Structure-guided T cell vaccine design for SARS-CoV-2 variants and sarbecoviruses.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants that escape convalescent and vaccine-induced antibody responses has renewed focus on the development of broadly protective T-cell-based vaccines. Here, we apply structure-based network analysis and assessments of HLA class I peptide stability to define mutationally constrained CD8+ T cell epitopes across the SARS-CoV-2 proteome. Highly networked residues are conserved temporally among circulating variants and sarbecoviruses and disproportionately impair spike pseudotyped lentivirus infectivity when mutated. Evaluation of HLA class I stabilizing activity for 18 globally prevalent alleles identifies CD8+ T cell epitopes within highly networked regions with limited mutational frequencies in circulating SARS-CoV-2 variants and deep-sequenced primary isolates. Moreover, these epitopes elicit demonstrable CD8+ T cell reactivity in convalescent individuals but reduced recognition in recipients of mRNA-based vaccines. These data thereby elucidate key mutationally constrained regions and immunogenic epitopes in the SARS-CoV-2 proteome for a global T-cell-based vaccine against emerging variants and SARS-like coronaviruses.

Functional impairment of HIV-specific CD8+ T cells precedes aborted spontaneous control of viremia.

Spontaneous control of HIV infection has been repeatedly linked to antiviral CD8+ T cells but is not always permanent. To address mechanisms of durable and aborted control of viremia, we evaluated immunologic and virologic parameters longitudinally among 34 HIV-infected subjects with differential outcomes. Despite sustained recognition of autologous virus, HIV-specific proliferative and cytolytic T cell effector functions became selectively and intrinsically impaired prior to aborted control. Longitudinal transcriptomic profiling of functionally impaired HIV-specific CD8+ T cells revealed altered expression of genes related to activation, cytokine-mediated signaling, and cell cycle regulation, including increased expression of the antiproliferative transcription factor KLF2 but not of genes associated with canonical exhaustion. Lymphoid HIV-specific CD8+ T cells also exhibited poor functionality during aborted control relative to durable control. Our results identify selective functional impairment of HIV-specific CD8+ T cells as prognostic of impending aborted HIV control, with implications for clinical monitoring and immunotherapeutic strategies.

Exon shuffling potentiates a diverse repertoire of brown algal NB-ARC-TPR candidate immune receptor proteins via alternative splicing.

Like other organisms, brown algae are subject to diseases caused by bacteria, fungi, and viruses. Brown algal immunity mechanisms are not well characterized; however, there is evidence suggesting that pathogen receptors exist in brown algae. One key protein family likely associated with brown algal innate immunity possesses an NB-ARC domain analogous to innate immune proteins in plants and animals. In this study, we conducted an extensive survey of NB-ARC genes in brown algae and obtained insights into the domain organization and evolutionary history of the encoded proteins. Our data show that brown algae possess an ancient NB-ARC-tetratricopeptide repeat (NB-TPR) domain architecture. We identified an N-terminal effector domain, the four-helix bundle, which was not previously found associated with NB-ARC domains. The phylogenetic tree including NB-ARC domains from all kingdoms of life suggests the three clades of brown algal NB-TPRs are likely monophyletic, whereas their TPRs seem to have distinct origins. One group of TPRs exhibit intense exon shuffling, with various alternative splicing and diversifying selection acting on them, suggesting exon shuffling is an important mechanism for evolving ligand-binding specificities. The reconciliation of gene duplication and loss events of the NB-ARC genes reveals that more independent gene gains than losses have occurred during brown algal evolution, and that tandem duplication has played a major role in the expansion of NB-ARC genes. Our results substantially enhance our understanding of the evolutionary history and exon shuffling mechanisms of the candidate innate immune repertoire of brown algae.

The NBS-LRR architectures of plant R-proteins and metazoan NLRs evolved in independent events.

