Antileishmanial Aminopyrazoles: Studies into Mechanisms and Stability of Experimental Drug Resistance.

Current antileishmanial treatment is hampered by limitations, such as drug toxicity and the risk of treatment failure, which may be related to parasitic drug resistance. Given the urgent need for novel drugs, the Drugs for Neglected Diseases initiative (DNDi) has undertaken a drug discovery program, which has resulted in the identification of aminopyrazoles, a highly promising antileishmanial chemical series. Multiple experiments have been performed to anticipate the propensity for resistance development. Resistance selection was performed by successive exposure of Leishmania infantum promastigotes (in vitro) and intracellular amastigotes (both in vitro and in golden Syrian hamsters). The stability of the resistant phenotypes was assessed after passage in mice and Lutzomyia longipalpis sandflies. Whole-genome sequencing (WGS) was performed to identify mutated genes, copy number variations (CNVs), and somy changes. The potential role of efflux pumps (the MDR and MRP efflux pumps) in the development of resistance was assessed by coincubation of aminopyrazoles with specific efflux pump inhibitors (verapamil, cyclosporine, and probenecid). Repeated drug exposure of amastigotes did not result in the emergence of drug resistance either in vitro or in vivo Selection at the promastigote stage, however, was able to select for parasites with reduced susceptibility (resistance index, 5.8 to 24.5). This phenotype proved to be unstable after in vivo passage in mice and sandflies, suggesting that nonfixed alterations are responsible for the elevated resistance. In line with this, single nucleotide polymorphisms and indels identified by whole-genome sequencing could not be directly linked to the decreased drug susceptibility. Copy number variations were absent, whereas somy changes were detected, which may have accounted for the transient acquisition of resistance. Finally, aminopyrazole activity was not influenced by the MDR and MRP efflux pump inhibitors tested. The selection performed does not suggest the rapid development of resistance against aminopyrazoles in the field. Karyotype changes may confer elevated levels of resistance, but these do not seem to be stable in the vertebrate and invertebrate hosts. MDR/MRP efflux pumps are not likely to significantly impact the activity of the aminopyrazole leads.

Transmission potential of paromomycin-resistant Leishmania infantum and Leishmania donovani.

OBJECTIVES: Former studies demonstrated quick selection of paromomycin resistance for Leishmania infantum and Leishmania donovani accompanied by increased fitness. The present study aimed to interpret these findings in an epidemiological context by comparing infection of WT and experimentally derived paromomycin-resistant strains in the sand fly vector. METHODS: Depending on the Leishmania species, Lutzomyia longipalpis and Phlebotomus perniciosus or Phlebotomus argentipes sand flies were artificially infected with procyclic promastigotes of WT and paromomycin-resistant L. infantum (MHOM/FR/96/LEM3323-cl4) or L. donovani (MHOM/NP/03/BPK275/0-cl18). The infection rate and gut/stomodeal valve colonization were determined to monitor parasite phenotypic behaviour within the vector. The impact of the previously described gain of fitness in the vertebrate host on infectivity for the vector was assessed by feeding L. longipalpis on Syrian golden hamsters heavily infected with either WT or paromomycin-resistant parasites. RESULTS: WT and paromomycin-resistant Leishmania of both species behaved similarly in terms of infection and parasite location within the studied sand fly species. Blood feeding on infected hamsters did not reveal differences in acquisition of WT and paromomycin-resistant parasites, despite the higher organ burdens observed for the paromomycin-resistant strain. Strains remained resistant after passage in the vector. CONCLUSIONS: Although paromomycin-resistant parasites show an increased parasite fitness in vitro and in laboratory rodents, the intrinsic infection potential of paromomycin-resistant parasites remains unaltered in the sand fly. Of importance is the fact that paromomycin-resistant Leishmania are able to complete development in the natural vectors and produce stomodeal infection with metacyclic forms, which clearly suggests their potential to spread and circulate in nature.

Need for sustainable approaches in antileishmanial drug discovery.

Leishmaniasis is a neglected parasitic disease for which the current antileishmania therapeutics are hampered by drug toxicity, high cost, need for parenteral administration, increasing treatment failure rates, and emergence of drug resistance. The R&D pipeline had run fairly dry for several years, but fortunately some new drug candidates are now under (pre)clinical development. Identification of novel drugs will nevertheless remain essential to adequately sustain and improve effective disease control in the future. In this review, a package of standard and accessible R&D approaches is discussed with expansion to some alternative strategies focusing on parasite-host and vector-host interactions.

Antibody-Induced Internalization of the Human Respiratory Syncytial Virus Fusion Protein.

