Affinity purification mass spectrometry analysis of PD-1 uncovers SAP as a new checkpoint inhibitor.

Programmed cell death-1 (PD-1) is an essential inhibitory receptor in T cells. Antibodies targeting PD-1 elicit durable clinical responses in patients with multiple tumor indications. Nevertheless, a significant proportion of patients do not respond to anti-PD-1 treatment, and a better understanding of the signaling pathways downstream of PD-1 could provide biomarkers for those whose tumors respond and new therapeutic approaches for those whose tumors do not. We used affinity purification mass spectrometry to uncover multiple proteins associated with PD-1. Among these proteins, signaling lymphocytic activation molecule-associated protein (SAP) was functionally and mechanistically analyzed for its contribution to PD-1 inhibitory responses. Silencing of SAP augmented and overexpression blocked PD-1 function. T cells from patients with X-linked lymphoproliferative disease (XLP), who lack functional SAP, were hyperresponsive to PD-1 signaling, confirming its inhibitory role downstream of PD-1. Strikingly, signaling downstream of PD-1 in purified T cell subsets did not correlate with PD-1 surface expression but was inversely correlated with intracellular SAP levels. Mechanistically, SAP opposed PD-1 function by acting as a molecular shield of key tyrosine residues that are targets for the tyrosine phosphatase SHP2, which mediates PD-1 inhibitory properties. Our results identify SAP as an inhibitor of PD-1 function and SHP2 as a potential therapeutic target in patients with XLP.

The PlyB Endolysin of Bacteriophage vB_BanS_Bcp1 Exhibits Broad-Spectrum Bactericidal Activity against Bacillus cereus Sensu Lato Isolates.

Lytic bacteriophages (or phages) drive bacterial mortality by elaborating exquisite abilities to bind, breach, and destroy bacterial cell membranes and subjugate critical bacterial cell functions. These antimicrobial activities make phages ideal candidates to serve as, or provide sources of, biological control measures for bacterial pathogens. In this study, we isolated the Myoviridae phage vB_BanS_Bcp1 (here referred to as Bcp1) from landfill soil, using a Bacillus anthracis host. The antimicrobial activities of both Bcp1 and its encoded endolysin, PlyB, were examined across different B. cereussensu lato group species, including B. cereussensu stricto, Bacillus thuringiensis, and Bacillus anthracis, with pathogenic potential in humans and multiple different uses in biotechnological applications. The Bcp1 phage infected only a subset (11 to 66%) of each B. cereussensu lato species group tested. In contrast, functional analysis of purified PlyB revealed a potent bacteriolytic activity against all B. cereussensu lato isolates tested (n = 79). PlyB was, furthermore, active across broad temperature, pH, and salt ranges, refractory to the development of resistance, bactericidal as a single agent, and synergistic with a second endolysin, PlyG. To confirm the potential for PlyB as an antimicrobial agent, we demonstrated the efficacy of a single intravenous treatment with PlyB alone or combination with PlyG in a murine model of lethal B. anthracis infection. Overall, our findings show exciting potential for the Bcp1 bacteriophage and the PlyB endolysin as potential new additions to the antimicrobial armamentarium.IMPORTANCE Organisms of the Bacillus cereussensu lato lineage are ubiquitous in the environment and are responsible for toxin-mediated infections ranging from severe food poisoning (B. cereussensu stricto) to anthrax (Bacillus anthracis). The increasing incidence of many of these infections, combined with the specter of antibiotic resistance, has created a need for novel antimicrobials with potent activity, including bacteriophages (or phages) and phage-encoded products (i.e., endolysins). In this study, we describe a broadly infective phage, Bcp1, and its encoded endolysin, PlyB, which exhibited a rapidly bacteriolytic effect against all B. cereussensu lato isolates tested with no evidence of evolving resistance. Importantly, PlyB was highly efficacious in a mouse model of lethal bacteremia with B. anthracis Both the Bcp1 phage and the PlyB endolysin represent novel mechanisms of action compared to antibiotics, with potential applications to address the evolving problem of antimicrobial resistance.

Novel phage lysin capable of killing the multidrug-resistant gram-negative bacterium Acinetobacter baumannii in a mouse bacteremia model.

Acinetobacter baumannii, a Gram-negative multidrug-resistant (MDR) bacterium, is now recognized as one of the more common nosocomial pathogens. Because most clinical isolates are found to be multidrug resistant, alternative therapies need to be developed to control this pathogen. We constructed a bacteriophage genomic library based on prophages induced from 13 A. baumannii strains and screened it for genes encoding bacteriolytic activity. Using this approach, we identified 21 distinct lysins with different activities and sequence diversity that were capable of killing A. baumannii. The lysin (PlyF307) displaying the greatest activity was further characterized and was shown to efficiently kill (>5-log-unit decrease) all tested A. baumannii clinical isolates. Treatment with PlyF307 was able to significantly reduce planktonic and biofilm A. baumannii both in vitro and in vivo. Finally, PlyF307 rescued mice from lethal A. baumannii bacteremia and as such represents the first highly active therapeutic lysin specific for Gram-negative organisms in an array of native lysins found in Acinetobacter phage.

