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Legionella pneumophila is an intracellular bacterial pathogen that uses a type IV secretion system (T4SS), termed Dot/Icm, to secrete more than 330 virulence effector proteins into the infected host cell. Many Dot/Icm effectors are involved in biogenesis of the Legionella-containing vacuole (LCV), which allows intracellular bacterial replication in environmental amoebae and alveolar macrophages. Through their activity, some effectors trigger the mammalian host immune response in a phenomenon termed effector-triggered immunity (ETI). Here, we describe a protocol to create and use L. pneumophila genome deletion mutants to identify effector(s) that alter pro-inflammatory cytokine production and bacterial clearance in the lungs of mice.

The unfolded protein response (UPR) is a homeostatic response to endoplasmic reticulum (ER) stress within eukaryotic cells. The UPR initiates transcriptional and post-transcriptional programs to resolve ER stress; or, if ER stress is severe or prolonged, initiates apoptosis. ER stress is a common feature of bacterial infection although the role of the UPR in host defense is only beginning to be understood. While the UPR is important for host defense against pore-forming toxins produced by some bacteria, other bacterial effector proteins hijack the UPR through the activity of translocated effector proteins that facilitate intracellular survival and proliferation. UPR-mediated apoptosis can limit bacterial replication but also often contributes to tissue damage and disease. Here, we discuss the dual nature of the UPR during infection and the implications of UPR activation or inhibition for inflammation and immunity as illustrated by different bacterial pathogens.

A vaccine that prevents hepatitis C virus (HCV) infection is urgently needed to support an emerging global elimination program. However, vaccine development has been confounded because of HCV's high degree of antigenic variability and the preferential induction of type-specific immune responses with limited potency against heterologous viral strains and genotypes. We showed previously that deletion of the three variable regions from the E2 receptor-binding domain (Δ123) increases the ability of human broadly neutralizing antibodies (bNAbs) to inhibit E2-CD81 receptor interactions, suggesting improved bNAb epitope exposure. In this study, the immunogenicity of Δ123 was examined. We show that high-molecular-weight forms of Δ123 elicit distinct antibody specificities with potent and broad neutralizing activity against all seven HCV genotypes. Antibody competition studies revealed that immune sera raised to high-molecular-weight Δ123 was poly specific, given that it inhibited the binding of human bNAbs directed to three major neutralization epitopes on E2. By contrast, the immune sera raised to monomeric Δ123 predominantly blocked the binding of a non-neutralizing antibody to Δ123, while having reduced ability to block bNAb binding to E2, and neutralization was largely toward the homologous genotype. This increased ability of oligomeric Δ123 to generate bNAbs correlates with occlusion of the non-neutralizing face of E2 in this glycoprotein form. Conclusion: The results from this study reveal new information on the antigenic and immunogenic potential of E2-based immunogens and provide a pathway for the development of a simple, recombinant protein-based prophylactic vaccine for HCV with potential for universal protection. (Hepatology 2017;65:1117-1131).

The hepatitis C virus (HCV) E2 glycoprotein is a major target of the neutralizing antibody (nAb) response, with multiple type-specific and broadly neutralizing antibody (bnAb) epitopes identified. The 412-to-423 region can generate bnAbs that block interaction with the cell surface receptor CD81, with activity toward multiple HCV genotypes. In this study, we reveal the structure of rodent monoclonal antibody 24 (MAb24) with an extensive contact area toward a peptide spanning the 412-to-423 region. The crystal structure of the MAb24–peptide 412-to-423 complex reveals the paratope bound to a peptide hairpin highly similar to that observed with human MAb HCV1 and rodent MAb AP33, but with a different angle of approach. In viral outgrowth experiments, we demonstrated three distinct genotype 2a viral populations that acquired resistance to MAb24 via N415D, N417S, and N415D/H386R mutations. Importantly, the MAb24-resistant viruses exhibited significant increases in sensitivity to the majority of bnAbs directed to epitopes within the 412-to-423 region and in additional antigenic determinants located within E2 and the E1E2 complex. This study suggests that modification of N415 causes a global change in glycoprotein structure that increases its vulnerability to neutralization by other antibodies. This finding suggests that in the context of an antibody response to viral infection, acquisition of escape mutations in the 412-to-423 region renders the virus more susceptible to neutralization by other specificities of nAbs, effectively reducing the immunological fitness of the virus. A vaccine for HCV that generates polyspecific humoral immunity with specificity for the 412-to-423 region and at least one other region of E2 is desirable. IMPORTANCE Understanding how antibodies neutralize hepatitis C virus (HCV) is essential for vaccine development. This study reveals for the first time that when HCV develops resistance to a major class of bnAbs targeting the 412-to-423 region of E2, this results in a concomitant increase in sensitivity to neutralization by a majority of other bnAb specificities. Vaccines for the prevention of HCV infection should therefore generate bnAbs directed toward the 412-to-423 region of E2 and additional bnAb epitopes within the viral glycoproteins.

