Activation Dynamics and Immunoglobulin Evolution of Pre-existing and Newly Generated Human Memory B cell Responses to Influenza Hemagglutinin.

Vaccine-induced memory B cell responses to evolving viruses like influenza A involve activation of pre-existing immunity and generation of new responses. To define the contribution of these two types of responses, we analyzed the response to H7N9 vaccination in H7N9-naive adults. We performed comprehensive comparisons at the single-cell level of the kinetics, Ig repertoire, and activation phenotype of established pre-existing memory B cells recognizing conserved epitopes and the newly generated memory B cells directed toward H7 strain-specific epitopes. The recall response to conserved epitopes on H7 HA involved a transient expansion of memory B cells with little observed adaptation. However, the B cell response to newly encountered epitopes was phenotypically distinct and generated a sustained memory population that evolved and affinity matured months after vaccination. These findings establish clear differences between newly generated and pre-existing memory B cells, highlighting the challenges in achieving long-lasting, broad protection against an ever-evolving virus.

Highly complete long-read genomes reveal pangenomic variation underlying yeast phenotypic diversity.

Understanding the genetic causes of trait variation is a primary goal of genetic research. One way that individuals can vary genetically is through variable pangenomic genes: genes that are only present in some individuals in a population. The presence or absence of entire genes could have large effects on trait variation. However, variable pangenomic genes can be missed in standard genotyping workflows, owing to reliance on aligning short-read sequencing to reference genomes. A popular method for studying the genetic basis of trait variation is linkage mapping, which identifies quantitative trait loci (QTLs), regions of the genome that harbor causative genetic variants. Large-scale linkage mapping in the budding yeast Saccharomyces cerevisiae has found thousands of QTLs affecting myriad yeast phenotypes. To enable the resolution of QTLs caused by variable pangenomic genes, we used long-read sequencing to generate highly complete de novo genome assemblies of 16 diverse yeast isolates. With these assemblies, we resolved QTLs for growth on maltose, sucrose, raffinose, and oxidative stress to specific genes that are absent from the reference genome but present in the broader yeast population at appreciable frequency. Copies of genes also duplicate onto chromosomes where they are absent in the reference genome, and we found that these copies generate additional QTLs whose resolution requires pangenome characterization. Our findings show the need for highly complete genome assemblies to identify the genetic basis of trait variation.

Systematic genetic characterization of the human PKR kinase domain highlights its functional malleability to escape a poxvirus substrate mimic.

Evolutionary arms races can arise at the contact surfaces between host and viral proteins, producing dynamic spaces in which genetic variants are continually pursued. However, the sampling of genetic variation must be balanced with the need to maintain protein function. A striking case is given by protein kinase R (PKR), a member of the mammalian innate immune system. PKR detects viral replication within the host cell and halts protein synthesis to prevent viral replication by phosphorylating eIF2α, a component of the translation initiation machinery. PKR is targeted by many viral antagonists, including poxvirus pseudosubstrate antagonists that mimic the natural substrate, eIF2α, and inhibit PKR activity. Remarkably, PKR has several rapidly evolving residues at this interface, suggesting it is engaging in an evolutionary arms race, despite the surface's critical role in phosphorylating eIF2α. To systematically explore the evolutionary opportunities available at this dynamic interface, we generated and characterized a library of 426 SNP-accessible nonsynonymous variants of human PKR for their ability to escape inhibition by the model pseudosubstrate inhibitor K3, encoded by the vaccinia virus gene K3L. We identified key sites in the PKR kinase domain that harbor K3-resistant variants, as well as critical sites where variation leads to loss of function. We find K3-resistant variants are readily available throughout the interface and are enriched at sites under positive selection. Moreover, variants beneficial against K3 were also beneficial against an enhanced variant of K3, indicating resilience to viral adaptation. Overall, we find that the eIF2α-binding surface of PKR is highly malleable, potentiating its evolutionary ability to combat viral inhibition.

