Non-random Escape Pathways from a Broadly Neutralizing Human Monoclonal Antibody Map to a Highly Conserved Region on the Hepatitis C Virus E2 Glycoprotein Encompassing Amino Acids 412–423

A challenge for hepatitis C virus (HCV) vaccine development is to define epitopes that are able to elicit protective antibodies against this highly diverse virus. The E2 glycoprotein region located at residues 412–423 is conserved and antibodies to 412–423 have broadly neutralizing activities. However, an adaptive mutation, N417S, is associated with a glycan shift in a variant that cannot be neutralized by a murine but by human monoclonal antibodies (HMAbs) against 412–423. To determine whether HCV escapes from these antibodies, we analyzed variants that emerged when cell culture infectious HCV virions (HCVcc) were passaged under increasing concentrations of a specific HMAb, HC33.1. Multiple nonrandom escape pathways were identified. Two pathways occurred in the context of an N-glycan shift mutation at N417T. At low antibody concentrations, substitutions of two residues outside of the epitope, N434D and K610R, led to variants having improved in vitro viral fitness and reduced sensitivity to HC33.1 binding and neutralization. At moderate concentrations, a S419N mutation occurred within 412–423 in escape variants that have greatly reduced sensitivity to HC33.1 but compromised viral fitness. Importantly, the variants generated from these pathways differed in their stability. N434D and K610R-associated variants were stable and became dominant as the virions were passaged. The S419N mutation reverted back to N419S when immune pressure was reduced by removing HC33.1. At high antibody concentrations, a mutation at L413I was observed in variants that were resistant to HC33.1 neutralization. Collectively, the combination of multiple escape pathways enabled the virus to persist under a wide range of antibody concentrations. Moreover, these findings pose a different challenge to vaccine development beyond the identification of highly conserved epitopes. It will be necessary for a vaccine to induce high potency antibodies that prevent the formation of escape variants, which can co-exist with lower potency or levels of neutralizing activities.     

Patient Perspectives on Post-Discharge Surgical Site Infections: Towards a Patient-Centered Mobile Health Solution

Background

Post-discharge surgical site infections (SSI) are a major source of morbidity, expense and anxiety for patients. However, patient perceptions about barriers experienced while seeking care for post-discharge SSI have not been assessed in depth. We explored patient experience of SSI and openness to a mobile health (mHealth) wound monitoring “app” as a novel solution to address this problem.

Methods

Mixed method design with semi-structured interviews and surveys. Participants were patients who had post-discharge surgical wound complications after undergoing operations with high risk of SSI, including open colorectal or ventral hernia repair surgery. The study was conducted at two affiliated teaching hospitals, including an academic medical center and a level 1 trauma center.

Results

From interviews with 13 patients, we identified 3 major challenges that impact patients'' ability to manage post-discharge surgical wound complications, including required knowledge for wound monitoring from discharge teaching, self-efficacy for wound monitoring at home, and accessible communication with their providers about wound concerns. Patients found an mHealth wound monitoring application highly acceptable and articulated its potential to provide more frequent, thorough, and convenient follow-up that could reduce post-discharge anxiety compared to the current practice. Major concerns with mHealth wound monitoring were lack of timely response from providers and inaccessibility due to either lack of an appropriate device or usability challenges.

Conclusions

Our findings reveal gaps and frustrations with post-discharge care after surgery which could negatively impact clinical outcomes and quality of life. To address these issues, we are developing mPOWEr, a patient-centered mHealth wound monitoring application for patients and providers to collaboratively bridge the care transition between hospital and home.     

Quantitative Genetic Analyses of Male Color Pattern and Female Mate Choice in a Pair of Cichlid Fishes of Lake Malawi,East Africa

The traits involved in sexual selection, such as male secondary sexual characteristics and female mate choice, often co-evolve which can promote population differentiation. However, the genetic architecture of these phenotypes can influence their evolvability and thereby affect the divergence of species. The extraordinary diversity of East African cichlid fishes is often attributed to strong sexual selection and thus this system provides an excellent model to test predictions regarding the genetic architecture of sexually selected traits that contribute to reproductive isolation. In particular, theory predicts that rapid speciation is facilitated when male sexual traits and female mating preferences are controlled by a limited number of linked genes. However, few studies have examined the genetic basis of male secondary sexual traits and female mating preferences in cichlids and none have investigated the genetic architecture of both jointly. In this study, we artificially hybridized a pair of behaviorally isolated cichlid fishes from Lake Malawi and quantified both melanistic color pattern and female mate choice. We investigated the genetic architecture of both phenotypes using quantitative genetic analyses. Our results suggest that 1) many non-additively acting genetic factors influence melanistic color patterns, 2) female mate choice may be controlled by a minimum of 1–2 non-additive genetic factors, and 3) F₂ female mate choice is not influenced by male courting effort. Furthermore, a joint analysis of color pattern and female mate choice indicates that the genes underlying these two traits are unlikely to be physically linked. These results suggest that reproductive isolation may evolve rapidly owing to the few genetic factors underlying female mate choice. Hence, female mate choice likely played an important role in the unparalleled speciation of East African cichlid fish.     

