Annalisa Macagno

A Neutralizing Antibody Selected from Plasma Cells That Binds to Group 1 and Group 2 Influenza A Hemagglutinins

An antibody able to broadly neutralize both group 1 and group 2 influenza A viruses—and its target epitope—are identified. The isolation of broadly neutralizing antibodies against influenza A viruses has been a long-sought goal for therapeutic approaches and vaccine design. Using a single-cell culture method for screening large numbers of human plasma cells, we isolated a neutralizing monoclonal antibody that recognized the hemagglutinin (HA) glycoprotein of all 16 subtypes and neutralized both group 1 and group 2 influenza A viruses. Passive transfer of this antibody conferred protection to mice and ferrets. Complexes with HAs from the group 1 H1 and the group 2 H3 subtypes analyzed by x-ray crystallography showed that the antibody bound to a conserved epitope in the F subdomain. This antibody may be used for passive protection and to inform vaccine design because of its broad specificity and neutralization potency.

Isolation of Human Monoclonal Antibodies That Potently Neutralize Human Cytomegalovirus Infection by Targeting Different Epitopes on the gH/gL/UL128-131A Complex

ABSTRACT Human cytomegalovirus (HCMV) is a widely circulating pathogen that causes severe disease in immunocompromised patients and infected fetuses. By immortalizing memory B cells from HCMV-immune donors, we isolated a panel of human monoclonal antibodies that neutralized at extremely low concentrations (90% inhibitory concentration [IC90] values ranging from 5 to 200 pM) HCMV infection of endothelial, epithelial, and myeloid cells. With the single exception of an antibody that bound to a conserved epitope in the UL128 gene product, all other antibodies bound to conformational epitopes that required expression of two or more proteins of the gH/gL/UL128-131A complex. Antibodies against gB, gH, or gM/gN were also isolated and, albeit less potent, were able to neutralize infection of both endothelial-epithelial cells and fibroblasts. This study describes unusually potent neutralizing antibodies against HCMV that might be used for passive immunotherapy and identifies, through the use of such antibodies, novel antigenic targets in HCMV for the design of immunogens capable of eliciting previously unknown neutralizing antibody responses.

Dendritic cells up-regulate immunoproteasomes and the proteasome regulator PA28 during maturation.

Dendritic cells (DC) are highly specialized professional antigen presenting cells which are pivotal for the initiation and control of the cytotoxic T cell response. Upon stimulation by cytokines, bacteria, or CD40L DC undergo a maturation process from an antigen‐receptive state to a state of optimal stimulation of T cells. We investigated the composition of proteasomes of DC derived from human peripheral blood monocytes before and after stimulation by CD40L, LPS, or proinflammatory cytokines (TNF‐α + IL‐6 + IL‐1β). Immunoprecipitation of proteasomes and analysis on two‐dimensional gels revealed that during maturation the inducible proteasome subunits LMP2, LMP7, and MECL‐1 are up‐regulated and that the neosynthesis of proteasomes is switched exclusively to the production of immunoproteasomes containing these subunits. The proteasome regulator PA28 is markedly up‐regulated in mature DC and in addition a so – far unidentified 21‐kDa protein co‐precipitates with the proteasome in LPS – stimulated DC. These changes in proteasome composition may be functionally linked to special properties of DC like MHC class I up‐regulation or cross‐priming. Our findings imply that the spectrum of class I‐bound peptides may change after DC maturation which could be relevant for the design of DC – based vaccines.

