Further characterization of the binding of human recombinant interleukin 2 to heparin and identification of putative binding sites

We have previously provided compelling evidence that human recombinant interleukin 2 (IL-2) binds to the sulfated polysaccharides heparin, highly sulfated heparan sulfate and fucoidan. Here we show that IL-2 binding is dependent on heparin chain length, but with fragments as small as 15-mers retaining binding activity. The addition of exogenous heparin has no effect on the in vitro biological activity of IL-2. In addition soluble IL-2 receptor alpha and beta polypeptides do not compete with heparin for the binding of IL-2. IL-2 bound by heparin is still recognized by two IL-2 specific monoclonal antibodies, 3H9 and H2- 8, whose epitopes lie in the amino terminal region. Murine IL-2 unlike its human counterpart fails to bind to heparin. Human IL-2 analogs with single amino acid substitutions at positions Lys43, Thr51, and Gln126 analogs no longer bind to heparin. By contrast the Arg38Ala analog retains heparin full heparin binding activity. These experimental findings together with molecular modeling studies suggest two putative heparin binding sites on human IL-2, one involving four basic residues, Lys48, Lys49, Lys54, and His55, and the other being a discontinuous site comprising Lys43, Lys64, Arg81, and Arg83. Neither of these two clusters is completely conserved in murine IL-2. Overall our data suggest that the binding of human IL-2 to heparin and heparan sulfate does not interfere with IL-2/IL-2 receptor interactions. Therefore, binding to glycosaminoglycan may be a mechanism for retaining the cytokine in an active form close to its site of secretion in the tissue, thus favoring a paracrine role for IL-2.      

Efflux pumps expression and its association with porin down-regulation and β-lactamase production among <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> causing bloodstream infections in Brazil

Background  

Multi-drug efflux pumps have been increasingly recognized as a major component of resistance in P. aeruginosa. We have investigated the expression level of efflux systems among clinical isolates of P. aeruginosa, regardless of their antimicrobial susceptibility profile.     

Aspidosperma (Apocynaceae) plant cytotoxicity and activity towards malaria parasites. Part II: experimental studies with Aspidosperma ramiflorum in vivo and in vitro

Several species of Aspidosperma plants are used to treat diseases in the tropics, including Aspidosperma ramiflorum, which acts against leishmaniasis, an activity that is experimentally confirmed. The species, known as guatambu-yellow, yellow peroba, coffee-peroba andmatiambu, grows in the Atlantic Forest of Brazil in the South to the Southeast regions. Through a guided biofractionation of A. ramiflorum extracts, the plant activity against Plasmodium falciparum was evaluated in vitro for toxicity towards human hepatoma G2 cells, normal monkey kidney cells and nonimmortalised human monocytes isolated from peripheral blood. Six of the seven extracts tested were active at low doses (half-maximal drug inhibitory concentration < 3.8 µg/mL); the aqueous extract was inactive. Overall, the plant extracts and the purified compounds displayed low toxicity in vitro. A nonsoluble extract fraction and one purified alkaloid isositsirikine (compound 5) displayed high selectivity indexes (SI) (= 56 and 113, respectively), whereas compounds 2 and 3 were toxic (SI < 10). The structure, activity and low toxicity of isositsirikine in vitro are described here for the first time in A. ramiflorum, but only the neutral and precipitate plant fractions were tested for activity, which caused up to 53% parasitaemia inhibition of Plasmodium berghei in mice with blood-induced malaria. This plant species is likely to be useful in the further development of an antimalarial drug, but its pharmacological evaluation is still required.     

A Novel Model of Lethal Hendra Virus Infection in African Green Monkeys and the Effectiveness of Ribavirin Treatment

