Blood-feeding ticks must control C activation or be damaged by the host inflammatory response. We report the characterization and expression of a novel, relatively small, broad-acting C inhibitory protein (termed OmCI) from the soft tick Ornithodoros moubata. The native 17-kDa nonglycosylated protein inhibits both human and guinea pig classical and alternative C activation pathways. The IC50 values for each pathway were 12 and 27 nM, respectively, in hemolytic assays using human serum diluted 40-fold. The cDNA encodes a protein of 168 aa, including an 18-aa secretion signal sequence that is absent in the mature form. The inhibitor has 46% amino acid identity with moubatin, a platelet aggregation inhibitor also from O. moubata that is an outlying member of the lipocalin family. Native OmCI had no inhibitory effect on the addition of C8 and C9 to preformed C5b-C7 and C5b-C8 to form the membrane attack complex and no effect on the rate of C3a production by the C3 convertase enzymes C4bC2a, C3(H2O)Bb, or C3bBb. Both recombinant and native OmCI abolish production of C5a by human classical (C4bC3bC2a) and alternative (C3bC3bBb) C5 convertases. Addition of excess C5 but not C3 competes away the inhibitory activity of OmCI, indicating that OmCI targets C5 itself rather than inhibiting the C5 convertase C4bC3bC2a itself. Direct binding of OmCI to C5 was demonstrated by Western blotting and gel filtration chromatography using 125I-labeled proteins. OmCI is the first lipocalin family member shown to inhibit C and also the first natural inhibitor that specifically targets the C5 activation step.

Activation of complement C5 generates the potent anaphylatoxin C5a and leads to pathogen lysis, inflammation and cell damage. The therapeutic potential of C5 inhibition has been demonstrated by eculizumab, one of the world's most expensive drugs. However, the mechanism of C5 activation by C5 convertases remains elusive, thus limiting development of therapeutics. Here we identify and characterize a new protein family of tick-derived C5 inhibitors. Structures of C5 in complex with the new inhibitors, the phase I and phase II inhibitor OmCI, or an eculizumab Fab reveal three distinct binding sites on C5 that all prevent activation of C5. The positions of the inhibitor-binding sites and the ability of all three C5-inhibitor complexes to competitively inhibit the C5 convertase conflict with earlier steric-inhibition models, thus suggesting that a priming event is needed for activation.

To provide insight into the structural and functional properties of human complement component 5 (C5), we determined its crystal structure at a resolution of 3.1 A. The core of C5 adopted a structure resembling that of C3, with the domain arrangement at the position corresponding to the C3 thioester being very well conserved. However, in contrast to C3, the convertase cleavage site in C5 was ordered and the C345C domain flexibly attached to the core of C5. Binding of the tick C5 inhibitor OmCI to C5 resulted in stabilization of the global conformation of C5 but did not block the convertase cleavage site. The structure of C5 may render possible a structure-based approach for the design of new selective complement inhibitors.

Mosquito-borne diseases cause substantial mortality and morbidity worldwide. These impacts are widely predicted to increase as temperatures warm and extreme precipitation events become more frequent, since mosquito biology and disease ecology are strongly linked to environmental conditions. However, direct evidence linking environmental change to changes in mosquito-borne disease is rare, and the ecological mechanisms that may underpin such changes are poorly understood. Environmental drivers, such as temperature, can have non-linear, opposing impacts on the demographic rates of different mosquito life cycle stages. As such, model frameworks that can deal with fluctuations in temperature explicitly are required to predict seasonal mosquito abundance, on which the intensity and persistence of disease transmission under different environmental scenarios depends. We present a novel, temperature-dependent, delay-differential equation model, which incorporates diapause and the differential effects of temperature on the duration and mortality of each life stage and demonstrates the sensitivity of seasonal abundance patterns to inter- and intra-annual changes in temperature. Likely changes in seasonal abundance and exposure to mosquitoes under projected changes in UK temperatures are presented, showing an increase in peak vector abundance with warming that potentially increases the risk of disease outbreaks.

BackgroundThe threat of pandemic influenza has focused attention and resources on virus surveillance, prevention, and containment. The World Health Organization has strongly recommended the use of the antiviral drug Tamiflu both to treat and prevent pandemic influenza infection. A major concern for the long-term efficacy of this strategy is to limit the development of Tamiflu-resistant influenza strains. However, in the event of a pandemic, hundreds of millions of courses of Tamiflu, stockpiled globally, will be rapidly deployed. Given its apparent resistance to biodegradation and hydrophilicity, oseltamivir carboxylate (OC), the active antiviral and metabolite of Tamiflu, is predicted to enter receiving riverwater from sewage treatment works in its active form.ObjectiveOur objective in this study was to determine the likely concentrations of OC released into U.S. and U.K. river catchments using hydrologic modeling and current assumptions about the course and management of an influenza pandemic.DiscussionWe predict that high concentrations of OC (micrograms per liter) capable of inhibiting influenza virus replication would be sustained for periods of several weeks, presenting an increased risk for the generation of antiviral resistance and genetic exchange between influenza viruses in wildfowl. Owing to the apparent recalcitrance of OC in sewage treatment works, widespread use of Tamiflu during an influenza pandemic also poses a potentially significant, uncharacterized, ecotoxicologic risk in each affected nation’s waterways.ConclusionTo gauge the hazard presented by Tamiflu use during a pandemic, we recommend a) direct measurement of Tamiflu persistence, biodegradation, and transformation in the environment; b) further modeling of likely drug concentrations in the catchments of countries where humans and waterfowl come into frequent close contact, and where significant Tamiflu deployment is envisaged; and c) further characterization of the risks of generating Tamiflu-resistant viruses in OC-exposed wildfowl.

