Cryo-ET of Env on intact HIV virions reveals structural variation and positioning on the Gag lattice.

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.

How a broadly neutralizing antibody grapples with antigenic and conformational diversity in dengue virus.

In this issue of Cell, two studies apply powerful structural approaches to probe the modes of interaction between a broadly neutralizing antibody and a conserved epitope found on four dengue virus serotypes and Zika virus. These findings offer new insights into how a broadly neutralizing antibody surmounts antigenic and conformational variation.

Elicitation of Potent Neutralizing Antibody Responses by Designed Protein Nanoparticle Vaccines for SARS-CoV-2.

A safe, effective, and scalable vaccine is needed to halt the ongoing SARS-CoV-2 pandemic. We describe the structure-based design of self-assembling protein nanoparticle immunogens that elicit potent and protective antibody responses against SARS-CoV-2 in mice. The nanoparticle vaccines display 60 SARS-CoV-2 spike receptor-binding domains (RBDs) in a highly immunogenic array and induce neutralizing antibody titers 10-fold higher than the prefusion-stabilized spike despite a 5-fold lower dose. Antibodies elicited by the RBD nanoparticles target multiple distinct epitopes, suggesting they may not be easily susceptible to escape mutations, and exhibit a lower binding:neutralizing ratio than convalescent human sera, which may minimize the risk of vaccine-associated enhanced respiratory disease. The high yield and stability of the assembled nanoparticles suggest that manufacture of the nanoparticle vaccines will be highly scalable. These results highlight the utility of robust antigen display platforms and have launched cGMP manufacturing efforts to advance the SARS-CoV-2-RBD nanoparticle vaccine into the clinic.

Induction of Potent Neutralizing Antibody Responses by a Designed Protein Nanoparticle Vaccine for Respiratory Syncytial Virus.

Respiratory syncytial virus (RSV) is a worldwide public health concern for which no vaccine is available. Elucidation of the prefusion structure of the RSV F glycoprotein and its identification as the main target of neutralizing antibodies have provided new opportunities for development of an effective vaccine. Here, we describe the structure-based design of a self-assembling protein nanoparticle presenting a prefusion-stabilized variant of the F glycoprotein trimer (DS-Cav1) in a repetitive array on the nanoparticle exterior. The two-component nature of the nanoparticle scaffold enabled the production of highly ordered, monodisperse immunogens that display DS-Cav1 at controllable density. In mice and nonhuman primates, the full-valency nanoparticle immunogen displaying 20 DS-Cav1 trimers induced neutralizing antibody responses ∼10-fold higher than trimeric DS-Cav1. These results motivate continued development of this promising nanoparticle RSV vaccine candidate and establish computationally designed two-component nanoparticles as a robust and customizable platform for structure-based vaccine design.

Single-Molecule Analysis of a Viral Fusion Protein Illuminates a Fusion-Active Intermediate State.

The influenza virus hemagglutinin (HA) fusion glycoprotein mediates viral entry into host cells through its receptor binding and membrane fusion activities. In this issue of Cell, Das et al. use single-molecule Förster resonance energy transfer (smFRET) to monitor HA conformational dynamics. Their study reveals this prototypical class I fusion protein to be a highly dynamic molecule capable of reversibly sampling multiple states, including on-pathway fusion intermediates between pre-fusion and post-fusion endpoints. These findings challenge long-held ideas for how HA functions and move the field closer to obtaining a mechanistic understanding of how class I fusion proteins mediate membrane fusion.

Vaccination with a structure-based stabilized version of malarial antigen Pfs48/45 elicits ultra-potent transmission-blocking antibody responses.

Malaria transmission-blocking vaccines (TBVs) aim to elicit human antibodies that inhibit sporogonic development of Plasmodium falciparum in mosquitoes, thereby preventing onward transmission. Pfs48/45 is a leading clinical TBV candidate antigen and is recognized by the most potent transmission-blocking monoclonal antibody (mAb) yet described; still, clinical development of Pfs48/45 antigens has been hindered, largely by its poor biochemical characteristics. Here, we used structure-based computational approaches to design Pfs48/45 antigens stabilized in the conformation recognized by the most potently inhibitory mAb, achieving >25°C higher thermostability compared with the wild-type protein. Antibodies elicited in mice immunized with these engineered antigens displayed on liposome-based or protein nanoparticle-based vaccine platforms exhibited 1-2 orders of magnitude superior transmission-reducing activity, compared with immunogens bearing the wild-type antigen, driven by improved antibody quality. Our data provide the founding principles for using molecular stabilization solely from antibody structure-function information to drive improved immune responses against a parasitic vaccine target.

