Generating sufficient antibody to block infection is a key challenge for vaccines against malaria. Here, we show that antibody titers to a key target, the repeat region of the Plasmodium falciparum ...circumsporozoite protein (PfCSP), plateaued after two immunizations in a clinical trial of the radiation-attenuated sporozoite vaccine. To understand the mechanisms limiting vaccine responsiveness, we developed immunoglobulin (Ig)-knockin mice with elevated numbers of PfCSP-binding B cells. We determined that recall responses were inhibited by antibody feedback, potentially via epitope masking of the immunodominant PfCSP repeat region. Importantly, the amount of antibody that prevents boosting is below the amount of antibody required for protection. Finally, while antibody feedback limited responses to the PfCSP repeat region in vaccinated volunteers, potentially protective subdominant responses to PfCSP C-terminal regions expanded with subsequent boosts. These data suggest that antibody feedback drives the diversification of immune responses and that vaccination for malaria will require targeting multiple antigens.
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•Human antibody and B cell responses to a malaria vaccine plateau after 2 immunizations•Anti-CSP Ig-knockin mice developed to probe regulatory mechanisms•Antibody feedback via likely epitope masking limits immunodominant B cell responses•Repeated boosting diversifies the immune response as subdominant responses expand
McNamara et al. show that B cell responses to the immunodominant repeat region of the Plasmodium circumsporozoite protein plateau rapidly in humans. However, additional boosts allow subdominant responses to expand. Using a knockin mouse model, they show that these vaccine responses are regulated by antibody feedback via potential epitope masking.
The diversity of circulating human B cells is unknown. We use single-cell RNA sequencing (RNA-seq) to examine the diversity of both antigen-specific and total B cells in healthy subjects and ...malaria-exposed individuals. This reveals two B cell lineages: a classical lineage of activated and resting memory B cells and an alternative lineage, which includes previously described atypical B cells. Although atypical B cells have previously been associated with disease states, the alternative lineage is common in healthy controls, as well as malaria-exposed individuals. We further track Plasmodium-specific B cells after malaria vaccination in naive volunteers. We find that alternative lineage cells are primed after the initial immunization and respond to booster doses. However, alternative lineage cells develop an atypical phenotype with repeated boosts. The data highlight that atypical cells are part of a wider alternative lineage of B cells that are a normal component of healthy immune responses.
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•Single-cell RNA-seq reveals two distinct B cell lineages•An alternative lineage contains CXCR3+ and atypical B cells•Alternative B cells are primed after primary vaccination and respond to boosters•Alternative B cells adopt a more atypical phenotype following repeated antigen exposure
Using single-cell RNA sequencing, Sutton et al. show that a population of “atypical” B cells, normally associated with disease, are part of a wider landscape of alternative B cells that participate in normal responses to vaccination.
The postendocytic recycling of signaling receptors is subject to multiple requirements. Why this is so, considering that many other proteins can recycle without apparent requirements, is a ...fundamental question. Here we show that cells can leverage these requirements to switch the recycling of the beta-2 adrenergic receptor (B2AR), a prototypic signaling receptor, between sequence-dependent and bulk recycling pathways, based on extracellular signals. This switch is determined by protein kinase A-mediated phosphorylation of B2AR on the cytoplasmic tail. The phosphorylation state of B2AR dictates its partitioning into spatially and functionally distinct endosomal microdomains mediating bulk and sequence-dependent recycling, and also regulates the rate of B2AR recycling and resensitization. Our results demonstrate that G protein-coupled receptor recycling is not always restricted to the sequence-dependent pathway, but may be reprogrammed as needed by physiological signals. Such flexible reprogramming might provide a versatile method for rapidly modulating cellular responses to extracellular signaling.
Precise and reliable cell-specific gene delivery remains technically challenging. Here we report a splicing-based approach for controlling gene expression whereby separate translational reading ...frames are coupled to the inclusion or exclusion of mutated, frameshifting cell-specific alternative exons. Candidate exons are identified by analyzing thousands of publicly available RNA sequencing datasets and filtering by cell specificity, conservation, and local intron length. This method, which we denote splicing-linked expression design (SLED), can be combined in a Boolean manner with existing techniques such as minipromoters and viral capsids. SLED can use strong constitutive promoters, without sacrificing precision, by decoupling the tradeoff between promoter strength and selectivity. AAV-packaged SLED vectors can selectively deliver fluorescent reporters and calcium indicators to various neuronal subtypes in vivo. We also demonstrate gene therapy utility by creating SLED vectors that can target PRPH2 and SF3B1 mutations. The flexibility of SLED technology enables creative avenues for basic and translational research.