Synopsis The saliva of blood-feeding parasites is a rich source of peptidase inhibitors that help overcome the host’s defense during host-parasite interactions. Using proteomic analysis, the cystatin OmC2 was demonstrated in the saliva of the soft tick Ornithodoros moubata, an important disease-vector transmitting African swine fever virus and the spirochaete Borrelia duttoni. A structural, biochemical and biological characterization of this peptidase inhibitor was undertaken. Recombinant OmC2 was screened against a panel of physiologically relevant peptidases and found to be an effective broad-specificity inhibitor of cysteine cathepsins, including endopeptidases (cathepsins L and S) and exopeptidases (cathepsins B, C and H). The crystal structure of OmC2 was determined at a resolution of 2.45 Å and used to describe the structure-inhibitory activity relationship. The biological impact of OmC2 was demonstrated both in vitro and in vivo. OmC2 affected the function of antigen-presenting mouse dendritic cells by reducing the production of the proinflammatory cytokines TNF-α and IL-12, and proliferation of antigen-specific CD4+ T cells. This suggests that OmC2 may suppress the host’s adaptive immune response. Immunization of mice with OmC2 significantly suppressed the survival of O. moubata in infestation experiments. We conclude that OmC2 is a promising target for the development of a novel anti-tick vaccine to control O. moubata populations and combat the spread of associated diseases.

Influenza virus A (IAV) causes annual epidemics and intermittent pandemics that affect millions of people worldwide. Potent inflammatory responses are commonly associated with severe cases of IAV infection. The complement system, an important mechanism of innate and humoral immune responses to infections, is activated during primary IAV infection and mediates, in association with natural IgM, viral neutralization by virion aggregation and coating of viral hemmagglutinin. Increased levels of the anaphylatoxin C5a were found in patients fatally infected with the most recent H1N1 pandemic virus. In this study, our aim was to evaluate whether targeting C5 activation alters inflammatory lung injury and viral load in a murine model of IAV infection. To address this question C57Bl/6j mice were infected intranasally with 104 PFU of the mouse adapted Influenza A virus A/WSN/33 (H1N1) or inoculated with PBS (Mock). We demonstrated that C5a is increased in bronchoalveolar lavage fluid (BALF) upon experimental IAV infection. To evaluate the role of C5, we used OmCI, a potent arthropod-derived inhibitor of C5 activation that binds to C5 and prevents release of C5a by complement. OmCI was given daily by intraperitoneal injection from the day of IAV infection until day 5. Treatment with OmCI only partially reduced C5a levels in BALF. However, there was significant inhibition of neutrophil and macrophage infiltration in the airways, Neutrophil Extracellular Traps (NETs) formation, death of leukocytes, lung epithelial injury and overall lung damage induced by the infection. There was no effect on viral load. Taken together, these data suggest that targeting C5 activation with OmCI during IAV infection could be a promising approach to reduce excessive inflammatory reactions associated with the severe forms of IAV infections.

The involvement of complement (C) in inflammatory diseases has driven the search for agents capable of inhibiting dysregulated complement activation. Many of these reagents inhibit the C3 convertases during the early stages of the cascade. However, a drawback of total systemic C inhibition, particularly in longterm treatment of chronic disease, is potentiation of infection and immune complex disease due to an inability to opsonize complexes and foreign cells and to lyse pathogens. Recent identification of a C5-binding protein in the salivary gland of the soft tick Ornithodoros moubata has enabled development of a terminal pathway-specific reagent, OmCI, with potential to ameliorate disease while leaving key physiological processes unaffected. Here we demonstrated that OmCI has broad cross-species activity. When given intravenously to rodents, OmCI totally ablated complement hemolytic activity, which gradually restored as C5 was resynthesized. The circulating half-life of OmCI was 30 h, demonstrating a much slower clearance than other small, biological agents. Using C5-sufficient and C5-deficient mice we showed that prolonged half-life was due to binding to plasma C5. Surface plasmon resonance analysis of C5 binding to OmCI confirmed a high binding affinity with a slow dissociation rate. OmCI was effective in preventing experimental autoimmune myasthenia gravis induced by passive transfer in normal Lewis rats. OmCI ablated clinical disease, reduced C3 and C9 deposition at the neuro-muscular junction, and effected a marked reduction in cellular infiltration at this site. These data offer exciting prospects for targeted treatment of complement-mediated diseases without the detrimental inhibition of the opsonic roles of complement.