HIV-1 Neutralizing Antibodies with Limited Hypermutation from an Infant.

HIV-1 broadly neutralizing antibodies (bnAbs) develop in a subset of infected adults and exhibit high levels of somatic hypermutation (SHM) due to years of affinity maturation. There is no precedent for eliciting highly mutated antibodies by vaccination, nor is it practical to wait years for a desired response. Infants develop broad responses early, which may suggest a more direct path to generating bnAbs. Here, we isolated ten neutralizing antibodies (nAbs) contributing to plasma breadth of an infant at ∼1 year post-infection, including one with cross-clade breadth. The nAbs bind to envelope trimer from the transmitted virus, suggesting that this interaction may have initiated development of the infant nAbs. The infant cross-clade bnAb targets the N332 supersite on envelope but, unlike adult bnAbs targeting this site, lacks indels and has low SHM. The identification of this infant bnAb illustrates that HIV-1-specific neutralization breadth can develop without prolonged affinity maturation and extensive SHM.

Immunogenicity of Stabilized HIV-1 Envelope Trimers with Reduced Exposure of Non-neutralizing Epitopes.

The envelope glycoprotein trimer mediates HIV-1 entry into cells. The trimer is flexible, fluctuating between closed and more open conformations and sometimes sampling the fully open, CD4-bound form. We hypothesized that conformational flexibility and transient exposure of non-neutralizing, immunodominant epitopes could hinder the induction of broadly neutralizing antibodies (bNAbs). We therefore modified soluble Env trimers to stabilize their closed, ground states. The trimer variants were indeed stabilized in the closed conformation, with a reduced ability to undergo receptor-induced conformational changes and a decreased exposure of non-neutralizing V3-directed antibody epitopes. In rabbits, the stabilized trimers induced similar autologous Tier-1B or Tier-2 NAb titers to those elicited by the corresponding wild-type trimers but lower levels of V3-directed Tier-1A NAbs. Stabilized, closed trimers might therefore be useful components of vaccines aimed at inducing bNAbs.

Visualizing intermediate stages of viral membrane fusion by cryo-electron tomography.

Protein-mediated membrane fusion is the dynamic process where specialized protein machinery undergoes dramatic conformational changes that drive two membrane bilayers together, leading to lipid mixing and opening of a fusion pore between previously separate membrane-bound compartments. Membrane fusion is an essential stage of enveloped virus entry that results in viral genome delivery into host cells. Recent studies applying cryo-electron microscopy techniques in a time-resolved fashion provide unprecedented glimpses into the interaction of viral fusion proteins and membranes, revealing fusion intermediate states from the initiation of fusion to release of the viral genome. In combination with complementary structural, biophysical, and computation modeling approaches, these advances are shedding new light on the mechanics and dynamics of protein-mediated membrane fusion.

Quadrivalent influenza nanoparticle vaccines induce broad protection.

Influenza vaccines that confer broad and durable protection against diverse viral strains would have a major effect on global health, as they would lessen the need for annual vaccine reformulation and immunization1. Here we show that computationally designed, two-component nanoparticle immunogens2 induce potently neutralizing and broadly protective antibody responses against a wide variety of influenza viruses. The nanoparticle immunogens contain 20 haemagglutinin glycoprotein trimers in an ordered array, and their assembly in vitro enables the precisely controlled co-display of multiple distinct haemagglutinin proteins in defined ratios. Nanoparticle immunogens that co-display the four haemagglutinins of licensed quadrivalent influenza vaccines elicited antibody responses in several animal models against vaccine-matched strains that were equivalent to or better than commercial quadrivalent influenza vaccines, and simultaneously induced broadly protective antibody responses to heterologous viruses by targeting the subdominant yet conserved haemagglutinin stem. The combination of potent receptor-blocking and cross-reactive stem-directed antibodies induced by the nanoparticle immunogens makes them attractive candidates for a supraseasonal influenza vaccine candidate with the potential to replace conventional seasonal vaccines3.

