Prophylactic and therapeutic activity of fully human monoclonal antibodies directed against Influenza A M2 protein

Influenza virus infection is a prevalent disease in humans. Antibodies against hemagglutinin have been shown to prevent infection and hence hemagglutinin is the major constituent of current vaccines. Antibodies directed against the highly conserved extracellular domain of M2 have also been shown to mediate protection against Influenza A infection in various animal models. Active vaccination is generally considered the best approach to combat viral diseases. However, passive immunization is an attractive alternative, particularly in acutely exposed or immune compromized individuals, young children and the elderly. We recently described a novel method for the rapid isolation of natural human antibodies by mammalian cell display. Here we used this approach to isolate human monoclonal antibodies directed against the highly conserved extracellular domain of the Influenza A M2 protein. The identified antibodies bound M2 peptide with high affinities, recognized native cell-surface expressed M2 and protected mice from a lethal influenza virus challenge. Moreover, therapeutic treatment up to 2 days after infection was effective, suggesting that M2-specific monoclonals have a great potential as immunotherapeutic agents against Influenza infection

Different Pattern of Immunoglobulin Gene Usage by HIV-1 Compared to Non-HIV-1 Antibodies Derived from the Same Infected Subject

A biased usage of immunoglobulin (Ig) genes is observed in human anti-HIV-1 monoclonal antibodies (mAbs) resulting probably from compensation to reduced usage of the VH3 family genes, while the other alternative suggests that this bias usage is due to antigen requirements. If the antigen structure is responsible for the preferential usage of particular Ig genes, it may have certain implications for HIV vaccine development by the targeting of particular Ig gene-encoded B cell receptors to induce neutralizing anti-HIV-1 antibodies. To address this issue, we have produced HIV-1 specific and non-HIV-1 mAbs from an infected individual and analyzed the Ig gene usage. Green-fluorescence labeled virus-like particles (VLP) expressing HIV-1 envelope (Env) proteins of JRFL and BaL and control VLPs (without Env) were used to select single B cells for the production of 68 recombinant mAbs. Ten of these mAbs were HIV-1 Env specific with neutralizing activity against V3 and the CD4 binding site, as well as non-neutralizing mAbs to gp41. The remaining 58 mAbs were non-HIV-1 Env mAbs with undefined specificities. Analysis revealed that biased usage of Ig genes was restricted only to anti-HIV-1 but not to non-HIV-1 mAbs. The VH1 family genes were dominantly used, followed by VH3, VH4, and VH5 among anti-HIV-1 mAbs, while non-HIV-1 specific mAbs preferentially used VH3 family genes, followed by VH4, VH1 and VH5 families in a pattern identical to Abs derived from healthy individuals. This observation suggests that the biased usage of Ig genes by anti-HIV-1 mAbs is driven by structural requirements of the virus antigens rather than by compensation to any depletion of VH3 B cells due to autoreactive mechanisms, according to the gp120 superantigen hypothesis

Isolation of Monoclonal Antibodies with Predetermined Conformational Epitope Specificity

Existing technologies allow isolating antigen-specific monoclonal antibodies (mAbs) from B cells. We devised a direct approach to isolate mAbs with predetermined conformational epitope specificity, using epitope mimetics (mimotopes) that reflect the three-dimensional structure of given antigen subdomains. We performed differential biopanning using bacteriophages encoding random peptide libraries and polyclonal antibodies (Abs) that had been affinity-purified with either native or denatured antigen. This strategy yielded conformational mimotopes. We then generated mimotope-fluorescent protein fusions, which were used as baits to isolate single memory B cells from rhesus monkeys (RMs). To amplify RM immunoglobulin variable regions, we developed RM-specific PCR primers and generated chimeric simian-human mAbs with predicted epitope specificity. We established proof-of-concept of our strategy by isolating mAbs targeting the conformational V3 loop crown of HIV Env; the new mAbs cross-neutralized viruses of different clades. The novel technology allows isolating mAbs from RMs or other hosts given experimental immunogens or infectious agents

IL-1α Mediated Chorioamnionitis Induces Depletion of FoxP3+ Cells and Ileal Inflammation in the Ovine Fetal Gut