There are intriguing parallels between plants and animals, with respect to the structures of their innate immune receptors, that suggest universal principles of innate immunity. The cytosolic nucleotide binding site-leucine rich repeat (NBS-LRR) resistance proteins of plants (R-proteins) and the so-called NOD-like receptors of animals (NLRs) share a domain architecture that includes a STAND (signal transduction ATPases with numerous domains) family NTPase followed by a series of LRRs, suggesting inheritance from a common ancestor with that architecture. Focusing on the STAND NTPases of plant R-proteins, animal NLRs, and their homologs that represent the NB-ARC (nucleotide-binding adaptor shared by APAF-1, certain R gene products and CED-4) and NACHT (named for NAIP, CIIA, HET-E, and TEP1) subfamilies of the STAND NTPases, we analyzed the phylogenetic distribution of the NBS-LRR domain architecture, used maximum-likelihood methods to infer a phylogeny of the NTPase domains of R-proteins, and reconstructed the domain structure of the protein containing the common ancestor of the STAND NTPase domain of R-proteins and NLRs. Our analyses reject monophyly of plant R-proteins and NLRs and suggest that the protein containing the last common ancestor of the STAND NTPases of plant R-proteins and animal NLRs (and, by extension, all NB-ARC and NACHT domains) possessed a domain structure that included a STAND NTPase paired with a series of tetratricopeptide repeats. These analyses reject the hypothesis that the domain architecture of R-proteins and NLRs was inherited from a common ancestor and instead suggest the domain architecture evolved at least twice. It remains unclear whether the NBS-LRR architectures were innovations of plants and animals themselves or were acquired by one or both lineages through horizontal gene transfer.

Preoperative Inferior Mesenteric Artery Embolization: A Valid Method to Reduce the Rate of Type II Endoleak after EVAR?

BACKGROUND: Type II endoleak is the most commonly encountered endoleak after endovascular abdominal aortic aneurysm repair (EVAR). Some have advocated preoperative inferior mesenteric artery (IMA) embolization as a valid method for reducing the incidence of this endoleak, but controversies exist. We sought to demonstrate the impact of IMA embolization using a meta-analysis of currently available studies combined with our own experience. METHODS: We conducted an institutional review board-approved, retrospective analysis of all patients undergoing IMA embolization before EVAR between the years 2010 and 2015 and used as a control a similar group of patients with patent IMA. We divided patients from our own experience and 5 other studies into 2 groups: those who did not undergo IMA embolization (control) before EVAR and those who did. Rates of type II endoleaks, aneurysm sac regression, and secondary interventions were analyzed. RESULTS: A total of 620 patients from 6 studies were analyzed, including 258 patients who underwent an attempted IMA embolization before EVAR with a cumulative success rate of 99.2% (range, 93.8% to 100%). There was 1 fatality associated with IMA embolization. A meta-analysis showed that preoperative IMA embolization protected against type II endoleaks compared to the control group (odds ratio [OR], 0.31 [0.17-0.57]; P < 0.001, I2 = 43%). Furthermore, the rate of secondary intervention was significantly lower in the treatment group (OR, 0.12 [0.004-0.36]; P < 0.001, I2 = 0%). After IMA embolization, type II endoleak resulted from patent lumbar arteries in all 62 patients with persistent endoleak. CONCLUSIONS: Preoperative embolization of the IMA protects against the development of type II endoleaks and secondary interventions and may potentially lead to a rapid aneurysm sac regression. The procedure can be performed with a high technical success rate and minimal complications and should be considered in patients with IMA >3 mm before EVAR. A randomized trial, however, is required to clearly delineate the clinical significance of this technique.

Trehalose biosynthesis promotes Pseudomonas aeruginosa pathogenicity in plants.