SARS-CoV-2-specific CD8+ T cells from people with long COVID establish and maintain effector phenotype and key TCR signatures over 2 years.

Long COVID occurs in a small but important minority of patients following COVID-19, reducing quality of life and contributing to healthcare burden. Although research into underlying mechanisms is evolving, immunity is understudied. SARS-CoV-2-specific T cell responses are of key importance for viral clearance and COVID-19 recovery. However, in long COVID, the establishment and persistence of SARS-CoV-2-specific T cells are far from clear, especially beyond 12 mo postinfection and postvaccination. We defined ex vivo antigen-specific B cell and T cell responses and their T cell receptors (TCR) repertoires across 2 y postinfection in people with long COVID. Using 13 SARS-CoV-2 peptide-HLA tetramers, spanning 11 HLA allotypes, as well as spike and nucleocapsid probes, we tracked SARS-CoV-2-specific CD8+ and CD4+ T cells and B-cells in individuals from their first SARS-CoV-2 infection through primary vaccination over 24 mo. The frequencies of ORF1a- and nucleocapsid-specific T cells and B cells remained stable over 24 mo. Spike-specific CD8+ and CD4+ T cells and B cells were boosted by SARS-CoV-2 vaccination, indicating immunization, in fully recovered and people with long COVID, altered the immunodominance hierarchy of SARS-CoV-2 T cell epitopes. Meanwhile, influenza-specific CD8+ T cells were stable across 24 mo, suggesting no bystander-activation. Compared to total T cell populations, SARS-CoV-2-specific T cells were enriched for central memory phenotype, although the proportion of central memory T cells decreased following acute illness. Importantly, TCR repertoire composition was maintained throughout long COVID, including postvaccination, to 2 y postinfection. Overall, we defined ex vivo SARS-CoV-2-specific B cells and T cells to understand primary and recall responses, providing key insights into antigen-specific responses in people with long COVID.

Identification of broad, potent antibodies to functionally constrained regions of SARS-CoV-2 spike following a breakthrough infection.

The antiviral benefit of antibodies can be compromised by viral escape especially for rapidly evolving viruses. Therefore, durable, effective antibodies must be both broad and potent to counter newly emerging, diverse strains. Discovery of such antibodies is critically important for SARS-CoV-2 as the global emergence of new variants of concern (VOC) has compromised the efficacy of therapeutic antibodies and vaccines. We describe a collection of broad and potent neutralizing monoclonal antibodies (mAbs) isolated from an individual who experienced a breakthrough infection with the Delta VOC. Four mAbs potently neutralize the Wuhan-Hu-1 vaccine strain, the Delta VOC, and also retain potency against the Omicron VOCs through BA.4/BA.5 in both pseudovirus-based and authentic virus assays. Three mAbs also retain potency to recently circulating VOCs XBB.1.5 and BQ.1.1 and one also potently neutralizes SARS-CoV-1. The potency of these mAbs was greater against Omicron VOCs than all but one of the mAbs that had been approved for therapeutic applications. The mAbs target distinct epitopes on the spike glycoprotein, three in the receptor-binding domain (RBD) and one in an invariant region downstream of the RBD in subdomain 1 (SD1). The escape pathways we defined at single amino acid resolution with deep mutational scanning show they target conserved, functionally constrained regions of the glycoprotein, suggesting escape could incur a fitness cost. Overall, these mAbs are unique in their breadth across VOCs, their epitope specificity, and include a highly potent mAb targeting a rare epitope outside of the RBD in SD1.

Human iN neuronal model of schizophrenia displays dysregulation of chromogranin B and related neuropeptide transmitter signatures.