Uterine cancers can be studied in mice due to the ease of handling and genetic manipulation in these models. However, these studies are often limited to assessing pathology post-mortem in animals ...euthanized at multiple time points in different cohorts, which increases the number of mice needed for a study. Imaging mice in longitudinal studies can track the progression of disease in individual animals, reducing the number of mice needed. Advances in ultrasound technology have allowed for the detection of micrometer-level changes in tissues. Ultrasound has been used to study follicle maturation in ovaries and xenograft growth but has not been applied to morphological changes in the mouse uterus. This protocol examines the juxtaposition of pathology with in vivo imaging comparisons in an induced endometrial cancer mouse model. The features observed by ultrasound were consistent with the degree of change seen by gross pathology and histology. Ultrasound was found to be highly predictive of the observed pathology, supporting the incorporation of ultrasonography into longitudinal studies of uterine diseases such as cancer in mice.
The functional consequences of signaling receptor endocytosis are determined by the endosomal sorting of receptors between degradation and recycling pathways. How receptors recycle efficiently, in a ...sequence-dependent manner that is distinct from bulk membrane recycling, is not known. Here, in live cells, we visualize the sorting of a prototypical sequence-dependent recycling receptor, the beta-2 adrenergic receptor, from bulk recycling proteins and the degrading delta-opioid receptor. Our results reveal a remarkable diversity in recycling routes at the level of individual endosomes, and indicate that sequence-dependent recycling is an active process mediated by distinct endosomal subdomains distinct from those mediating bulk recycling. We identify a specialized subset of tubular microdomains on endosomes, stabilized by a highly localized but dynamic actin machinery, that mediate this sorting, and provide evidence that these actin-stabilized domains provide the physical basis for a two-step kinetic and affinity-based model for protein sorting into the sequence-dependent recycling pathway.
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► B2AR is sorted into a specialized subset of tubules in the endosome and recycled ► A highly localized actin-based machinery stabilizes this subset of tubular domains ► This allows selective sequence-dependent entry of B2AR by regulated diffusion ► Actin binding via PDZ domains concentrates B2AR in these recycling domains
The common marmoset (Callithrix jacchus), a New World NHP, has emerged as important animal model in multiple areas of translational biomedical research. The quality of translational research in ...marmosets depends on early diagnosis, treatment, and prevention of their spontaneous
diseases. Here, we characterize an outbreak of infectious cholangiohepatitis that affected 7 adult common marmosets in a single building over a 10-mo period. Marmosets presented for acute onset of lethargy, dull mentation, weight loss, dehydration, hyporexia, and hypothermia. Blood chemistries
at presentation revealed markedly elevated hepatic and biliary enzymes, but mild neutrophilia was detected in only 1 of the 7. Affected marmosets were unresponsive to rigorous treatment and died or were euthanized within 48 h of presentation. Gross and histopathologic examinations revealed
severe, necrosuppurative cholangiohepatitis and proliferative cholecystitis with bacterial colonies and an absence of gallstones. Perimortem and postmortem cultures revealed single or dual isolates of Escherichia coli and Pseudomonas aeruginosa. Other postmortem findings included
bile duct hyperplasia, periportal hepatitis, bile peritonitis, ulcerative gastroenteritis, and typhlitis. Environmental contamination of water supply equipment with Pseudomonas spp. was identified as the source of infection, but pathogenesis remains unclear. This type of severe, infectious
cholangiohepatitis with proliferative cholecystitis with Pseudomonas spp. had not been reported previously in marmosets, and we identified and here describe several contributing factors in addition to contaminated drinking water.