Pseudomonas aeruginosa strain PA14 is a multi-host pathogen that infects plants, nematodes, insects, and vertebrates. Many PA14 factors are required for virulence in more than one of these hosts. Noting that plants have a fundamentally different cellular architecture from animals, we sought to identify PA14 factors that are specifically required for plant pathogenesis. We show that synthesis by PA14 of the disaccharide trehalose is required for pathogenesis in Arabidopsis, but not in nematodes, insects, or mice. In-frame deletion of two closely-linked predicted trehalose biosynthetic operons, treYZ and treS, decreased growth in Arabidopsis leaves about 50 fold. Exogenously co-inoculated trehalose, ammonium, or nitrate, but not glucose, sulfate, or phosphate suppressed the phenotype of the double ΔtreYZΔtreS mutant. Exogenous trehalose or ammonium nitrate does not suppress the growth defect of the double ΔtreYZΔtreS mutant by suppressing the plant defense response. Trehalose also does not function intracellularly in P. aeruginosa to ameliorate a variety of stresses, but most likely functions extracellularly, because wild-type PA14 rescued the in vivo growth defect of the ΔtreYZΔtreS in trans. Surprisingly, the growth defect of the double ΔtreYZΔtreS double mutant was suppressed by various Arabidopsis cell wall mutants that affect xyloglucan synthesis, including an xxt1xxt2 double mutant that completely lacks xyloglucan, even though xyloglucan mutants are not more susceptible to pathogens and respond like wild-type plants to immune elicitors. An explanation of our data is that trehalose functions to promote the acquisition of nitrogen-containing nutrients in a process that involves the xyloglucan component of the plant cell wall, thereby allowing P. aeruginosa to replicate in the intercellular spaces in a leaf. This work shows how P. aeruginosa, a multi-host opportunistic pathogen, has repurposed a highly conserved "house-keeping" anabolic pathway (trehalose biosynthesis) as a potent virulence factor that allows it to replicate in the intercellular environment of a leaf.

Genome-wide identification of Pseudomonas aeruginosa virulence-related genes using a Caenorhabditis elegans infection model.

Pseudomonas aeruginosa strain PA14 is an opportunistic human pathogen capable of infecting a wide range of organisms including the nematode Caenorhabditis elegans. We used a non-redundant transposon mutant library consisting of 5,850 clones corresponding to 75% of the total and approximately 80% of the non-essential PA14 ORFs to carry out a genome-wide screen for attenuation of PA14 virulence in C. elegans. We defined a functionally diverse 180 mutant set (representing 170 unique genes) necessary for normal levels of virulence that included both known and novel virulence factors. Seven previously uncharacterized virulence genes (ABC transporters PchH and PchI, aminopeptidase PepP, ATPase/molecular chaperone ClpA, cold shock domain protein PA0456, putative enoyl-CoA hydratase/isomerase PA0745, and putative transcriptional regulator PA14_27700) were characterized with respect to pigment production and motility and all but one of these mutants exhibited pleiotropic defects in addition to their avirulent phenotype. We examined the collection of genes required for normal levels of PA14 virulence with respect to occurrence in P. aeruginosa strain-specific genomic regions, location on putative and known genomic islands, and phylogenetic distribution across prokaryotes. Genes predominantly contributing to virulence in C. elegans showed neither a bias for strain-specific regions of the P. aeruginosa genome nor for putatively horizontally transferred genomic islands. Instead, within the collection of virulence-related PA14 genes, there was an overrepresentation of genes with a broad phylogenetic distribution that also occur with high frequency in many prokaryotic clades, suggesting that in aggregate the genes required for PA14 virulence in C. elegans are biased towards evolutionarily conserved genes.

Exon shuffling and alternative splicing of ROCO genes in brown algae enables a diverse repertoire of candidate immune receptors.

The ROCO family is a family of GTPases characterized by a central ROC-COR tandem domain. Interest in the structure and function of ROCO proteins has increased with the identification of their important roles in human disease. Nevertheless, the functions of most ROCO proteins are still unknown. In the present study, we characterized the structure, evolution, and expression of ROCOs in four species of brown algae. Brown algae have a larger number of ROCO proteins than other organisms reported to date. Phylogenetic analyses showed that ROCOs have an ancient origin, likely originated in prokaryotes. ROCOs in brown algae clustered into four groups and showed no strong relationship with red algae or green algae. Brown algal ROCOs retain the ancestral LRR-ROC-COR domain arrangement, which is found in prokaryotes, plants and some basal metazoans. Remarkably, individual LRR motifs in ROCO genes are each encoded by separate exons and exhibit intense exon shuffling and diversifying selection. Furthermore, the tandem LRR exons exhibit alternative splicing to generate multiple transcripts. Both exon shuffling and alternative splicing of LRR repeats may be important mechanisms for generating diverse ligand-binding specificities as immune receptors. Besides their potential immune role, expression analysis shows that many ROCO genes are responsive to other stress conditions, suggesting they could participate in multiple signal pathways, not limited to the immune response. Our results substantially enhance our understanding of the structure and function of this mysterious gene family.