Schizophrenia (SZ) is a serious mental illness and neuropsychiatric brain disorder with behavioral symptoms that include hallucinations, delusions, disorganized behavior, and cognitive impairment. Regulation of such behaviors requires utilization of neurotransmitters released to mediate cell-cell communication which are essential to brain functions in health and disease. We hypothesized that SZ may involve dysregulation of neurotransmitters secreted from neurons. To gain an understanding of human SZ, induced neurons (iNs) were derived from SZ patients and healthy control subjects to investigate peptide neurotransmitters, known as neuropeptides, which represent the major class of transmitters. The iNs were subjected to depolarization by high KCl in the culture medium and the secreted neuropeptides were identified and quantitated by nano-LC-MS/MS tandem mass spectrometry. Several neuropeptides were identified from schizophrenia patient-derived neurons, including chromogranin B (CHGB), neurotensin, and natriuretic peptide. Focusing on the main secreted CHGB neuropeptides, results revealed differences in SZ iNs compared to control iN neurons. Lower numbers of distinct CHGB peptides were found in the SZ secretion media compared to controls. Mapping of the peptides to the CHGB precursor revealed peptides unique to either SZ or control, and peptides common to both conditions. Also, the iNs secreted neuropeptides under both KCl and basal (no KCl) conditions. These findings are consistent with reports that chromogranin B levels are reduced in the cerebrospinal fluid and specific brain regions of SZ patients. These findings suggest that iNs derived from SZ patients can model the decreased CHGB neuropeptides observed in human SZ.

Structural dynamics reveal subtype-specific activation and inhibition of influenza virus hemagglutinin.

Influenza hemagglutinin (HA) is a prototypical class 1 viral entry glycoprotein, responsible for mediating receptor binding and membrane fusion. Structures of its prefusion and postfusion forms, embodying the beginning and endpoints of the fusion pathway, have been extensively characterized. Studies probing HA dynamics during fusion have begun to identify intermediate states along the pathway, enhancing our understanding of how HA becomes activated and traverses its conformational pathway to complete fusion. HA is also the most variable, rapidly evolving part of influenza virus, and it is not known whether mechanisms of its activation and fusion are conserved across divergent viral subtypes. Here, we apply hydrogen-deuterium exchange mass spectrometry to compare fusion activation in two subtypes of HA, H1 and H3. Our data reveal subtype-specific behavior in the regions of HA that undergo structural rearrangement during fusion, including the fusion peptide and HA1/HA2 interface. In the presence of an antibody that inhibits the conformational change (FI6v3), we observe that acid-induced dynamic changes near the epitope are dampened, but the degree of protection at the fusion peptide is different for the two subtypes investigated. These results thus provide new insights into variation in the mechanisms of influenza HA's dynamic activation and its inhibition.

Cytoplasmic Tail Truncation Stabilizes S1-S2 Association and Enhances S Protein Incorporation into SARS-CoV-2 Pseudovirions.

Truncations of the cytoplasmic tail (CT) of entry proteins of enveloped viruses dramatically increase the infectivity of pseudoviruses (PVs) bearing these proteins. Several mechanisms have been proposed to explain this enhanced entry, including an increase in cell surface expression. However, alternative explanations have also been forwarded, and the underlying mechanisms for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) S protein remain undetermined. Here, we show that the partial or complete deletion of the CT (residues 19 to 35) does not modify SARS-CoV-2 S protein expression on the cell surface when the S2 subunit is measured, whereas it is significantly increased when the S1 subunit is measured. We also show that the higher level of S1 in these CT-truncated S proteins reflects the decreased dissociation of the S1 subunit from the S2 subunit. In addition, we demonstrate that CT truncation further promotes S protein incorporation into PV particles, as indicated by biochemical analyses and cryo-electron microscopy. Thus, our data show that two distinct mechanisms contribute to the markedly increased infectivity of PVs carrying CT-truncated SARS-CoV-2 S proteins and help clarify the interpretation of the results of studies employing such PVs. IMPORTANCE Various forms of PVs have been used as tools to evaluate vaccine efficacy and study virus entry steps. When PV infectivity is inherently low, such as that of SARS-CoV-2, a CT-truncated version of the viral entry glycoprotein is widely used to enhance PV infectivity, but the mechanism underlying this enhanced PV infectivity has been unclear. Here, our study identified two mechanisms by which the CT truncation of the SARS-CoV-2 S protein dramatically increases PV infectivity: a reduction of S1 shedding and an increase in S protein incorporation into PV particles. An understanding of these mechanisms can clarify the mechanistic bases for the differences observed among various assays employing such PVs.

Pretreatment characteristics associated with symptom reduction during group cognitive processing therapy versus exposure therapy for PTSD: an exploratory study of Veterans.

