Development of strategies for induction of HIV-1 broadly neutralizing antibodies (bnAbs) by vaccines is a priority. Determining the steps of bnAb induction in HIV-1-infected individuals who make ...bnAbs is a key strategy for immunogen design. Here, we study the B cell response in a bnAb-producing individual and report cooperation between two B cell lineages to drive bnAb development. We isolated a virus-neutralizing antibody lineage that targeted an envelope region (loop D) and selected virus escape mutants that resulted in both enhanced bnAb lineage envelope binding and escape mutant neutralization—traits associated with increased B cell antigen drive. Thus, in this individual, two B cell lineages cooperated to induce the development of bnAbs. Design of vaccine immunogens that simultaneously drive both helper and broadly neutralizing B cell lineages may be important for vaccine-induced recapitulation of events that transpire during the maturation of neutralizing antibodies in HIV-1-infected individuals.
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•The steps in induction of HIV-1 broadly neutralizing antibodies have been mapped•The steps involve cooperation between two distinct B cell antibody lineages•One lineage induced escape mutants with enhanced binding to bnAb precursors
The molecular identification of two B cell lineages that cooperate for the generation of broadly neutralizing antibodies offers new insights into the design of antigens that can be used to elicit protective immunity to HIV infection.
Head motion estimates in functional magnetic resonance imaging (fMRI) scans appear qualitatively different with sub-second image sampling rates compared to the multi-second sampling rates common in ...the past. Whereas formerly the head appeared still for much of a scan with brief excursions from baseline, the head now appears to be in constant motion, and motion estimates often seem to divulge little information about what is happening in a scan. This constant motion has been attributed to respiratory oscillations that do not alias at faster sampling rates, and investigators are divided on the extent to which such motion is “real” motion or only “apparent” pseudomotion. Some investigators have abandoned the use of motion estimates entirely due to these considerations. Here we investigate the properties of motion in several fMRI datasets sampled at rates between 720 and 1160 ms, and describe 5 distinct kinds of respiratory motion: 1) constant real respiratory motion in the form of head nodding most evident in vertical position and pitch, which can be very large; 2) constant pseudomotion at the same respiratory rate as real motion, occurring only in the phase encode direction; 3) punctate real motions occurring at times of very deep breaths; 4) a low-frequency pseudomotion in only the phase encode direction at and after very deep breaths; 5) slow modulation of vertical and anterior-posterior head position by the respiratory envelope. We reformulate motion estimates in light of these considerations and obtain good concordance between motion estimates, physiologic records, image quality measures, and events evident in the fMRI signals. We demonstrate how variables describing respiration or body habitus separately scale with distinct kinds of head motion. We also note heritable aspects of respiration and motion.
•Examines several fast-TR datasets with sampling rates of 720–1160 ms•Identifies 7 kinds of motion in fMRI scans, 5 of them related to respiration.•Identifies 2 forms of pseudomotion occurring only in the phase encode direction.•Pseudomotion is a function of soft tissue mass, not lung volume.•Reformulates motion estimates to draw out particular kinds of motion.
Passive immunization with HIV-1-neutralizing monoclonal antibodies (mAbs) is being considered for prevention and treatment of HIV-1 infection. As therapeutic agents, mAbs could be used to suppress ...active virus replication, maintain suppression induced by antiretroviral therapy (ART), and/or decrease the size of the persistent virus reservoir. We assessed the impact of VRC01, a potent human mAb targeting the HIV-1 CD4 binding site, on ART-treated and untreated HIV-1-infected subjects. Among six ART-treated individuals with undetectable plasma viremia, two infusions of VRC01 did not reduce the peripheral blood cell-associated virus reservoir measured 4 weeks after the second infusion. In contrast, six of eight ART-untreated, viremic subjects infused with a single dose of VRC01 experienced a 1.1 to 1.8 log10 reduction in plasma viremia. The two subjects with minimal responses to VRC01 were found to have predominantly VRC01-resistant virus before treatment. Notably, two subjects with plasma virus load <1000 copies/ml demonstrated virus suppression to undetectable levels for over 20 days until VRC01 levels declined. Among the remaining four subjects with baseline virus loads between 3000 and 30,000 copies, viremia was only partially suppressed by mAb infusion, and we observed strong selection pressure for the outgrowth of less neutralization-sensitive viruses. In summary, a single infusion of mAb VRC01 significantly decreased plasma viremia and preferentially suppressed neutralization-sensitive virus strains. These data demonstrate the virological effect of this neutralizing antibody and highlight the need for combination strategies to maintain virus suppression.