Impact of HLA class I functional divergence on HIV control.

Heterozygosity of Human leukocyte antigen (HLA) class I genes is linked to beneficial outcomes after HIV infection, presumably through greater breadth of HIV epitope presentation and cytotoxic T cell response. Distinct allotype pairs, however, differ in the extent to which they bind shared sets of peptides. We developed a functional divergence metric that measures pairwise complementarity of allotype-associated peptide binding profiles. Greater functional divergence for pairs of HLA-A and/or HLA-B allotypes was associated with slower AIDS progression and independently with enhanced viral load control. The metric predicts immune breadth at the peptide level rather than gene level and redefines HLA heterozygosity as a continuum differentially affecting disease outcome. Functional divergence may affect response to additional infections, vaccination, immunotherapy, and other diseases where HLA heterozygote advantage occurs.

Cytolytic CD8+ T cells infiltrate germinal centers to limit ongoing HIV replication in spontaneous controller lymph nodes.

Follicular CD8+ T cells (fCD8) mediate surveillance in lymph node (LN) germinal centers against lymphotropic infections and cancers, but the precise mechanisms by which these cells mediate immune control remain incompletely resolved. To address this, we investigated functionality, clonotypic compartmentalization, spatial localization, phenotypic characteristics, and transcriptional profiles of LN-resident virus-specific CD8+ T cells in persons who control HIV without medications. Antigen-induced proliferative and cytolytic potential consistently distinguished spontaneous controllers from noncontrollers. T cell receptor analysis revealed complete clonotypic overlap between peripheral and LN-resident HIV-specific CD8+ T cells. Transcriptional analysis of LN CD8+ T cells revealed gene signatures of inflammatory chemotaxis and antigen-induced effector function. In HIV controllers, the cytotoxic effectors perforin and granzyme B were elevated among virus-specific CXCR5+ fCD8s proximate to foci of HIV RNA within germinal centers. These results provide evidence consistent with cytolytic control of lymphotropic infection supported by inflammatory recruitment, antigen-specific proliferation, and cytotoxicity of fCD8s.

Molecular basis of differential HLA class I-restricted T cell recognition of a highly networked HIV peptide.

Cytotoxic-T-lymphocyte (CTL) mediated control of HIV-1 is enhanced by targeting highly networked epitopes in complex with human-leukocyte-antigen-class-I (HLA-I). However, the extent to which the presenting HLA allele contributes to this process is unknown. Here we examine the CTL response to QW9, a highly networked epitope presented by the disease-protective HLA-B57 and disease-neutral HLA-B53. Despite robust targeting of QW9 in persons expressing either allele, T cell receptor (TCR) cross-recognition of the naturally occurring variant QW9_S3T is consistently reduced when presented by HLA-B53 but not by HLA-B57. Crystal structures show substantial conformational changes from QW9-HLA to QW9_S3T-HLA by both alleles. The TCR-QW9-B53 ternary complex structure manifests how the QW9-B53 can elicit effective CTLs and suggests sterically hindered cross-recognition by QW9_S3T-B53. We observe populations of cross-reactive TCRs for B57, but not B53 and also find greater peptide-HLA stability for B57 in comparison to B53. These data demonstrate differential impacts of HLAs on TCR cross-recognition and antigen presentation of a naturally arising variant, with important implications for vaccine design.

A defined transposon mutant library and its use in identifying motility genes in Vibrio cholerae.

Defined mutant libraries allow for efficient genome-scale screening and provide a convenient collection of mutations in almost any nonessential gene of interest. Here, we present a near-saturating transposon insertion library in Vibrio cholerae strain C6706, a clinical isolate belonging to the O1 El Tor biotype responsible for the current cholera pandemic. Automated sequencing analysis of 23,312 mutants allowed us to build a 3,156-member subset library containing a representative insertion in every disrupted ORF. Because uncharacterized mutations that affect motility have shown utility in attenuating V. cholerae live vaccines, we used this genome-wide subset library to define all genes required for motility and to further assess the accuracy and purity of the library. In this screen, we identified the hypothetical gene VC2208 (flgT) as essential for motility. Flagellated cells were very rare in a flgT mutant, and transcriptional analysis showed it was specifically stalled at the class III/IV assembly checkpoint of the V. cholerae flagellar regulatory system. Because FlgT is predicted to have structural homology to TolB, a protein involved in determining outer membrane architecture, and the sheath of the V. cholerae flagellum appears to be derived from the cell's outer membrane, FlgT may play a direct role in flagellar sheath formation.