The 1.6 A crystal structure of the catalytic domain of PlyB, a bacteriophage lysin active against Bacillus anthracis.

Lysins are peptidoglycan hydrolases that are produced by bacteriophage and act to lyse the bacterial host cell wall during progeny phage release. Here, we describe the structure and function of a novel bacteriophage-derived lysin, PlyB, which displays potent lytic activity against the Bacillus anthracis-like strain ATCC 4342. This molecule comprises an N-terminal catalytic domain (PlyB(cat)) and a C-terminal bacterial SH3-like domain, SH3b. It is shown that both domains are required for effective catalytic activity against ATCC 4342. Further, PlyB has specific activity comparable to the phage lysin PlyG, an amidase being developed as a therapeutic against anthrax. In contrast to PlyG, however, the 1.6 A X-ray crystal structure of PlyB(cat) reveals that the catalytic domain adopts the glycosyl hydrolase (GH)-25, rather than phage T7 lysozyme-like fold. PlyB therefore represents a new class of anthrax lysin and a new defensive tool in the armament against anthrax-mediated bioterrorism.

Hierarchy of human IgG recognition within the Staphylococcus aureus immunome.

Staphylococcus aureus is an opportunistic pathogen that causes a range of serious infections associated with significant morbidity, by strains increasingly resistant to antibiotics. However, to date all candidate vaccines have failed to induce protective immune responses in humans. We need a more comprehensive understanding of the antigenic targets important in the context of human infection. To investigate infection-associated immune responses, patients were sampled at initial presentation and during convalescence from three types of clinical infection; skin and soft tissue infection (SSTI), prosthetic joint infection (PJI) and pediatric hematogenous osteomyelitis (PHO). Reactivity of serum IgG was tested with an array of recombinant proteins, representing over 2,652 in-vitro-translated open reading frames (ORFs) from a community-acquired methicillin-resistant S. aureus USA300 strain. High-level reactivity was demonstrated for 104 proteins with serum IgG in all patient samples. Overall, high-level IgG-reactivity was most commonly directed against a subset of secreted proteins. Although based on limited surveys, we found subsets of S. aureus proteins with differential reactivity with serum samples from patients with different clinical syndromes. Together, our studies have revealed a hierarchy within the diverse proteins of the S. aureus "immunome", which will help to advance efforts to develop protective immunotherapeutic agents.

Human Memory B Cells Targeting Staphylococcus aureus Exotoxins Are Prevalent with Skin and Soft Tissue Infection.

Staphylococcus aureus is a Gram-positive opportunistic pathogen that causes superficial and invasive infections in the hospital and community. High mortality from infection emphasizes the need for improved methods for prevention and treatment. Although S. aureus possesses an arsenal of virulence factors that contribute to evasion of host defenses, few studies have examined long-term humoral and B-cell responses. Adults with acute-phase skin and soft tissue infections were recruited; blood samples were obtained; and S. aureus isolates, including methicillin-resistant strains, were subjected to genomic sequence analysis. In comparisons of acute-phase sera with convalescent-phase sera, a minority (37.5%) of patients displayed 2-fold or greater increases in antibody titers against three or more S. aureus antigens, whereas nearly half exhibited no changes, despite the presence of toxin genes in most infecting strains. Moreover, enhanced antibody responses waned over time, which could reflect a defect in B-cell memory or long-lived plasma cells. However, memory B cells reactive with a range of S. aureus antigens were prevalent at both acute-phase and convalescent-phase time points. While some memory B cells exhibited toxin-specific binding, those cross-reactive with structurally related leucocidin subunits were dominant across patients, suggesting the targeting of conserved epitopes. Memory B-cell reactivity correlated with serum antibody levels for selected S. aureus exotoxins, suggesting a relationship between the cellular and humoral compartments. Overall, although there was no global defect in the representation of anti-S. aureus memory B cells, there was evidence of restrictions in the range of epitopes recognized, which may suggest potential therapeutic approaches for augmenting host defenses.IMPORTANCE The contribution of B-cell memory and long-term antibody responses to host defenses against S. aureus exotoxins remains poorly understood. Our studies confirmed that infection did not commonly lead to enhanced long-term humoral responses. Whereas circulating memory B cells against S. aureus secreted exotoxins were prevalent, they were dominated by cross-reactivity with structurally related leucocidin subunits, consistent with recognition of conserved epitopes. These findings also provide the first evidence of a relationship between the reactivity of antistaphylococcal circulating memory B cells and serum antibody levels. In general, infection was not associated with a global defect in B-cell memory for S. aureus secreted factors, and responses were highly dominated by cross-reactivity to structurally related exotoxins, which arguably may alone be suboptimal in providing host defenses. Our studies illuminate aspects of the S. aureus-host relationship that may better inform strategies for the development of an effective protective vaccine.