HIV-1-neutralizing antibodies develop in most HIV-1-infected individuals, although highly effective antibodies are generally observed only after years of chronic infection. Here, we characterize the ...rate of maturation and extent of diversity for the lineage that produced the broadly neutralizing antibody VRC01 through longitudinal sampling of peripheral B cell transcripts over 15 years and co-crystal structures of lineage members. Next-generation sequencing identified VRC01-lineage transcripts, which encompassed diverse antibodies organized into distinct phylogenetic clades. Prevalent clades maintained characteristic features of antigen recognition, though each evolved binding loops and disulfides that formed distinct recognition surfaces. Over the course of the study period, VRC01-lineage clades showed continuous evolution, with rates of ∼2 substitutions per 100 nucleotides per year, comparable to that of HIV-1 evolution. This high rate of antibody evolution provides a mechanism by which antibody lineages can achieve extraordinary diversity and, over years of chronic infection, develop effective HIV-1 neutralization.
The site on the HIV-1 gp120 glycoprotein that binds the CD4 receptor is recognized by broadly reactive antibodies, several of which neutralize over 90% of HIV-1 strains. To understand how antibodies ...achieve such neutralization, we isolated CD4-binding-site (CD4bs) antibodies and analyzed 16 co-crystal structures –8 determined here– of CD4bs antibodies from 14 donors. The 16 antibodies segregated by recognition mode and developmental ontogeny into two types: CDR H3-dominated and VH-gene-restricted. Both could achieve greater than 80% neutralization breadth, and both could develop in the same donor. Although paratope chemistries differed, all 16 gp120-CD4bs antibody complexes showed geometric similarity, with antibody-neutralization breadth correlating with antibody-angle of approach relative to the most effective antibody of each type. The repertoire for effective recognition of the CD4 supersite thus comprises antibodies with distinct paratopes arrayed about two optimal geometric orientations, one achieved by CDR H3 ontogenies and the other achieved by VH-gene-restricted ontogenies.
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•Population-level analysis revealed only two types of effective CD4bs antibodies•Each type, CDR H3-dominated or VH-gene-restricted, had distinct ontogenies•Both types could neutralize effectively, each with an optimal angle of approach•Despite geometric similarities, paratope chemistries were extremely diverse
A population-level analysis of antibodies that recognize the CD4-binding site on the HIV-1 gp120 glycoprotein, the target of some of the most potent and broadly neutralizing antibodies, defines structural geometries, binding chemistries, and developmental pathways of antibody recognition, providing a catalog from which to choose optimal templates for vaccine design.
Breathing rate and depth influence the concentration of carbon dioxide in the blood, altering cerebral blood flow and thus functional magnetic resonance imaging (fMRI) signals. Such respiratory ...fluctuations can have substantial influence in studies of fMRI signal covariance in subjects at rest, the so-called “resting state functional connectivity” technique. If respiration is monitored during fMRI scanning, it is typically done using a belt about the subject’s abdomen to record abdominal circumference. Several measures have been derived from these belt records, including the windowed envelope of the waveform (ENV), the windowed variance in the waveform (respiration variation, RV), and a measure of the amplitude of each breath divided by the cycle time of the breath (respiration volume per time, RVT). Any attempt to gauge respiratory contributions to fMRI signals requires a respiratory measure, but little is known about how these measures compare to each other, or how they perform beyond the small studies in which they were initially proposed. Here, we examine the properties of these measures in hundreds of healthy young adults scanned for an hour each at rest, a subset of the Human Connectome Project chosen for having high-quality physiological records. We find: 1) ENV, RV, and RVT are all correlated, and ENV and RV are more highly correlated to each other than to RVT; 2) respiratory events like deep breaths exhibit characteristic heart rate elevations, fMRI signal changes, head motions, and image quality abnormalities time-locked to large deflections in the belt traces; 3) all measures can “miss” deep breaths; 4) RVT “misses” deep breaths more than ENV or RV; 5) all respiratory measures change systematically over the course of a 14.4-min scan. We discuss the implications of these findings for the literature and ways to move forward in modeling respiratory influences on fMRI scans.
•Data are resting state fMRI scans in healthy young adults.•Examines respiratory belt records and derived measures ENV, RV, and RVT.•All respiratory measures “miss” deep breaths, RVT more than others.•Deep breaths exhibit characteristic heart rate and fMRI signal changes.•All respiratory measures change systematically over 14.4 min scans.