The hepatitis C virus (HCV) envelope glycoprotein E2 is the major target of broadly neutralizing antibodies in vivo and is the focus of efforts in the rational design of a universal B cell vaccine against HCV. The E2 glycoprotein exhibits a high degree of amino acid variability which localizes to three discrete regions: hypervariable region 1 (HVR1), hypervariable region 2 (HVR2), and the intergenotypic variable region (igVR). All three variable regions contribute to immune evasion and/or isolate-specific structural variations, both important considerations for vaccine design. A high-resolution structural definition of the intact HCV envelope glycoprotein complex containing E1 and E2 remains to be elucidated, while crystallographic structures of a recombinant E2 ectodomain failed to resolve HVR1, HVR2, and a major neutralization determinant adjacent to HVR1. To obtain further information on E2, we characterized the role of all three variable regions in E2 ectodomain folding and function in the context of a recombinant ectodomain fragment (rE2). We report that removal of the variable regions accelerates binding to the major host cell receptor CD81 and that simultaneous deletion of HVR2 and the igVR is required to maintain wild-type CD81-binding characteristics. The removal of the variable regions also rescued the ability of rE2 to form a functional homodimer. We propose that the rE2 core provides novel insights into the role of the variable motifs in the higher-order assembly of the E2 ectodomain and may have implications for E1E2 structure on the virion surface. IMPORTANCE Hepatitis C virus (HCV) infection affects ∼2% of the population globally, and no vaccine is available. HCV is a highly variable virus, and understanding the presentation of key antigenic sites at the virion surface is important for the design of a universal vaccine. This study investigates the role of three surface-exposed variable regions in E2 glycoprotein folding and function in the context of a recombinant soluble ectodomain. Our data demonstrate the variable motifs modulate binding of the E2 ectodomain to the major host cell receptor CD81 and have an impact on the formation of an E2 homodimer with high-affinity binding to CD81.

Transcription of type I interferon genes during RNA virus infection requires signal communication between several pattern recognition receptor (PRR)-adaptor complexes located at distinct subcellular membranous compartments and a central cytoplasmic TBK1-interferon regulatory factor 3 (IRF3) kinase-transcription factor module. However, how the cell integrates signal transduction through spatially distinct modules of antiviral signaling pathways is less defined. RIG-I is a major cytosolic PRR involved in the control of several RNA viruses. Here we identify ArfGAP domain-containing protein 2 (ADAP2) as a key novel scaffolding protein that integrates different modules of the RIG-I pathway, located at distinct subcellular locations, and mediates cellular antiviral type I interferon production. ADAP2 served to bridge the mitochondrial membrane-bound upstream RIG-I adaptor MAVS and the downstream cytosolic complex of NEMO (regulatory subunit of TBK1), TBK1, and IRF3, leading to IRF3 phosphorylation. Furthermore, independently, ADAP2 also functioned as a major orchestrator of the interaction of TBK1 with NEMO and IRF3. Mutational and in vitro cell-free reconstituted RIG-I signaling assay-based analyses identified that the ArfGAP domain of ADAP2 mediates the interferon response. TRAF3 acted as a trigger for ADAP2 to recruit RIG-I pathway component proteins into a single macromolecular complex. This study provides important novel insights into the assembly and integration of different modules of antiviral signaling cascades.

The ER (endoplasmic reticulum) is the protein folding ‘factory’ of the secretory pathway. Virtually all proteins destined for the plasma membrane, the extracellular space or other secretory compartments undergo folding and maturation within the ER. The ER hosts a unique PQC (protein quality control) system that allows specialized modifications such as glycosylation and disulfide bond formation essential for the correct folding and function of many secretory proteins. It is also the major checkpoint for misfolded or aggregation-prone proteins that may be toxic to the cell or extracellular environment. A failure of this system, due to aging or other factors, has therefore been implicated in a number of serious human diseases. In this article, we discuss several key features of ER PQC that maintain the health of the cellular secretome.

