Characterization of human monoclonal antibodies is providing considerable insight into mechanisms of broad HIV-1 neutralization. Here we report an HIV-1 gp41 membrane-proximal external region (MPER)-specific antibody, named 10E8, which neutralizes ~98% of tested viruses. An analysis of sera from 78 healthy HIV-1-infected donors demonstrated that 27% contained MPER-specific antibodies and 8% contained 10E8-like specificities. In contrast to other neutralizing MPER antibodies, 10E8 did not bind phospholipids, was not autoreactive, and bound cell-surface envelope. The structure of 10E8 in complex with the complete MPER revealed a site-of-vulnerability comprising a narrow stretch of highly conserved gp41-hydrophobic residues and a critical Arg/Lys just prior to the transmembrane region. Analysis of resistant HIV-1 variants confirmed the importance of these residues for neutralization. The highly conserved MPER is a target of potent, non-self-reactive neutralizing antibodies, suggesting that HIV-1 vaccines should aim to induce antibodies to this region of HIV-1 Env.

The isolation of human monoclonal antibodies (mAbs) is providing important insights regarding the specificities that underlie broad neutralization of HIV-1 (reviewed in1). Here we report a broad and extremely potent HIV-specific mAb, termed 35O22, which binds novel HIV-1 envelope glycoprotein (Env) epitope. 35O22 neutralized 62% of 181 pseudoviruses with an IC50<50 μg/ml. The median IC50 of neutralized viruses was 0.033 μg/ml, among the most potent thus far described. 35O22 did not bind monomeric forms of Env tested, but did bind the trimeric BG505 SOSIP.664. Mutagenesis and a reconstruction by negative-stain electron microscopy of the Fab in complex with trimer revealed it to bind a conserved epitope, which stretched across gp120 and gp41. The specificity of 35O22 represents a novel site of vulnerability on HIV Env, which serum analysis indicates to be commonly elicited by natural infection. Binding to this new site of vulnerability may thus be an important complement to current mAb-based approaches to immunotherapies, prophylaxis, and vaccine design.

Isolation of monoclonal antibodies is an important technique for understanding the specificities and characteristics of antibodies that underlie the humoral immune response to a given antigen. Here we describe a technique for isolating monoclonal antibodies from human peripheral blood mononuclear cells. The protocol includes strategies for the isolation of switch-memory B cells from peripheral blood, the culture of B cells, the removal of the supernatant for screening and the lysis of B cells in preparation for immunoglobulin heavy-chain and light-chain amplification and cloning. We have observed that the addition of cytokines IL-2, IL-21 and irradiated 3T3-msCD40L feeder cells can successfully stimulate switch-memory B cells to produce high concentrations of IgG in the supernatant. The supernatant may then be screened by appropriate assays for binding or for other functions. This protocol can be completed in 2 weeks. It is adaptable to use in other species and enables the efficient isolation of antibodies with a desired functional characteristic without prior knowledge of specificity.

The aryl hydrocarbon receptor (AHR) mediates the toxic effects of environmental contaminants, such as 2, 3,7,. Frogs are very insensitive to TCDD toxicity, and AHRs from Xenopus laevis (African clawed frog) bind TCDD with >20-fold lower affinity than mouse AHR b-1 . Frog AHRs may nonetheless be highly responsive to structurally distinct compounds, especially putative endogenous ligands. We sought to determine the responsiveness of an X. laevis cell line, XLK-WG, to the candidate endogenous AHR ligand 6-formylindolo[3,2-b]carbazole (FICZ), a tryptophan photoproduct that exhibits high potency in mammalian systems. FICZ readily induced mRNAs for CYP1A6 and CYP1A7. Cells exposed to FICZ for 3 hours expressed up to 5-fold greater quantities of CYP1A6/7 mRNAs than those exposed for 24 hours, suggesting FICZ is metabolized following rapid enzyme induction. FICZ appeared more potent than TCDD. Following a 3-hr exposure, the EC 50 for CYP1A6 mRNA induction by FICZ was ~6 nM, while the TCDD response was greater than 174 nM. These potencies were lower than those determined for mouse hepatoma cells (Hepa1c1c7; EC 50 = ~0.06 nM each). The difference in ligand potency between cell lines was confirmed by induction of ethoxyresorufin-O-deethylase (EROD) activity. mRNA from XLK-WG cells treated with 100 nM FICZ, 100 nM TCDD, or vehicle was also analyzed on expression microarrays. FICZ altered the expression of 105 more transcripts than TCDD, and common targets were altered more dramatically by FICZ. Overall, these studies demonstrate that although FICZ is a less potent CYP1A inducer in frog cells than in mouse cells, the reduction is much less than for TCDD. Relative conservation of the FICZ response in a TCDD-insensitive species suggests its physiological importance as an AHR ligand.

Antibodies directed against the HIV-1 Envelope glycoprotein are routinely assessed for in vitro viral neutralization. The most commonly used assay is based on infection of a cell line that expresses luciferase upon infection by HIV-1. Recently, several groups have published techniques for single-cell culture of B cells in 384-well plates. The standard 96 well neutralization assay format is not suitable for screening the large number of low-volume samples produced in such cultures. Therefore, we adapted the assays to a "microneutralization" format, which allows the use of 20 ul of undiluted antibody sample and permits an assessment of the relative potency of each sample. The method can be adapted for assays by hand or using robotic pipetting devices.

Ofek et al.: 454 pyrosequencing and bioinformatics analysis of the lineage of the broad and potent gp41-directed antibody 10e8. Retrovirology 2012 9(Suppl 2):O40.

Introduction Pancreatic pseudocysts are abnormal mature collections of pancreatic fluid that can develop in association with acute or chronic pancreatitis. Here, we share the discovery of an infected hepatic subcapsular pseudocyst of the pancreas causing septic shock following endoscopic retrograde cholangiopancreatography (ERCP). Presentation of Case A 55-year-old woman with ethanol-related chronic pancreatitis and biliary stricture was transferred to the ICU for hypotension 8 hours following ERCP. Examination revealed mild right upper quadrant tenderness without sign of peritonitis. Laboratory studies were notable for leukocytosis (14.6 k/L) and slightly elevated serum lipase (489 U/L). Abdominal CT scan revealed a previously undescribed subcapsular fluid collection. She underwent CT-guided percutaneous subcapsular drainage with return of opaque yellowish fluid. Fluid analysis showed elevated lipase of 62,901 U/L with cultures positive for ESBL Escherichia coli , Streptococcus constellatus , and Enterococcus faecium . Discussion A majority of pancreatic pseudocysts develop in peripancreatic regions, while, in a recent study, over a quarter of cases were found in usual sites. The management of subcapsular pseudocysts has not been standardized and often involves endoscopic or percutaneous drainage. Operative intervention is reserved for severe infection or rupture in patients with intrahepatic pseudocysts. Rarely do subcapsular pseudocysts become infected. In this case, we postulate the pseudocyst became seeded by bacteria during ERCP resulting in infection and then sepsis. Conclusion This case report highlights an atypical presentation of pancreatic pseudocyst as well as a rare septic complication of ERCP.