Exposure and cognitive-based therapies are both effective for PTSD, but knowledge of which intervention is best for which patient is lacking. This lack of knowledge is particularly noticeable for group treatments, as no study has examined whether responses to different group therapies are associated with different pretreatment characteristics. Here, we explored whether pretreatment levels of three types of psychological characteristics-PTSD symptom clusters, posttraumatic cognitions, and emotion regulation difficulties-were associated with symptom reduction during group-delivered cognitive versus exposure-based PTSD treatment. Participants were Veterans with PTSD drawn from two previous clinical trials: one of group CPT (GCPT; n = 32) and the other of group-based exposure therapy (GBET; n = 21). Growth curve modeling was used to identify pretreatment variables that predicted weekly PTSD symptom changes during each therapy. Higher posttraumatic cognitions at pretreatment predicted steeper PTSD symptom reduction during GCPT but not GBET. Additionally, symptom reduction during each therapy was associated with different pretreatment emotion regulation difficulties: difficulties with goal-directed behavior for GBET and lack of emotional clarity and limited access to emotion regulation strategies for GCPT. These findings suggest that assigning Veterans to a group PTSD therapy that better matches their pretreatment psychological profile might facilitate a better therapeutic response.

Cholinergic depolarization recruits a persistent Ca2+ current in Aplysia bag cell neurons.

Many behaviors and types of information storage are mediated by lengthy changes in neuronal activity. In bag cell neurons of the hermaphroditic sea snail Aplysia californica, a transient cholinergic synaptic input triggers an ∼30-min afterdischarge. This causes these neuroendocrine cells to release egg laying hormone and elicit reproductive behavior. When acetylcholine is pressure-ejected onto a current-clamped bag cell neuron, the evoked depolarization is far longer than the current evoked by acetylcholine under voltage clamp, suggesting recruitment of another conductance. Our earlier studies found bag cell neurons to display a voltage-dependent persistent Ca2+ current. Hence, we hypothesized that this current is activated by the acetylcholine-induced depolarization and sought a selective Ca2+ current blocker. Rapid Ca2+ current evoked by 200-ms depolarizing steps in voltage-clamped cultured bag cell neurons demonstrated a concentration-dependent sensitivity to Ni2+, Co2+, Zn2+, and verapamil but not Cd2+ or ω-conotoxin GIVa. Leak subtraction of Ca2+ current evoked by 10-s depolarizing steps using the IC100 (concentration required to eliminate maximal current) of Ni2+, Co2+, Zn2+, or verapamil revealed persistent Ca2+ current, demonstrating persistent current block. Only Co2+ and Zn2+ did not suppress the acetylcholine-induced current, although Zn2+ appeared to impact additional channels. When Co2+ was applied during an acetylcholine-induced depolarization, the amplitude was reduced; furthermore, protein kinase C activation, previously established to enhance the persistent Ca2+ current, extended the depolarization. Therefore, the persistent Ca2+ current sustains the acetylcholine-induced depolarization and may translate brief cholinergic input into afterdischarge initiation. This could be a general mechanism of triggering long-term change in activity with a short-lived input.NEW & NOTEWORTHY Ionotropic acetylcholine receptors mediate brief synaptic communication, including in bag cell neurons of the sea snail Aplysia. However, this study demonstrates that cholinergic depolarization can open a voltage-gated persistent Ca2+ current, which extends the bag cell neuron response to acetylcholine. Bursting in these neuroendocrine cells results in hormone release and egg laying. Thus, this emphasizes the role of ionotropic signaling in reaching a depolarized level to engage Ca2+ influx and perpetuating the activity necessary for behavior.

Use-dependent facilitation of electrical transmission involves changes to postsynaptic K+ current.