Systematic characterization of the local evolutionary space available to human PKR and vaccinia virus K3.

The interfaces between host and viral proteins can be dynamic spaces in which genetic variants are continually pursued, giving rise to evolutionary arms races. One such scenario is found between the mammalian innate immunity protein PKR (protein kinase R) and the poxvirus antagonist K3. Once activated, PKR phosphorylates the natural substrate eIF2α, which halts protein synthesis within the cell and prevents viral replication. K3 acts as a pseudosubstrate antagonist against PKR by directly antagonizing this halt in protein synthesis, enabling poxviruses to replicate in the cell. Exploring the impact of genetic variants in both PKR and K3 is necessary not only to highlight residues of evolutionary constraint and opportunity but also to elucidate the mechanism by which human PKR is able to subvert a rapidly evolving viral antagonist. To systematically explore this dynamic interface, we have generated a combinatorial library of PKR and K3 missense variants to be co-expressed and characterized in a high-throughput yeast selection assay. This assay allows us to characterize hundreds of thousands of unique PKR-K3 genetic combinations in a single pooled experiment. Our results highlight specific missense variants available to PKR that subvert the K3 antagonist. We find that improved PKR variants are readily available at sites under positive selection, with limited opportunity at sites interfacing with K3 and eIF2α. Additionally, we find many variants that improve and disable K3 antagonism, suggesting a pliable interface. We reason that this approach can be leveraged to explore the evolutionary plasticity of many other host-virus interfaces.

Plasmodium falciparum-specific IgM B cells dominate in children, expand with malaria, and produce functional IgM.

IgG antibodies play a role in malaria immunity, but whether and how IgM protects from malaria and the biology of Plasmodium falciparum (Pf)-specific IgM B cells is unclear. In a Mali cohort spanning infants to adults, we conducted longitudinal analyses of Pf- and influenza-specific B cells. We found that Pf-specific memory B cells (MBCs) are disproportionally IgM+ and only gradually shift to IgG+ with age, in contrast to influenza-specific MBCs that are predominantly IgG+ from infancy to adulthood. B cell receptor analysis showed Pf-specific IgM MBCs are somatically hypermutated at levels comparable to influenza-specific IgG B cells. During acute malaria, Pf-specific IgM B cells expand and upregulate activation/costimulatory markers. Finally, plasma IgM was comparable to IgG in inhibiting Pf growth and enhancing phagocytosis of Pf by monocytes in vitro. Thus, somatically hypermutated Pf-specific IgM MBCs dominate in children, expand and activate during malaria, and produce IgM that inhibits Pf through neutralization and opsonic phagocytosis.

Convergent Evolution in Breadth of Two VH6-1-Encoded Influenza Antibody Clonotypes from a Single Donor.

Understanding how broadly neutralizing antibodies (bnAbs) to influenza hemagglutinin (HA) naturally develop in humans is critical to the design of universal influenza vaccines. Several classes of bnAbs directed to the conserved HA stem were found in multiple individuals, including one encoded by heavy-chain variable domain VH6-1. We describe two genetically similar VH6-1 bnAb clonotypes from the same individual that exhibit different developmental paths toward broad neutralization activity. One clonotype evolved from a germline precursor recognizing influenza group 1 subtypes to gain breadth to group 2 subtypes. The other clonotype recognized group 2 subtypes and developed binding to group 1 subtypes through somatic hypermutation. Crystal structures reveal that the specificity differences are primarily mediated by complementarity-determining region H3 (CDR H3). Thus, while VH6-1 provides a framework for development of HA stem-directed bnAbs, sequence differences in CDR H3 junctional regions during VDJ recombination can alter reactivity and evolutionary pathways toward increased breadth.

High temperature effects on water loss and survival examining the hardiness of female adults of the spider beetles, Mezium affine and Gibbium aequinoctiale.