The Intact Proviral DNA Assay (IPDA) was developed to address the critical need for a scalable method for intact HIV-1 reservoir quantification. This droplet digital PCR-based assay simultaneously ...targets two HIV-1 regions to distinguish genomically intact proviruses against a large background of defective ones, and its application has yielded insights into HIV-1 persistence. Reports of assay failures however, attributed to HIV-1 polymorphism, have recently emerged. Here, we describe a diverse North American cohort of people with HIV-1 subtype B, where the IPDA yielded a failure rate of 28% due to viral polymorphism. We further demonstrate that within-host HIV-1 diversity can lead the IPDA to underestimate intact reservoir size, and provide examples of how this phenomenon could lead to erroneous interpretation of clinical trial data. While the IPDA represents a major methodological advance, HIV-1 diversity should be addressed before its widespread adoption as a principal readout in HIV-1 remission trials.
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•The longer HIV-1 actively replicates before ART, the more complex the reservoir.•Questions remain about the efficacy of bNAb therapy in tissues.•Phenotype but not genotype of ...provirus can predict virus rebound during ATI.•In vitro potency is a relative metric that affects in vivo efficacy.•Multiple strategies exist to overcome the genetic diversity of the HIV-1 reservoir.
Genetic diversity in the latent proviral reservoir of HIV-1 infected individuals poses a challenge to cure strategies. It has become increasingly evident that diversity increases proportionally with length of active infection, and that functional and/or sterilizing cure strategies will need to overcome this obstacle in individuals who initiated antiretroviral therapy (ART) during chronic infection. Analyzing the results of analytic treatment interruption (ATI) has allowed for the evaluation of such therapeutic strategies in HIV+ individuals. Strategies to overcome the genetic diversity of the HIV-1 reservoir include antibody combinations, pre-screening individuals for bNAb sensitivity, focusing on low-diversity individuals as well as targeting host proteins.
Broadly neutralizing antibodies (bNAbs) have been isolated from selected HIV-1-infected individuals and shown to bind to conserved sites on the envelope glycoprotein (Env). However, circulating ...plasma virus in these donors is usually resistant to autologous isolated bNAbs, indicating that during chronic infection, HIV-1 can escape from even broadly cross-reactive antibodies. Here, we evaluate if such viral escape is associated with an impairment of viral replication. Antibodies of the VRC01 class target the functionally conserved CD4 binding site and share a structural mode of gp120 recognition that includes mimicry of the CD4 receptor. We examined naturally occurring VRC01-sensitive and -resistant viral strains, as well as their mutated sensitive or resistant variants, and tested point mutations in the backbone of the VRC01-sensitive isolate YU2. In several cases, VRC01 resistance was associated with a reduced efficiency of CD4-mediated viral entry and diminished viral replication. Several mutations, alone or in combination, in the loop D or β23-V5 region of Env conferred a high level of resistance to VRC01 class antibodies, suggesting a preferred escape pathway. We further mapped the VRC01-induced escape pathway in vivo using Envs from donor 45, from whom antibody VRC01 was isolated. Initial escape mutations, including the addition of a key glycan, occurred in loop D and were associated with impaired viral replication; however, compensatory mutations restored full replicative fitness. These data demonstrate that escape from VRC01 class antibodies can diminish viral replicative fitness, but compensatory changes may explain the limited impact of neutralizing antibodies during the course of natural HIV-1 infection.
Some antibodies that arise during natural HIV-1 infection bind to conserved regions on the virus envelope glycoprotein and potently neutralize the majority of diverse HIV-1 strains. The VRC01 class of antibodies blocks the conserved CD4 receptor binding site interaction that is necessary for viral entry, raising the possibility that viral escape from antibody neutralization might exert detrimental effects on viral function. Here, we show that escape from VRC01 class antibodies can be associated with impaired viral entry and replication; however, during the course of natural infection, compensatory mutations restore the ability of the virus to replicate normally.
Resting state functional connectivity magnetic resonance imaging (fMRI) is a tool for investigating human brain organization. Here we identify, visually and algorithmically, two prevalent influences ...on fMRI signals during 440 h of resting state scans in 440 healthy young adults, both caused by deviations from normal breathing which we term deep breaths and bursts. The two respiratory patterns have distinct influences on fMRI signals and signal covariance, distinct timescales, distinct cardiovascular correlates, and distinct tendencies to manifest by sex. Deep breaths are not sex-biased. Bursts, which are serial taperings of respiratory depth typically spanning minutes at a time, are more common in males. Bursts share features of chemoreflex-driven clinical breathing patterns that also occur primarily in males, with notable neurological, psychiatric, medical, and lifespan associations. These results identify common breathing patterns in healthy young adults with distinct influences on functional connectivity and an ability to differentially influence resting state fMRI studies.
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