Persistence of Disease-Associated Anti-Citrullinated Protein Antibody-Expressing Memory B Cells in Rheumatoid Arthritis in Clinical Remission.

OBJECTIVE: In rheumatoid arthritis (RA), autoreactive B cells are pathogenic drivers and sources of anti-citrullinated protein antibodies (ACPAs) that are a diagnostic biomarker and predictor of worse long-term prognosis. Yet, the immunobiologic significance of persistent ACPA production at the cellular level is poorly understood. This study was undertaken to investigate the representation of ACPA-expressing switched-memory B cells in RA. METHODS: In a cross-sectional study of RA patients, we investigated the presence of continued defects in immune homeostasis as a function of disease activity. Using an enzyme-linked immunosorbent assay (ELISA) and a sensitive multiplex bead-based immunoassay, we characterized fine binding antibody specificities in sera, synovial fluid (SF), and B cell culture supernatants. In this manner, we determined the frequency and epitope reactivity patterns of ACPAs produced by SF B cells and switched-memory blood B cells and compared the latter to serum ACPA levels and disease activity scores. RESULTS: Cultured B cells from SF were shown to spontaneously secrete ACPAs, while constitutive IgG autoantibody production by peripheral blood mononuclear cells (PBMCs) was substantially less frequent. After in vitro stimulation, PBMCs secreted IgG ACPA that was overwhelmingly from switched-memory B cells, across all patient groups treated with methotrexate and/or a tumor necrosis factor inhibitor. Intriguingly, the frequencies of ACPA-expressing switched-memory B cells significantly correlated with serum IgG anti-cyclic citrullinated peptide 3 (r = 0.57, P = 0.003). Moreover, treatment-induced clinical remission had little or no effect on the circulating burden of switched-memory ACPA-expressing B cells, in part explaining the continued dysregulation of humoral immunity. CONCLUSION: Our findings rationalize why therapeutic cessation most often results in disease reactivation and clinical flare. Hence, a clinical disease activity score is not a reliable indicator of the resolution of pathologic recirculating B cell autoimmunity.

Anti-carbamylated Protein Antibody Levels Correlate with Anti-Sa (Citrullinated Vimentin) Antibody Levels in Rheumatoid Arthritis.

OBJECTIVE: The presence of anticitrullinated protein antibodies (ACPA) in rheumatoid arthritis (RA) indicates a breach in immune tolerance. Recent studies indicate that this breach extends to homocitrullination of lysines with the formation of anti-carbamylated protein (anti-CarP) antibodies. We analyzed the clinical and serologic relationships of anti-CarP in 2 RA cohorts. METHODS: Circulating levels of immunoglobulin G anti-CarP antibodies were determined by ELISA in established (Dartmouth-Hitchcock Medical Center) and early (Sherbrooke University Hospital Center) cohorts and evaluated for anticyclic citrullinated peptide antibodies (anti-CCP), specific ACPA, and rheumatoid factor (RF) levels using the Student t test and correlation analysis. RESULTS: We identified elevated anti-CarP antibodies titers in 47.0% of seropositive patients (Dartmouth, n = 164), with relationships to anti-CCP (p < 0.0001) and IgM-RF (p = 0.001). Similarly, 38.2% of seropositive patients from the Sherbrooke cohort (n = 171) had elevated anti-CarP antibodies; titers correlated to anti-CCP (p = 0.01) but not IgM-RF (p = 0.09). A strong correlation with anti-Sa was observed: 47.9% anti-Sa+ patients were anti-CarP antibodies+ versus only 25.4% anti-Sa- in the Sherbrooke cohort (p = 0.0002), and 62.6% anti-Sa+ patients versus 26.9% anti-Sa- were anti-CarP antibodies+ in Dartmouth (p < 0.0001). We found a more variable response for reactivity to citrullinated fibrinogen or to citrullinated peptides from fibrinogen and α enolase. CONCLUSION: In 2 North American RA cohorts, we observed a high prevalence of anti-CarP antibody positivity. We also describe a surprising and unexpected association of anti-CarP with anti-Sa antibodies that could not be explained by cross-reactivity. Further, considerable heterogeneity exists between anti-CarP reactivity and other citrullinated peptide reactivity, raising the question of how the pathogenesis of antibody responses for carbamylated proteins and citrullinated proteins may be linked in vivo.