Vasopressin antagonism in heart failure: a review of the hemodynamic studies and major clinical trials.

For decades, plasma arginine vasopressin (AVP) levels have been known to be elevated in patients with congestive heart failure (HF). Excessive AVP signaling at either or both the V1a and V2 receptors could contribute to the pathophysiology of HF by several mechanisms. V1a activation could cause vasoconstriction and/or direct myocardial hypertrophy as intracellular signaling pathways are closely related to those for angiotensin II. V2 activation could cause fluid retention and hyponatremia. A hemodynamic study with the pure V2 antagonist tolvaptan (TV) showed minimal hemodynamic effects. Compared with furosemide in another study, the renal and neurohormonal effects of TV were favorable. Several clinical trials with TV as adjunctive therapy in acute HF have shown beneficial effects on fluid balance and dyspnea, with no worsening of renal function or neurohormonal stimulation. Two smaller studies, one in acute and one in chronic HF, have shown comparable clinical and more favorable renal and neurohormonal effects of TV compared with loop diuretics. However, long-term treatment with TV did not alter outcomes in acute HF. No data are available other than single-dose studies of an intravenous pure V1a antagonist, which showed a vasodilating effect if plasma AVP levels were elevated. One hemodynamic study and one short-duration clinical trial with the balanced intravenous V1a/V2 antagonist conivaptan (CV) showed hemodynamic and clinical effects largely similar to those with TV in similar studies. A new orally effective balanced V1/V2 antagonist (pecavaptan) is currently undergoing phase II study as both adjunctive and alternative therapy during and after hospitalization for acute HF. The purpose of this review is to summarize what we have learned from the clinical experience with TV and CV, and to suggest implications of these findings for future work with newer agents.

A Pericardial Pin: Embolization of an Inferior Vena Cava Filter Strut Presenting as Acute Pericarditis.

A 39-year-old man presented with chest pain initially attributed to viral pericarditis. He was found to have an embolized inferior vena cava filter strut that perforated the right ventricle. Inferior vena cava filter fracture and embolization should be considered in patients with chest pain and pericardial effusion. (Level of Difficulty: Beginner.).

HLA class-I-peptide stability mediates CD8+ T cell immunodominance hierarchies and facilitates HLA-associated immune control of HIV.

Defining factors that govern CD8+ T cell immunodominance is critical for the rational design of vaccines for viral pathogens. Here, we assess the contribution of human leukocyte antigen (HLA) class-I-peptide stability for 186 optimal HIV epitopes across 18 HLA alleles using transporter associated with antigen processing (TAP)-deficient mono-allelic HLA-expressing cell lines. We find that immunodominant HIV epitopes increase surface stabilization of HLA class-I molecules in comparison to subdominant epitopes. HLA class-I-peptide stability is also strongly correlated with overall immunodominance hierarchies, particularly for epitopes from high-abundance proteins (e.g., Gag). Moreover, HLA alleles associated with HIV protection are preferentially stabilized by epitopes derived from topologically important viral regions at a greater frequency than neutral and risk alleles. These findings indicate that relative stabilization of HLA class-I is a key factor for CD8+ T cell epitope immunodominance hierarchies, with implications for HIV control and the design of T-cell-based vaccines.

Tablet vs. station-based laptop ultrasound devices increases internal medicine resident point-of-care ultrasound performance: a prospective cohort study.