A cyanobacterial LPS antagonist prevents endotoxin shock and blocks sustained TLR4 stimulation required for cytokine expression

Toll-like receptors (TLRs) function as primary sensors that elicit coordinated innate immune defenses through recognition of microbial products and induction of immune and proinflammatory genes. Here we report the identification and biological characterization of a lipopolysaccharide (LPS)-like molecule extracted from the cyanobacterium Oscillatoria Planktothrix FP1 (cyanobacterial product [CyP]) that is not stimulatory per se but acts as a potent and selective antagonist of bacterial LPS. CyP binds to MD-2 and efficiently competes with LPS for binding to the TLR4–MD-2 receptor complex. The addition of CyP together with LPS completely inhibited both MyD88- and TRIF-dependent pathways and suppressed the whole LPS-induced gene transcription program in human dendritic cells (DCs). CyP protected mice from endotoxin shock in spite of a lower capacity to inhibit LPS stimulation of mouse DCs. Interestingly, the delayed addition of CyP to DCs responding to LPS strongly inhibited signaling and cytokine production by immediate down-regulation of inflammatory cytokine mRNAs while not affecting other aspects of DC maturation, such as expression of major histocompatibility complex molecules, costimulatory molecules, and CCR7. Collectively, these results indicate that CyP is a potent competitive inhibitor of LPS in vitro and in vivo and reveal the requirement of sustained TLR4 stimulation for induction of cytokine genes in human DCs.

Pronounced up‐regulation of the PA28α/β proteasome regulator but little increase in the steady‐state content of immunoproteasome during dendritic cell maturation

Dendritic cells (DC) are professional antigen‐presenting cells that activate CTL by presenting MHC class I‐restricted peptides that are processed through the proteasome pathway. Previously, wereported that upon DC maturation the synthesis is switched towards the exclusive production of immunoproteasomes containing the active site subunits LMP2, LMP7 and MECL‐1. In this study we investigated the mechanism by which proteasome assembly is regulated in mature DC. Quantitative analysis of mRNA expression showed very limited transcriptional induction of LMP7, MECL‐1 and UMP1 in mature DC and a moderate mRNA increment for LMP2 and PA28α and β. We investigated a role of PA28α/β in regulating proteasome assembly in DC. PA28α/β coprecipitated with 13S/16S proteasome precursor complexes but associated with mature constitutive and immunoproteasomes to the same extent. Furthermore, we determined the steady‐state proteasome subunit composition in DC. Replacement of constitutive proteasomes by immunoproteasomes in maturing DC was very slow and occurred only to a minor extent. Our data suggest that the limited turnover of 20S proteasomes in mature DC probably contributes little to recently reported marked differences in antigen presentation between immature and mature DC and that alternative mechanisms may be responsible for this phenomenon.

A Cyanobacterial Lipopolysaccharide Antagonist Inhibits Cytokine Production Induced by Neisseria meningitidis in a Human Whole-Blood Model of Septicemia

ABSTRACT Septicemia caused by Neisseria meningitidis is characterized by increasing levels of meningococcal lipopolysaccharide (Nm-LPS) and cytokine production in the blood. We have used an in vitro human whole-blood model of meningococcal septicemia to investigate the potential of CyP, a selective Toll-like receptor 4 (TLR4)-MD-2 antagonist derived from the cyanobacterium Oscillatoria planktothrix FP1, for reducing LPS-mediated cytokine production. CyP (≥1 μg/ml) inhibited the secretion of the proinflammatory cytokines tumor necrosis factor alpha, interleukin-1β (IL-1β), and IL-6 (by >90%) and chemokines IL-8 and monocyte chemoattractant protein 1 (by ∼50%) induced by the treatment of blood with pure Nm-LPS, by isolated outer membranes, and after infection with live meningococci of different serogroups. In vitro studies with human dendritic cells and TLR4-transfected Jurkat cells demonstrated that CyP competitively inhibited Nm-LPS interactions with TLR4 and subsequent NF-κB activation. These data demonstrate that CyP is a potent antagonist of meningococcal LPS and could be considered a new adjunctive therapy for treating septicemia.