Anti-α4 Antibody Treatment Blocks Virus Traffic to the Brain and Gut Early,and Stabilizes CNS Injury Late in Infection

Four SIV-infected monkeys with high plasma virus and CNS injury were treated with an anti-α4 blocking antibody (natalizumab) once a week for three weeks beginning on 28 days post-infection (late). Infection in the brain and gut were quantified, and neuronal injury in the CNS was assessed by MR spectroscopy, and compared to controls with AIDS and SIV encephalitis. Treatment resulted in stabilization of ongoing neuronal injury (NAA/Cr by 1H MRS), and decreased numbers of monocytes/macrophages and productive infection (SIV p28⁺, RNA⁺) in brain and gut. Antibody treatment of six SIV infected monkeys at the time of infection (early) for 3 weeks blocked monocyte/macrophage traffic and infection in the CNS, and significantly decreased leukocyte traffic and infection in the gut. SIV – RNA and p28 was absent in the CNS and the gut. SIV DNA was undetectable in brains of five of six early treated macaques, but proviral DNA in guts of treated and control animals was equivalent. Early treated animals had low-to-no plasma LPS and sCD163. These results support the notion that monocyte/macrophage traffic late in infection drives neuronal injury and maintains CNS viral reservoirs and lesions. Leukocyte traffic early in infection seeds the CNS with virus and contributes to productive infection in the gut. Leukocyte traffic early contributes to gut pathology, bacterial translocation, and activation of innate immunity.     

FliZ induces a kinetic switch in flagellar gene expression

FliZ is an activator of class 2 flagellar gene expression in Salmonella enterica. To understand its role in flagellar assembly, we investigated how FliZ affects gene expression dynamics. We demonstrate that FliZ participates in a positive-feedback loop that induces a kinetic switch in class 2 gene expression.     

Rapid generation of rotavirus-specific human monoclonal antibodies from small-intestinal mucosa

The gut mucosal surface is efficiently protected by Abs, and this site represents one of the richest compartments of Ab-secreting cells in the body. A simple and effective method to generate Ag-specific human monoclonal Abs (hmAbs) from such cells is lacking. In this paper, we describe a method to generate hmAbs from single Ag-specific IgA- or IgM-secreting cells of the intestinal mucosa. We found that CD138-positive plasma cells from the duodenum expressed surface IgA or IgM. Using eGFP-labeled virus-like particles, we harnessed the surface Ig expression to detect rotavirus-specific plasma cells at low frequency (0.03-0.35%) in 9 of 10 adult subjects. Single cells were isolated by FACS, and as they were viable, further testing of secreted Abs by ELISPOT and ELISA indicated a highly specific selection procedure. Ab genes from single cells of three donors were cloned, sequenced, and expressed as recombinant hmAbs. Of 26 cloned H chain Ab genes, 22 were IgA and 4 were IgM. The genes were highly mutated, and there was an overrepresentation of the VH4 family. Of 10 expressed hmAbs, 8 were rotavirus-reactive (6 with K(d) < 1 × 10(-10)). Importantly, our method allows generation of hmAbs from cells implicated in the protection of mucosal surfaces, and it can potentially be used in passive vaccination efforts and for discovery of epitopes directly relevant to human immunity.     

Examining Hedgehog pathway genes GLI3, SHH, and PTCH1 and the p53 target GLIPR1/GLIPR1L1/GLIPR1L2 gene cluster using fluorescence in situ hybridization uncovers GLIPR1/GLIPR1L1/GLIPR1L2 deletion in 9% of patients with multiple myeloma

Mutations in genes regulating cell cycle and apoptosis are considered major culprits for the malignant transformation of cancer cells. Aberrant activation of the Hedgehog (HH) signaling pathway which primarily regulates genes involved in cell growth, proliferation, survival and apoptosis has been demonstrated in multiple myeloma. Mutations resulting in defective components of the p53 pathway, which serves a critical role in mediating cellular stress response by triggering DNA repair, cell cycle arrest, senescence and apoptosis, have also been identified. This study focuses on detecting copy number variations for the GLIPR1/GLIPR1L1/GLIPR1L2 gene cluster of the p53 pathway and three elements of the HH pathway, SHH, PTCH1 and GLI3 in multiple myeloma (MM) using fluorescence in situ hybridization (FISH). In eighteen samples, there was no evidence of abnormal copy number for PTCH1, GLI3 or SHH. Thus, it is unlikely that copy number variations of these genes are linked to multiple myeloma. However, a deletion of the GLIPR1/GLIPR1L1/ GLIPR1L2 gene cluster, all p53 targets, was found in three of 32 samples (9.4%) indicating that these deleted genes may have significant implications in MM. Further studies should be performed to determine the role of the GLIPR1/GLIPR1L1/GLIPR1L2 gene cluster in the pathogenesis of multiple myeloma.     

Defining the Ligand Specificity of the Deleted in Colorectal Cancer (DCC) Receptor

The growth and guidance of many axons in the developing nervous system require Netrin-mediated activation of Deleted in Colorectal Cancer (DCC) and other still unknown signaling cues. Commissural axon guidance defects are more severe in DCC mutant mice than Netrin-1 mutant mice, suggesting additional DCC activating signals besides Netrin-1 are involved in proper axon growth. Here we report that interaction screens on extracellular protein microarrays representing over 1,000 proteins uniquely identified Cerebellin 4 (CBLN4), a member of the C1q-tumor necrosis factor (TNF) family, and Netrin-1 as extracellular DCC-binding partners. Immunofluorescence and radio-ligand binding studies demonstrate that Netrin-1 competes with CBLN4 binding at an overlapping site within the membrane-proximal fibronectin domains (FN) 4–6 of DCC and binds with ∼5-fold higher affinity. CBLN4 also binds to the DCC homolog, Neogenin-1 (NEO1), but with a lower affinity compared to DCC. CBLN4-null mice did not show a defect in commissural axons of the developing spinal cord but did display a transient increase in the number of wandering axons in the brachial plexus, consistent with a role in axon guidance. Overall, the data solidifies CBLN4 as a bona fide DCC ligand and strengthens its implication in axon guidance.