The henipaviruses, Hendra virus (HeV) and Nipah virus (NiV), are emerging zoonotic paramyxoviruses that can cause severe and often lethal neurologic and/or respiratory disease in a wide variety of mammalian hosts, including humans. There are presently no licensed vaccines or treatment options approved for human or veterinarian use. Guinea pigs, hamsters, cats, and ferrets, have been evaluated as animal models of human HeV infection, but studies in nonhuman primates (NHP) have not been reported, and the development and approval of any vaccine or antiviral for human use will likely require efficacy studies in an NHP model. Here, we examined the pathogenesis of HeV in the African green monkey (AGM) following intratracheal inoculation. Exposure of AGMs to HeV produced a uniformly lethal infection, and the observed clinical signs and pathology were highly consistent with HeV-mediated disease seen in humans. Ribavirin has been used to treat patients infected with either HeV or NiV; however, its utility in improving outcome remains, at best, uncertain. We examined the antiviral effect of ribavirin in a cohort of nine AGMs before or after exposure to HeV. Ribavirin treatment delayed disease onset by 1 to 2 days, with no significant benefit for disease progression and outcome. Together our findings introduce a new disease model of acute HeV infection suitable for testing antiviral strategies and also demonstrate that, while ribavirin may have some antiviral activity against the henipaviruses, its use as an effective standalone therapy for HeV infection is questionable.Hendra virus (HeV) and Nipah virus (NiV) are members of the genus Henipavirus (family Paramyxoviridae) that can cause severe respiratory illness and/or encephalitis in a wide variety of mammals, including horses, pigs, and humans (7, 23). HeV was identified as the causative agent of an acute respiratory disease in horses in 1994 in Queensland, Australia (23), and to date there have been 14 outbreaks in Australia since, with at least one occurrence per year since 2006, most recently in May 2010 (ProMed-mail no. 20100522.1699 [International Society for Infectious Diseases, http://www.promedmail.org]). Every outbreak of HeV has involved horses as the initial infected host, and there have been a total of seven human cases arising from exposure to infected horses. Four human fatalities have occurred (22), with the most recent occurring in August of 2009 (ProMed-mail no. 20090826.2998 and 20090903.3098). All patients initially presented with influenza-like illnesses (ILIs) after an incubation period of 7 to 16 days. While two individuals recovered from ILI, one patient developed pneumonitis and died from multiorgan failure. Three of the lethal cases developed encephalitic manifestations (mild confusion and ataxia), with two patients experiencing seizures (22, 23, 27).Data on the histopathology of fatal human HeV cases are limited, but the pathology includes small necrotic plaques in the cerebrum and cerebellum, in addition to mild parenchymal inflammation (21, 27). Severe parenchymal inflammation and necrosis were observed in the lungs. More extensive histopathologic data are available from 32 autopsies of fatal human NiV cases (28). Similarly to the HeV cases, pathology was characterized by systemic vasculitis and parenchymal necrosis in the central nervous system (CNS), while in the lung, pathological findings mainly included vasculitis, fibrinoid necrosis, alveolar hemorrhage, pulmonary edema, and aspiration pneumonia. Other organs that were affected included heart, kidney, and spleen and showed generally mild or focal inflammation. The development of syncytial multinucleated endothelial cells is characteristic of both HeV and NiV (27, 28). At present, the details of the pathogenesis and histopathological changes mediated by either HeV or NiV infection in humans are naturally derived from only the late phases of the disease course, and therefore a relevant animal model is needed that mimics the disease progression seen in humans.Pteropid fruit bats, commonly known as flying foxes in the family Pteropodidae, are the principle natural reservoirs for both NiV and HeV (reviewed in reference 3). However, these henipaviruses display a broad species tropism, and in addition to bats, horses and humans, natural and/or experimental infection of HeV has been demonstrated in guinea pigs, hamsters, pigs, cats, and ferrets (25). Experimental infections of Syrian hamsters with HeV is lethal, and animals show disease similar to that of human cases, including respiratory and neurological symptoms, depending on the dose (11; unpublished data). In this model, viral RNA can be detected in various organs of infected hamsters, including brain, lung, kidney, heart, liver, and spleen. The main histopathological findings included parenchymal infection in various organs, including the brain, with vasculitis and syncytial multinucleated endothelial cells in many blood vessels (11). While this model is useful in studying pathogenesis, it is limited in the availability of reagents to do so.There are currently no vaccines or treatments licensed for human use. Several in vitro studies have shown that ribavirin is effective against both HeV and NiV infection (1, 2, 29). An open-label ribavirin treatment trial was run during an outbreak of NiV in Malaysia in 1998 and reported to reduce mortality by 36% (6). Of the seven recorded human HeV cases, three patients were treated with ribavirin, one of whom survived (22). In the most recent outbreak of HeV in Australia, three additional people received ribavirin treatment in combination with chloroquine after suspected exposure to HeV-contaminated secretions from infected horses. While all three individuals survived, infection was not confirmed, and therefore it remains unknown whether the treatment had any beneficiary effect (ProMed-mail no. 20090826.2998). In addition, two animal studies in hamsters showed that ribavirin treatment delays but does not prevent death from NiV or HeV infection (8, 10). Therefore, an animal model with greater relevance to humans and that recapitulates the disease processes seen in human cases of HeV is needed to get a better answer to whether ribavirin might be effective against henipavirus infections. In addition, the U.S. FDA implemented the “Animal Efficacy Rule,” which specifically applies to the development of therapeutic products when human efficacy studies are not possible or ethical, such as is often the case with highly virulent pathogens like HeV (24). Essentially, this rule allows for the evaluation of vaccines or therapeutics using data derived from studies carried out in at least two animal models. The licensure of any therapeutic modalities for HeV will require a thorough evaluation of HeV pathogenesis in nonhuman primates (NHPs).In the present study, we report the development and characterization of a new nonhuman primate (NHP) model of lethal HeV infection in the African green monkey (AGM). The pathogenesis and disease progression in the AGM upon HeV infection essentially mirrored the lethal disease episodes seen among human cases of HeV. Using this new model, the efficacy of ribavirin treatment against lethal challenge with HeV was examined. Here we have shown that ribavirin treatment can significantly delay but not prevent death of AGMs from lethal HeV infection. In addition to severe respiratory symptoms in all animals, prolonged disease progression in ribavirin-treated animals was also marked by the appearance of neurological symptoms.     