Hepatitis C virus glycoprotein E2 contains 18 conserved cysteines predicted to form nine disulfide pairs. In this study, a comprehensive cysteine-alanine mutagenesis scan of all 18 cysteine residues was performed in E1E2-pseudotyped retroviruses (HCVpp) and recombinant E2 receptor-binding domain (E2 residues 384 to 661 [E2 661 ]). All 18 cysteine residues were absolutely required for HCVpp entry competence. The phenotypes of individual cysteines and pairwise mutation of disulfides were largely the same for retrovirion-incorporated E2 and E2 661 , suggesting their disulfide arrangements are similar. However, the contributions of each cysteine residue and the nine disulfides to E2 structure and function varied. Individual Cys-to-Ala mutations revealed discordant effects, where removal of one Cys within a pair had minimal effect on H53 recognition and CD81 binding (C486 and C569) while mutation of its partner abolished these functions (C494 and C564). Removal of disulfides at C581-C585 and C452-C459 significantly reduced the amount of E1 coprecipitated with E2, while all other disulfides were absolutely required for E1E2 heterodimerization. Remarkably, E2 661 tolerates the presence of four free cysteines, as simultaneous mutation of C452A, C486A, C569A, C581A, C585A, C597A, and C652A (M+C597A) retained wild-type CD81 binding. Thus, only one disulfide from each of the three predicted domains, C429-C552 (DI), C503-C508 (DII), and C607-C644 (DIII), is essential for the assembly of the E2 661 CD81-binding site. Furthermore, the yield of total monomeric E2 increased to 70% in M+C597A. These studies reveal the contribution of each cysteine residue and the nine disulfide pairs to E2 structure and function.

The three variable regions of hepatitis C virus (HCV) glycoprotein E2 can be removed simultaneously from the E2 ectodomain (residues 384-661) without affecting folding or CD81 binding. In this study, we show that deletion of hypervariable region (HVR) 2 or the intergenotypic variable region (igVR) in the context of the E1E2 polyprotein eliminates formation of heterodimers, reduces CD81 binding and abolishes virus entry. The replication competence of genomic RNA transcribed from the JFH1 infectious HCV clone was not affected by the HVR1, HVR2 or igVR deletions in transfected Huh7.5 cells. However, infectivity of the resultant cell-culture-derived HCV (HCVcc) was abolished by HVR2 or igVR deletions, while deletion of HVR1 led to a 5- to 10-fold reduction in infectivity. Serial passage of cells transfected with genomes lacking HVR1 generated reverted viruses with wild-type levels of infectivity. Sequencing of viral cDNA obtained after full reversion revealed mutations in E1 (I262L) and E2 (N415D) that were present in 35 and 27 % of clones, respectively. Insertion of N415D into HVR1-deleted HCV genomes conferred wild-type levels of infectivity, while I262L increased infectivity by 2.5-fold. These results suggest that HVR2 and the igVR, but not HVR1, are essential for structural integrity and function of the HCV glycoprotein heterodimer.