BACKGROUND: Point-of-care ultrasound (POCUS) is becoming an important part of internal medicine (IM) residency training. Achieving competency requires performing a large volume of clinical exams which can be difficult within the constraints of residency. Often-cited barriers include insufficient resident time and the interruption of daily workflow. Despite availability of hospital station-based laptop ultrasound machines, we hypothesized that the addition of ward team-based tablet ultrasound devices would lower barriers and increase clinical POCUS volume within an IM residency POCUS curriculum at a 670-bed, quaternary care, teaching hospital. IM resident POCUS volumes and characteristics during an 18-mo. baseline (station-based laptop devices only) period were compared to matched months during the intervention (station-based + tablet). RESULTS: Total patients examined with POCUS by 6 inpatient resident teams during the 18-mo. baseline and intervention periods were 1386 and 1853, respectively. Patients examined per month increased during the intervention by 34% (77 vs. 103, p = 0.002). The number of areas (e.g., abdominal, cardiac) and items (e.g., bladder, pericardial effusion) examined per month increased by 27% (p = 0.021) and 23% (p = 0.073), respectively. CONCLUSIONS: A combined program infrastructure of station-based laptop and "in-the-pocket" tablet ultrasound devices lowered common POCUS barriers of inadequate time and workflow disruption for IM residents and resulted in a meaningful increase of exams within a longitudinal residency-based training program where station-based laptop devices already existed.

Computed Tomographic Angiography-Derived Risk Factors for Vascular Complications in Percutaneous Transfemoral Transcatheter Aortic Valve Implantation.

Transfemoral aortic valve implantation (TAVI) has become a viable alternative to surgical valve implantation, particularly for higher risk patients; however, vascular complications (VCs) remain a concern in transfemoral TAVI. We aimed to determine clinical and computed tomographic angiography-derived risk factors associated with Valve Academic Research Consortium (VARC)-2 criteria VCs in patients who underwent TAVI. From 2011 to 2017, 481 patients underwent percutaneous transfemoral TAVI at the Minneapolis Heart Institute and were screened for procedural and postprocedural access-related VC according to VARC-2 criteria. Clinical and clinical and computed tomographic angiography-derived data were collected to establish risk factors for VC. A total of 99 (21%) patients had VARC-2 VCs. Closure device failure (CDF) occurred in 56 of 99 (57%), minor VCs in 37 of 99 (37%), and major VCs occurred in 6 of 99 (6%). Access site-related VCs were preceded by CDF in 18 of 43 (42%) patients and the risk of major/minor VCs was 14 times greater in patients who experienced closure complications. The incidence of CDF was higher in common femoral artery (CFA) access sites with circumferential vessel wall calcification of more than 90° (p = 0.02) and when skin-surface to CFA access-site distance at an optimal access angle of 45° exceeded 80 mm (p = 0.03). In conclusion, both the degree of circumferential CFA access site calcification and distance to skin surface at an optimal access angle may improve risk stratification of access planning in patients who underwent percutaneous transfemoral TAVI.

Structural topology defines protective CD8+ T cell epitopes in the HIV proteome.

Mutationally constrained epitopes of variable pathogens represent promising targets for vaccine design but are not reliably identified by sequence conservation. In this study, we employed structure-based network analysis, which applies network theory to HIV protein structure data to quantitate the topological importance of individual amino acid residues. Mutation of residues at important network positions disproportionately impaired viral replication and occurred with high frequency in epitopes presented by protective human leukocyte antigen (HLA) class I alleles. Moreover, CD8+ T cell targeting of highly networked epitopes distinguished individuals who naturally control HIV, even in the absence of protective HLA alleles. This approach thereby provides a mechanistic basis for immune control and a means to identify CD8+ T cell epitopes of topological importance for rational immunogen design, including a T cell-based HIV vaccine.

Comparing insertion libraries in two Pseudomonas aeruginosa strains to assess gene essentiality.

Putative essential genes can be identified by comparing orthologs not disrupted in multiple near-saturated transposon insertion mutation libraries in related strains of the same bacterial species. Methods for identifying all orthologs between two bacterial strains and putative essential orthologs are described. In addition, protocols detailing near-saturation transposon insertion mutagenesis of bacteria are presented, including (1) conjugation-mediated mutagenesis, (2) automated colony picking and liquid handling of mutant cultures, and (3) arbitrary polymerase chain reaction amplification and sequencing of genomic DNA adjacent to transposon insertion sites.