Discovering potent human monoclonal antibodies (mAbs) targeting the Plasmodium falciparum circumsporozoite protein (PfCSP) on sporozoites (SPZ) and elucidating their mechanisms of neutralization will ...facilitate translation for passive prophylaxis and aid next-generation vaccine development. Here, we isolated a neutralizing human mAb, L9 that preferentially bound NVDP minor repeats of PfCSP with high affinity while cross-reacting with NANP major repeats. L9 was more potent than six published neutralizing human PfCSP mAbs at mediating protection against mosquito bite challenge in mice. Isothermal titration calorimetry and multiphoton microscopy showed that L9 and the other most protective mAbs bound PfCSP with two binding events and mediated protection by killing SPZ in the liver and by preventing their egress from sinusoids and traversal of hepatocytes. This study defines the subdominant PfCSP minor repeats as neutralizing epitopes, identifies an in vitro biophysical correlate of SPZ neutralization, and demonstrates that the liver is an important site for antibodies to prevent malaria.
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•The subdominant NVDP minor repeats of PfCSP are neutralizing epitopes•Potent mAbs with junctional and NANP cross-reactivity bind PfCSP in two steps•Cytotoxic PfCSP mAbs kill sporozoites in the liver•PfCSP mAbs prevent sporozoites from exiting liver sinusoids to infect hepatocytes
Wang et al. isolate a potent neutralizing human mAb, L9 that preferentially binds NVDP minor repeats of Plasmodium falciparum circumsporozoite protein (PfCSP) on sporozoites with high affinity while cross-reacting with NANP major repeats. Their findings define the subdominant PfCSP minor repeats as neutralizing epitopes and demonstrate that the liver is an important site for antibodies to prevent malaria.
The common marmoset ( Callithrix jacchus ), a New World NHP, has emerged as important animal model in multiple areas of translational biomedical research. The quality of translational research in ...marmosets depends on early diagnosis, treatment, and prevention of their spontaneous diseases. Here, we characterize an outbreak of infectious cholangiohepatitis that affected 7 adult common marmosets in a single building over a 10-mo period. Marmosets presented for acute onset of lethargy, dull mentation, weight loss, dehydration, hyporexia, and hypothermia. Blood chemistries at presentation revealed markedly elevated hepatic and biliary enzymes, but mild neutrophilia was detected in only 1 of the 7. Affected marmosets were unresponsive to rigorous treatment and died or were euthanized within 48 h of presentation. Gross and histopathologic examinations revealed severe, necrosuppurative cholangiohepatitis and proliferative cholecystitis with bacterial colonies and an absence of gallstones. Perimortem and postmortem cultures revealed single or dual isolates of Escherichia coli and Pseudomonas aeruginosa . Other postmortem findings included bile duct hyperplasia, periportal hepatitis, bile peritonitis, ulcerative gastroenteritis, and typhlitis. Environmental contamination of water supply equipment with Pseudomonas spp. was identified as the source of infection, but pathogenesis remains unclear. This type of severe, infectious cholangiohepatitis with proliferative cholecystitis with Pseudomonas spp. had not been reported previously in marmosets, and we identified and here describe several contributing factors in addition to contaminated drinking water.
The recycling of internalized signaling receptors, which has direct functional consequences, is subject to multiple sequence and biochemical requirements. Why signaling receptors recycle via a ...specialized pathway, unlike many other proteins that recycle by bulk, is a fundamental unanswered question. Here, we show that these specialized pathways allow selective control of signaling receptor recycling by heterologous signaling. Using assays to visualize receptor recycling in living cells, we show that the recycling of the beta‐2 adrenergic receptor (B2AR), a prototypic signaling receptor, is regulated by Src family kinases. The target of Src is cortactin, an essential factor for B2AR sorting into specialized recycling microdomains on the endosome. Phosphorylation of a single cortactin residue, Y466, regulates the rate of fission of B2AR recycling vesicles from these microdomains and, therefore, the rate of delivery of B2AR to the cell surface. Together, our results indicate that actin‐stabilized microdomains that mediate signaling receptor recycling can serve as a functional point of convergence for crosstalk between signaling pathways.
Why are signaling receptors recycled via a specialized ‘sequence‐dependent’ pathway, when many proteins can recycle by ‘bulk’ without apparent requirements? This study shows that sequence‐dependent recycling provides control points for heterologous signaling pathways to regulate receptor recycling and resensitization. Specifically, we show that Src kinase regulates G protein‐coupled receptor recycling without affecting bulk recycling, by phosphorylating cortactin and controlling the dynamics of specialized sequence‐dependent endosomal microdomains. Such a mechanism might be generally applicable for signaling crosstalk.
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