Biochemical, Conformational, and Immunogenic Analysis of Soluble Trimeric Forms of Henipavirus Fusion Glycoproteins

The henipaviruses, Hendra virus (HeV) and Nipah virus (NiV), are paramyxoviruses discovered in the mid- to late 1990s that possess a broad host tropism and are known to cause severe and often fatal disease in both humans and animals. HeV and NiV infect cells by a pH-independent membrane fusion mechanism facilitated by their attachment (G) and fusion (F) glycoproteins. Here, several soluble forms of henipavirus F (sF) were engineered and characterized. Recombinant sF was produced by deleting the transmembrane (TM) and cytoplasmic tail (CT) domains and appending a glycosylphosphatidylinositol (GPI) anchor signal sequence followed by GPI-phospholipase D digestion, appending a trimeric coiled-coil (GCNt) domain (sF_GCNt), or deleting the TM, CT, and fusion peptide domain. These sF glycoproteins were produced as F₀ precursors, and all were apparent stable trimers recognized by NiV-specific antisera. Surprisingly, however, only the GCNt-appended constructs (sF_GCNt) could elicit cross-reactive henipavirus-neutralizing antibody in mice. In addition, sF_GCNt constructs could be triggered in vitro by protease cleavage and heat to transition from an apparent prefusion to postfusion conformation, transitioning through an intermediate that could be captured by a peptide corresponding to the C-terminal heptad repeat domain of F. The pre- and postfusion structures of sF_GCNt and non-GCNt-appended sF could be revealed by electron microscopy and were distinguishable by F-specific monoclonal antibodies. These data suggest that only certain sF constructs could serve as potential subunit vaccine immunogens against henipaviruses and also establish important tools for further structural, functional, and diagnostic studies on these important emerging viruses.     

Ephrin-B2 and ephrin-B3 as functional henipavirus receptors

Members of the ephrin cell-surface protein family interact with the Eph receptors, the largest family of receptor tyrosine kinases, mediating bi-directional signaling during tumorogenesis and various developmental events. Surprisingly, ephrin-B2 and -B3 were recently identified as entry receptors for henipaviruses, emerging zoonotic paramyxoviruses responsible for repeated outbreaks in humans and animals in Australia, Southeast Asia, India and Bangladesh. Nipah virus (NiV) and Hendra virus (HeV) are the only two identified members in the henipavirus genus. While the initial human infection cases came from contact with infected pigs (NiV) or horses (HeV), in the more recent outbreaks of NiV both food-borne and human-to-human transmission were reported. These characteristics, together with high mortality and morbidity rates and lack of effective anti-viral therapies, make the henipaviruses a potential biological-agent threat. Viral entry is an important target for the development of anti-viral drugs. The entry of henipavirus is initiated by the attachment of the viral G envelope glycoprotein to the host cell receptors ephrin-B2 and/or -B3, followed by activation of the F fusion protein, which triggers fusion between the viral envelop and the host membrane. We review recent progress in the study of henipavirus entry, particularly the identification of ephrins as their entry receptors, and the structural characterization of the ephrin/Henipa-G interactions.     

Cross-reactive human immunodeficiency virus type 1-neutralizing human monoclonal antibody that recognizes a novel conformational epitope on gp41 and lacks reactivity against self-antigens

Broadly cross-reactive human immunodeficiency virus (HIV)-neutralizing antibodies are infrequently elicited in infected humans. The two best-characterized gp41-specific cross-reactive neutralizing human monoclonal antibodies, 4E10 and 2F5, target linear epitopes in the membrane-proximal external region (MPER) and bind to cardiolipin and several other autoantigens. It has been hypothesized that, because of such reactivity to self-antigens, elicitation of 2F5 and 4E10 and similar antibodies by vaccine immunogens based on the MPER could be affected by tolerance mechanisms. Here, we report the identification and characterization of a novel anti-gp41 monoclonal antibody, designated m44, which neutralized most of the 22 HIV type 1 (HIV-1) primary isolates from different clades tested in assays based on infection of peripheral blood mononuclear cells by replication-competent virus but did not bind to cardiolipin and phosphatidylserine in an enzyme-linked immunosorbent assay and a Biacore assay nor to any protein or DNA autoantigens tested in Luminex assays. m44 bound to membrane-associated HIV-1 envelope glycoproteins (Envs), to recombinant Envs lacking the transmembrane domain and cytoplasmic tail (gp140s), and to gp41 structures containing five-helix bundles and six-helix bundles, but not to N-heptad repeat trimers, suggesting that the C-heptad repeat is involved in m44 binding. In contrast to 2F5, 4E10, and Z13, m44 did not bind to any significant degree to denatured gp140 and linear peptides derived from gp41, suggesting a conformational nature of the epitope. This is the first report of a gp41-specific cross-reactive HIV-1-neutralizing human antibody that does not have detectable reactivity to autoantigens. Its novel conserved conformational epitope on gp41 could be helpful in the design of vaccine immunogens and as a target for therapeutics.