Endotoxin induced chorioamnionitis increases IL-1 and provokes an inflammatory response in the fetal ileum that interferes with intestinal maturation. In the present study, we tested in an ovine chorioamnionitis model whether IL-1 is a major cytokine driving the inflammatory response in the fetal ileum.Sheep bearing singleton fetuses received a single intraamniotic injection of recombinant ovine IL-1α at 7, 3 or 1 d before caesarian delivery at 125 days gestational age (term = 150 days).3 and 7 d after IL-1α administration, intestinal mRNA levels for IL-4, IL-10, IFN-γ and TNF-α were strongly elevated. Numbers of CD3+ and CD4+ T-lymphocytes and myeloidperoxidase+ cells were increased whereas FoxP3+ T-cells were detected at low frequency. This increased proinflammatory state was associated with ileal mucosal barrier loss as demonstrated by decreased levels of the intestinal fatty acid binding protein and disruption of the tight junctional protein ZO-1.Intraamniotic IL-1α causes an acute detrimental inflammatory response in the ileum, suggesting that induction of IL-1 is a critical element in the pathophysiological effects of endotoxin induced chorioamnionitis. A disturbed balance between T-effector and FoxP3+ cells may contribute to this process

[3H]Adenine is a suitable radioligand for the labeling of G protein-coupled adenine receptors but shows high affinity to bacterial contaminations in buffer solutions

[3H]Adenine has previously been used to label the newly discovered G protein-coupled murine adenine receptors. Recent reports have questioned the suitability of [3H]adenine for adenine receptor binding studies because of curious results, e.g. high specific binding even in the absence of mammalian protein. In this study, we showed that specific [3H]adenine binding to various mammalian membrane preparations increased linearly with protein concentration. Furthermore, we found that Tris-buffer solutions typically used for radioligand binding studies (50 mM, pH 7.4) that have not been freshly prepared but stored at 4°C for some time may contain bacterial contaminations that exhibit high affinity binding for [3H]adenine. Specific binding is abolished by heating the contaminated buffer or filtering it through 0.2-μm filters. Three different, aerobic, gram-negative bacteria were isolated from a contaminated buffer solution and identified as Achromobacter xylosoxidans, A. denitrificans, and Acinetobacter lwoffii. A. xylosoxidans, a common bacterium that can cause nosocomial infections, showed a particularly high affinity for [3H]adenine in the low nanomolar range. Structure–activity relationships revealed that hypoxanthine also bound with high affinity to A. xylosoxidans, whereas other nucleobases (uracil, xanthine) and nucleosides (adenosine, uridine) did not. The nature of the labeled site in bacteria is not known, but preliminary results indicate that it may be a high-affinity purine transporter. We conclude that [3H]adenine is a well-suitable radioligand for adenine receptor binding studies but that bacterial contamination of the employed buffer solutions must be avoided

Cockroaches Probably Cleaned Up after Dinosaurs

Dinosaurs undoubtedly produced huge quantities of excrements. But who cleaned up after them? Dung beetles and flies with rapid development were rare during most of the Mesozoic. Candidates for these duties are extinct cockroaches (Blattulidae), whose temporal range is associated with herbivorous dinosaurs. An opportunity to test this hypothesis arises from coprolites to some extent extruded from an immature cockroach preserved in the amber of Lebanon, studied using synchrotron X-ray microtomography. 1.06% of their volume is filled by particles of wood with smooth edges, in which size distribution directly supports their external pre-digestion. Because fungal pre-processing can be excluded based on the presence of large particles (combined with small total amount of wood) and absence of damages on wood, the likely source of wood are herbivore feces. Smaller particles were broken down biochemically in the cockroach hind gut, which indicates that the recent lignin-decomposing termite and cockroach endosymbionts might have been transferred to the cockroach gut upon feeding on dinosaur feces

Influenza A virus-associated acute necrotizing encephalopathy in the United States

During the past several years, influenza-associated acute necrotizing encephalopathy has been well-recognized in Asia but has not yet been reported in the United States. We describe a 28-month-old patient who displayed the classical clinical features of acute necrotizing encephalopathy in association with a documented influenza A infection. This disease is characterized by fever, a rapid alteration in consciousness and seizures, with radiologic involvement of the bilateral thalami and cerebellum