A remarkable ability to tolerate temperatures as high as 52 degrees C for Mezium affine Boieldieu and 56 degrees C for Gibbium aequinoctiale Boieldieu (Coleoptera: Anobiidae) was discovered as part of a water balance study that was conducted to determine whether desiccation-resistance (xerophilic water balance classification) is linked to survival at high temperature. Characteristics of the heat shock response were an intermediate, reversible level of injury, appearing as though dead; greater recovery from heat shock by G. aequinoctiale (57%) than M. affine (30%) that supplemented higher temperature survival by G. aequinoctiale; and lack of protection generated by conditioning at sublethal temperature. Heatinduced mortality is attributed to an abrupt, accelerated water loss at 50 degrees C for M. affine and 54 degrees C for G. aequinoctiale, not to the species (M. affine) that loses water the slowest and has the lower activation energy, E(a) as a measure of cuticular boundary effectiveness. These temperatures where water loss increases sharply are not critical transition temperatures because Arrhenius analysis causes them to be erased (uninterrupted Boltzmann function) and E(a) fails to change when cuticular lipid from these beetles is removed. Our conclusion is that the temperature thresholds for survival and accelerated water loss closely match, and the key survival element in hot and dry environments contributing to wide distribution of G. aequinoctiale and M. affine derives from rising temperature prompting entry into quiescence and a resistance in cuticular lipid fluidity.

Preferential induction of cross-group influenza A hemagglutinin stem-specific memory B cells after H7N9 immunization in humans.

Antigenic drift and shift of influenza strains underscore the need for broadly protective influenza vaccines. One strategy is to design immunogens that elicit B cell responses against conserved epitopes on the hemagglutinin (HA) stem. To better understand the elicitation of HA stem-targeted B cells to group 1 and group 2 influenza subtypes, we compared the memory B cell response to group 2 H7N9 and group 1 H5N1 vaccines in humans. Upon H7N9 vaccination, almost half of the HA stem-specific response recognized the group 1 and group 2 subtypes, whereas the response to H5N1 was largely group 1-specific. Immunoglobulin repertoire analysis of HA-specific B cells indicated that the H7N9 and H5N1 vaccines induced genetically similar cross-group HA stem-binding B cells, albeit at a much higher frequency upon H7N9 vaccination. These data suggest that a group 2-based stem immunogen could prove more effective than a group 1 immunogen at eliciting broad cross-group protection in humans.

Reducing restraint use in a public psychiatric inpatient service.

The use of behavioral restraint in psychiatric inpatients can have physically and emotionally damaging effects. However, staff may view the use of restraint as a routine and acceptable means of maintaining safety. The goal of this project was to reduce the use of restraint in a public psychiatric inpatient service that serves an economically disadvantaged urban population. Six interventions that primarily involved changing staff behavior were made to reduce the use of restraint. These included better identification of restraint-prone patients, a stress/anger management group for patients, staff training on crisis intervention, development of a crisis response team, daily review of all restraints, and an incentive system for the staff. The rate of restraint use (number of restraints/1000 patient-days) during the 3 years before the interventions was compared with the rate during the 2 years after the interventions. There was a significant decrease in the rate of restraint use after the restraint reduction initiatives were implemented. The reduction was not accompanied by a sustained increase in incidents of assault, suicidal behavior, or self-injury.

First case report of SAM(r) Junctional tourniquet use in Afghanistan to control inguinal hemorrhage on the battlefield.

Junctional hemorrhage, bleeding that occurs at the junction of the trunk and its appendages, is the most common preventable cause of death from compressible hemorrhage on the battlefield. As of January 2014, four types of junctional tourniquets have been developed and cleared by the U.S. Food and Drug Administration (FDA). Successful use of the Abdominal Aortic Tourniquet (AAT™) and Combat Ready Clamp (CRoC™) has already been reported. We report here the first known prehospital use of the SAM® Junctional Tourniquet (SJT) for a battlefield casualty with inguinal junctional hemorrhage.