The monomer-dimer equilibrium of stromal cell-derived factor-1 (CXCL 12) is altered by pH, phosphate, sulfate, and heparin.

Chemokines, like stromal cell-derived factor-1 (SDF1/CXCL12), are small secreted proteins that signal cells to migrate. Because SDF1 and its receptor CXCR4 play important roles in embryonic development, cancer metastasis, and HIV/AIDS, this chemokine signaling system is the subject of intense study. However, it is not known whether the monomeric or dimeric structure of SDF1 is responsible for signaling in vivo. Previous structural studies portrayed the SDF1 structure as either strictly monomeric in solution or dimeric when crystallized. Here, we report two-dimensional NMR, pulsed-field gradient diffusion and fluorescence polarization measurements at various SDF1 concentrations, solution conditions, and pH. These results demonstrate that SDF1 can form a dimeric structure in solution, but only at nonacidic pH when stabilizing counterions are present. Thus, while the previous NMR structural studies were performed under acidic conditions that strongly promote the monomeric state, crystallographic studies used nonacidic buffer conditions that included divalent anions shown here to promote dimerization. This pH-sensitive aggregation behavior is explained by a dense cluster of positively charged residues at the SDF1 dimer interface that includes a histidine side chain at its center. A heparin disaccharide shifts the SDF1 monomer-dimer equilibrium in the same manner as other stabilizing anions, suggesting that glycosaminoglycan binding may be coupled to SDF1 dimerization in vivo.

Complete Genome Sequence of Bacillus cereus Sensu Lato Bacteriophage Bcp1.

Bacillus cereus sensu lato organisms are an ecologically diverse group that includes etiologic agents of food poisoning, periodontal disease, and anthrax. The recently identified Bcp1 bacteriophage infects B. cereus sensu lato and is being developed as a therapeutic decontamination agent and diagnostic countermeasure. We announce the complete genome sequence of Bcp1.

Use of a bacteriophage lysin to identify a novel target for antimicrobial development.

We identified an essential cell wall biosynthetic enzyme in Bacillus anthracis and an inhibitor thereof to which the organism did not spontaneously evolve measurable resistance. This work is based on the exquisite binding specificity of bacteriophage-encoded cell wall-hydrolytic lysins, which have evolved to recognize critical receptors within the bacterial cell wall. Focusing on the B. anthracis-specific PlyG lysin, we first identified its unique cell wall receptor and cognate biosynthetic pathway. Within this pathway, one biosynthetic enzyme, 2-epimerase, was required for both PlyG receptor expression and bacterial growth. The 2-epimerase was used to design a small-molecule inhibitor, epimerox. Epimerox prevented growth of several Gram-positive pathogens and rescued mice challenged with lethal doses of B. anthracis. Importantly, resistance to epimerox was not detected (<10(-11) frequency) in B. anthracis and S. aureus. These results describe the use of phage lysins to identify promising lead molecules with reduced resistance potential for antimicrobial development.

An engineered second disulfide bond restricts lymphotactin/XCL1 to a chemokine-like conformation with XCR1 agonist activity.

Chemokines adopt a conserved tertiary structure stabilized by two disulfide bridges and direct the migration of leukocytes. Lymphotactin (Ltn) is a unique chemokine in that it contains only one disulfide and exhibits large-scale structural heterogeneity. Under physiological solution conditions (37 degrees C and 150 mM NaCl), Ltn is in equilibrium between the canonical chemokine fold (Ltn10) and a distinct four-stranded beta-sheet (Ltn40). Consequently, it has not been possible to address the biological significance of each structural species independently. To stabilize the Ltn10 structure in a manner independent of specific solution conditions, Ltn variants containing a second disulfide bridge were designed. Placement of the new cysteines was based on a sequence alignment of Ltn with either the first (Ltn-CC1) or third disulfide (Ltn-CC3) in the CC chemokine, HCC-2. NMR data demonstrate that both CC1 and CC3 retain the Ltn10 chemokine structure and no longer exhibit structural rearrangement. The ability of each mutant to activate the Ltn receptor, XCR1, has been tested using an intracellular Ca2+ flux assay. These data support the conclusion that the chemokine fold of Ltn10 is responsible for receptor activation. We also examined the role of amino- and carboxyl-terminal residues in Ltn-mediated receptor activation. In contrast to previous reports, we find that the 25 residues comprising the novel C-terminal extension do not participate in receptor activation, while the native N-terminus is absolutely required for Ltn function.