Rapid Structural Characterization of Human Antibody–Antigen Complexes through Experimentally Validated Computational Docking

If we understand the structural rules governing antibody (Ab)-antigen (Ag) interactions in a given virus, then we have the molecular basis to attempt to design and synthesize new epitopes to be used as vaccines or optimize the antibodies themselves for passive immunization. Comparing the binding of several different antibodies to related Ags should also further our understanding of general principles of recognition. To obtain and compare the three-dimensional structure of a large number of different complexes, however, we need a faster method than traditional experimental techniques. While biocomputational docking is fast, its results might not be accurate. Combining experimental validation with computational prediction may be a solution. As a proof of concept, here we isolated a monoclonal Ab from the blood of a human donor recovered from dengue virus infection, characterized its immunological properties, and identified its epitope on domain III of dengue virus E protein through simple and rapid NMR chemical shift mapping experiments. We then obtained the three-dimensional structure of the Ab/Ag complex by computational docking, using the NMR data to drive and validate the results. In an attempt to represent the multiple conformations available to flexible Ab loops, we docked several different starting models and present the result as an ensemble of models equally agreeing with the experimental data. The Ab was shown to bind a region accessible only in part on the viral surface, explaining why it cannot effectively neutralize the virus.

Cyanobacterial LPS antagonist (CyP)-A novel and efficient inhibitor of Escherichia coli LPS-induced cytokine response in the pig

Toll-like receptors are essential pattern-recognition receptors of the innate immune system. They recognize a range of conserved molecules of invading microorganisms. The innate immune system is developed to protect the host, but can be deleterious if activated uncontrolled or inappropriate, such as in sepsis with Gram-negative bacteria. New approaches for treatment, like inhibition of innate immune responses, may be beneficial for the outcome of such conditions. Toll-like receptor 4 associated with CD14 and MD-2, is the lipopolysaccharide (LPS)-receptor and one of the candidates for such intervention. We investigated the newly described cyanobacterial LPS analogue CyP as a potential inhibitor of Escherichia coli (E. coli) LPS-induced inflammatory response in porcine whole blood. Pro-inflammatory cytokines and soluble terminal complement complex, sC5b-9, were used as read-outs. CyP, in contrast to E. coli LPS, did not induce cytokine production using doses up to 1mug/mL whole blood, indicating a lack of agonistic effect of CyP. In contrast, CyP was an efficient LPS antagonist, dose-dependently and completely inhibiting E. coli LPS-induced TNF-alpha, IL-1beta and IL-8 production. CyP was a modest activator of porcine complement compared to LPS from other Gram-negative bacteria. When CyP was pre-incubated in porcine whole blood before adding whole E. coli bacteria, a modest, variable and non-significant inhibition of cytokines were seen, reaching an average inhibition of 44% for IL-1beta. We have demonstrated for the first time that the cyanobacterial LPS analogue, CyP, is an efficient inhibitor of E. coli LPS-induced cytokines in whole blood and may be a candidate for therapeutic LPS-inhibition.

Pneumocystis carinii Does Not Induce Maturation of Human Dendritic Cells

Pneumocystis carinii is an opportunistic pulmonary pathogen that causes significant clinical complications in patients with immunodeficiency diseases. In particular, P. carinii pneumonia (PCP) is a severe complication in subjects with HIV infection. The importance of alveolar macrophages as a f i t line of host defense against P. carinii, the involvement of antigen-specific CD4+ and CDS+ T cells in the clearance of P. carinii, and inflammation-associated respiratory impairment during PCP have been demonstrated [5,6,10]. On the other hand, the ability to initiate primary immune responses is a unique property of dendritic cells (DCs). which are found in an immature state at the periphery, that function as sentinels of the immune system. Upon uptake of antigens, DCs undergo a maturation process that induces their migration to the draining lymph nodes where they present peptides and efficiently stimulate naTve T cells. DCs determine Thl versus Th2 polarization of CD4+ cells in coordination with activation of CD8+ cytotoxic T cells and therefore play a central role in the regulation of specific immune responses. On this basis, we decided to investigate the potential involvement of DCs in the host response against P. carinii infection.