"Are we adequately addressing the emotional side of learning for our students?" When the faculty members of Webb Bridge Middle School in Alpharetta, Georgia, seriously asked ourselves that question, we began a process that culminated in starting an advisory program. Our initial answers to the question made evident the inadequacies of'our then current efforts to deal with the personal and social concerns of our students and their development as persons of good character. Yes, we did have "word of the week," which we thought addressed character education, but other than student agendas and posters, there really was no meaningful support. Yes, we loosely called our homeroom an "advisory" time, but we knew that this was not a valuable or meaningful period of time; it was too filled with paperwork and announcements to really connect with students. Yes, we did have a record of academic excellence, bul how were we addressing the issues of academic honesty and personal social behavior? Yes, we did have organizational structures and processes that addressed building needs, but how were we addressing such matters as anger management or kindness? How were we as a middle school addressing overall developmental responsiveness and social equity? Were we truly addressing the needs of all our young learners if we were celebrating our academic accomplishments, while some students could have been wandering our halls without an adult recognizing that personal dilemmas were consuming them? Were we really assisting our youth in this key period of character formation? Reaching way back As we opened up this important aspect of an education for honest consideration and delved into the advisory model and the purposes of character education, we quickly realized that character development could not be viewed in isolation, as its own history stretches back to before biblical times. Within character or moral education, there has been an emphasis on the education of the whole person, dealing with the physical, moral, spiritual, and cultural aspects of human life (Best, 2000). These considerations have been present throughout the history of American education. In the early 20th century, John Dewey defined character as the "interpretation of habits" and an important cornerstone in the development of character education. While character education has been evolving for centuries, more recent concerns for moral education appeared in the early 1960s. Not surprisingly, with the "social and political upheaval of the 1960s, a resurgent interest in this age-old challenge arose in response to societal questioning of moral values and policies, numerous approaches to socializing our youth were proposed" (Althof & Berkowitz, 2006). Best (2000) has traced the history of attempts in England to legislate the development of spiritual, moral, social, and cultural growth of students through a comprehensive national curriculum. He concluded: "To improve performance across the academic curriculum, we need to protect and extend opportunities for children to engage the world emotionally, aesthetically, and morally, in the hope that they come to understand much better themselves, their fellow human beings, and that greater whole of which we are all part." The contemporary observations of Anfara (2006) point toward the realization that when especially designed programs are combined with other components of the middle school concept, student self-concept improves, dropout rates decrease, and school climate becomes more positive. With this in mind, it is not surprising that in her congressional testimony, Principal Aldredge of Fairfax County Public Schools in Virginia opened by recognizing "what Socrates, Kohlberg, Likona, and so many others have taught us throughout the years; that is, the importance of developing moral character in our youth" (Aldredge, 2006). These observations draw on a very similar theme running through American education: Learning extends beyond the traditional classroom to the "life experiences" of learners. …

Hepatitis C virus (HCV) envelope glycoproteins E1 and E2 form a heterodimer and mediate receptor interactions and viral fusion. Both E1 and E2 are targets of the neutralizing antibody (NAb) response and are candidates for the production of vaccines that generate humoral immunity. Previous studies demonstrated that N-terminal hypervariable region 1 (HVR1) can modulate the neutralization potential of monoclonal antibodies (MAbs), but no information is available on the influence of HVR2 or the intergenotypic variable region (igVR) on antigenicity. In this study, we examined how the variable regions influence the antigenicity of the receptor binding domain of E2 spanning HCV polyprotein residues 384 to 661 (E2 661 ) using a panel of MAbs raised against E2 661 and E2 661 lacking HVR1, HVR2, and the igVR (Δ123) and well-characterized MAbs isolated from infected humans. We show for a subset of both neutralizing and nonneutralizing MAbs that all three variable regions decrease the ability of MAbs to bind E2 661 and reduce the ability of MAbs to inhibit E2-CD81 interactions. In addition, we describe a new MAb directed toward the region spanning residues 411 to 428 of E2 (MAb24) that demonstrates broad neutralization against all 7 genotypes of HCV. The ability of MAb24 to inhibit E2-CD81 interactions is strongly influenced by the three variable regions. Our data suggest that HVR1, HVR2, and the igVR modulate exposure of epitopes on the core domain of E2 and their ability to prevent E2-CD81 interactions. These studies suggest that the function of HVR2 and the igVR is to modulate antibody recognition of glycoprotein E2 and may contribute to immune evasion. IMPORTANCE This study reveals conformational and antigenic differences between the Δ123 and intact E2 661 glycoproteins and provides new structural and functional data about the three variable regions and their role in occluding neutralizing and nonneutralizing epitopes on the E2 core domain. The variable regions may therefore function to reduce the ability of HCV to elicit NAbs directed toward the conserved core domain. Future studies aimed at generating a three-dimensional structure for intact E2 containing HVR1, and the adjoining NAb epitope at residues 412 to 428, together with HVR2, will reveal how the variable regions modulate antigenic structure.

The hepatitis C virus glycoprotein E2 receptor-binding domain is encompassed by amino acids 384 to 661 (E2661) and contains two hypervariable sequences, HVR1 and HVR2. E2 sequence comparisons revealed a third variable region, located between residues 570 and 580, that varies widely between genotypes, designated here as igVR, the intergenotypic variable region. A secreted E2661glycoprotein with simultaneous deletions of the three variable sequences retained its ability to bind CD81 and conformation-dependent monoclonal antibodies (MAbs) and displayed enhanced binding to a neutralizing MAb directed to E2 immunogenic domain B. Our data provide insights into the E2 structure by suggesting that the three variable regions reside outside a conserved E2 core.