Activity-dependent modulation of electrical transmission typically involves Ca2+ influx acting directly on gap junctions or initiating Ca2+-dependent pathways that in turn modulate coupling. We now describe short-term use-dependent facilitation of electrical transmission between bag cell neurons from the hermaphroditic snail, Aplysia californica, that is instead mediated by changes in postsynaptic responsiveness. Bag cell neurons secrete reproductive hormone during a synchronous afterdischarge of action potentials coordinated by electrical coupling. Here, recordings from pairs of coupled bag cell neurons in culture showed that nonjunctional currents influence electrical transmission in a dynamic manner. Under a dual whole cell voltage-clamp, the junctional current was linear and largely voltage-independent, while in current-clamp, the coupling coefficient was similar regardless of the extent of presynaptic hyperpolarization. Moreover, a train stimulus of action potential-like waveforms, in a voltage-clamped presynaptic neuron, elicited electrotonic potentials, in a current-clamped postsynaptic neuron, that facilitated over time when delivered at a frequency approximating the afterdischarge. Junctional current remained constant over the train stimulus, as did postsynaptic voltage-gated Ca2+ current. However, postsynaptic voltage-gated K+ current underwent cumulative inactivation, suggesting that K+ current run-down facilitates the electrotonic potential by boosting the response to successive junctional currents. Accordingly, preventing run-down by blocking postsynaptic K+ channels occluded facilitation. Finally, stimulation of bursts in coupled pairs resulted in synchronous firing, where active neurons could recruit silent partners through short-term use-dependent facilitation. Thus, potentiation of electrical transmission may promote synchrony in bag cell neurons and, by extension, reproductive function.NEW & NOTEWORTHY The understanding of how activity can facilitate electrical transmission is incomplete. We found that electrotonic potentials between electrically coupled neuroendocrine bag cell neurons facilitated in a use-dependent fashion. Rather than changes to the junctional current, facilitation was associated with cumulative inactivation of postsynaptic K+ current, presumably augmenting responsiveness. When made to burst, neurons synchronized their spiking, in part by use-dependent facilitation bringing quiescent cells to the threshold. Facilitation may foster en masse firing and neurosecretion.

Social isolation produces a sex- and brain region-specific alteration of microglia state.

Social isolation is a profound form of psychological stress that impacts the mental health of a large proportion of society. Other experimental models of stress have demonstrated a microglia response that serves either a protective or pathological function. However, the effect of adult social isolation on microglia has not been thoroughly investigated. We measured microglia territory, branching, end points and phagocytic-lysosomal activity in group housed C57Bl/6 mice and mice that were socially isolated for 2 weeks. Our results show that the dorsomedial hypothalamus and hippocampal CA2 region of adult male mice undergo increased microglia volume, territory and endpoints following social isolation, whereas females exhibit this increase in the hypothalamus only. Males exhibited decreases in the phagocytic-lysosomal marker CD68 in microglia in these regions, whereas females showed an increase in CD68 in the hypothalamus suggesting sexually dimorphic and brain region-specific change in microglia state in response to social isolation. The prefrontal cortex, central amygdala, nucleus accumbens shell and visual cortex did not exhibit changes in microglia structure in either male or female mice. These data show that microglia in different brain regions undergo a distinct response to social isolation which may account for changes in cognition and behaviour associated with this prevalent form of psychological stress.

Structurally related but genetically unrelated antibody lineages converge on an immunodominant HIV-1 Env neutralizing determinant following trimer immunization.

Understanding the molecular mechanisms by which antibodies target and neutralize the HIV-1 envelope glycoprotein (Env) is critical in guiding immunogen design and vaccine development aimed at eliciting cross-reactive neutralizing antibodies (NAbs). Here, we analyzed monoclonal antibodies (mAbs) isolated from non-human primates (NHPs) immunized with variants of a native flexibly linked (NFL) HIV-1 Env stabilized trimer derived from the tier 2 clade C 16055 strain. The antibodies displayed neutralizing activity against the autologous virus with potencies ranging from 0.005 to 3.68 µg/ml (IC50). Structural characterization using negative-stain EM and X-ray crystallography identified the variable region 2 (V2) of the 16055 NFL trimer to be the common epitope for these antibodies. The crystal structures revealed that the V2 segment adopts a ß-hairpin motif identical to that observed in the 16055 NFL crystal structure. These results depict how vaccine-induced antibodies derived from different clonal lineages penetrate through the glycan shield to recognize a hypervariable region within V2 (residues 184-186) that is unique to the 16055 strain. They also provide potential explanations for the potent autologous neutralization of these antibodies, confirming the immunodominance of this site and revealing that multiple angles of approach are permissible for affinity/avidity that results in potent neutralizing capacity. The structural analysis reveals that the most negatively charged paratope correlated with the potency of the mAbs. The atomic level information is of interest to both define the means of autologous neutralization elicited by different tier 2-based immunogens and facilitate trimer redesign to better target more conserved regions of V2 to potentially elicit cross-neutralizing HIV-1 antibodies.