds with the viral lipid membrane, where the epitope of the HIV-1 glycoprotein Env is located. Chemical modification of 10E8 with aromatic acetamides facilitates the productive and specific recognition of the native antigen, partially buried in the crowded environment of the viral membrane, resulting in a dramatic increase of its capacity to block viral infection. These observations support the harnessing of interfacial affinity through site-selective chemical modification to optimize the function of antibodies that target membrane-proximal epitopes.

The transmembrane (TM) domain of HIV glycoprotein gp41 anchors the envelope (Env) spike in the viral membrane and is highly conserved. The mid-span arginine 696 is particularly conserved, and the only other residue found in this position is lysine. Seeking to examine the role of this conserved positive charge in the structure and function of the gp41 TM domain, we synthesized a series of peptides corresponding to this region. Analysis of the peptides in a previously validated fluorescence assay in model membranes showed that the native TM domain is trimeric. Peptides in which the intramembrane arginine was mutated to alanine showed significantly lower trimerization propensity. In contrast, this mutation in the context of infectious pseudovirus caused only modest decreases in viral stability and infectivity. We propose a model to explain the importance of this charge to gp41 structure and to HIV infection.