Respiratory syncytial virus (RSV) infections remain a major cause of respiratory disease and hospitalizations among infants. Infection recurs frequently and establishes a weak and short-lived immunity. To date, RSV immunoprophylaxis and vaccine research is mainly focused on the RSV fusion (F) protein, but a vaccine remains elusive. The RSV F protein is a highly conserved surface glycoprotein and is the main target of neutralizing antibodies induced by natural infection. Here, we analyzed an internalization process of antigen-antibody complexes after binding of RSV-specific antibodies to RSV antigens expressed on the surface of infected cells. The RSV F protein and attachment (G) protein were found to be internalized in both infected and transfected cells after the addition of either RSV-specific polyclonal antibodies (PAbs) or RSV glycoprotein-specific monoclonal antibodies (MAbs), as determined by indirect immunofluorescence staining and flow-cytometric analysis. Internalization experiments with different cell lines, well-differentiated primary bronchial epithelial cells (WD-PBECs), and RSV isolates suggest that antibody internalization can be considered a general feature of RSV. More specifically for RSV F, the mechanism of internalization was shown to be clathrin dependent. All RSV F-targeted MAbs tested, regardless of their epitopes, induced internalization of RSV F. No differences could be observed between the different MAbs, indicating that RSV F internalization was epitope independent. Since this process can be either antiviral, by affecting virus assembly and production, or beneficial for the virus, by limiting the efficacy of antibodies and effector mechanism, further research is required to determine the extent to which this occurs in vivo and how this might impact RSV replication.IMPORTANCE Current research into the development of new immunoprophylaxis and vaccines is mainly focused on the RSV F protein since, among others, RSV F-specific antibodies are able to protect infants from severe disease, if administered prophylactically. However, antibody responses established after natural RSV infections are poorly protective against reinfection, and high levels of antibodies do not always correlate with protection. Therefore, RSV might be capable of interfering, at least partially, with antibody-induced neutralization. In this study, a process through which surface-expressed RSV F proteins are internalized after interaction with RSV-specific antibodies is described. One the one hand, this antigen-antibody complex internalization could result in an antiviral effect, since it may interfere with virus particle formation and virus production. On the other hand, this mechanism may also reduce the efficacy of antibody-mediated effector mechanisms toward infected cells.

Evaluating drug resistance in visceral leishmaniasis: the challenges.

For decades antimonials were the drugs of choice for the treatment of visceral leishmaniasis (VL), but the recent emergence of resistance has made them redundant as first-line therapy in the endemic VL region in the Indian subcontinent. The application of other drugs has been limited due to adverse effects, perceived high cost, need for parenteral administration and increasing rate of treatment failures. Liposomal amphotericin B (AmB) and miltefosine (MIL) have been positioned as the effective first-line treatments; however, the number of monotherapy MIL-failures has increased after a decade of use. Since no validated molecular resistance markers are yet available, monitoring and surveillance of changes in drug sensitivity and resistance still depends on standard phenotypic in vitro promastigote or amastigote susceptibility assays. Clinical isolates displaying defined MIL- or AmB-resistance are still fairly scarce and fundamental and applied research on resistance mechanisms and dynamics remains largely dependent on laboratory-generated drug resistant strains. This review addresses the various challenges associated with drug susceptibility and -resistance monitoring in VL, with particular emphasis on the choice of strains, susceptibility model selection and standardization of procedures with specific read-out parameters and well-defined threshold criteria. The latter are essential to support surveillance systems and safeguard the limited number of currently available antileishmanial drugs.

In vitro 'time-to-kill' assay to assess the cidal activity dynamics of current reference drugs against Leishmania donovani and Leishmania infantum.

OBJECTIVES: Despite a continued search for novel antileishmanial drugs, treatment options remain restricted to a few standard drugs, e.g. antimonials, miltefosine, amphotericin B and paromomycin. Although these drugs have now been used for several decades, their mechanism of action still remains partly hypothetical and their dynamics of cidal action and time-to-kill are still poorly documented. METHODS: An in vitro time-to-kill assay on intracellular amastigotes of the laboratory reference strains Leishmania donovani (MHOM/ET/67/L82) and Leishmania infantum [MHOM/MA(BE)/67/ITMAP263] evaluated the cidal action dynamics of the listed reference drugs at three different concentrations: at IC50, 2 × IC50 and the near cytotoxic dose level (CC90: determined on MRC-5 cells). This assay focused on identifying the minimal exposure time needed to completely eliminate viable intracellular amastigotes, using the standard microscopic Giemsa assay and the promastigote back-transformation assay. RESULTS: While 100% reduction was microscopically apparent for most drugs, the promastigote back-transformation assay clearly demonstrated a concentration- and time-dependent cidal mechanism. The time-to-kill at 2 × IC50 was ≥240 h for pentavalent antimony (77 µg eq./mL), 96 h for trivalent antimony (44 µg eq./mL), 168 to >240 h for miltefosine (10 µM), 168 h for paromomycin (100 µM) and >240 h for amphotericin B (2 µM). No differences were noted between both Leishmania species. CONCLUSIONS: Evaluation of the concentration- and time-dependent cidal activity using the promastigote back-transformation assay revealed striking differences in efficacy of the different antileishmanial reference drugs. This assay should allow in-depth pharmacodynamic evaluation of novel drug leads in comparison with the existing antileishmanial drug repertoire.

Mouse models for pathogenic African trypanosomes: unravelling the immunology of host-parasite-vector interactions.

African trypanosomiasis is a parasitic disease that affects a variety of mammals, including humans, on the sub-Saharan African continent. To understand the diverse parameters that govern the host-parasite-vector interactions, mouse models for the disease have proven to be a cornerstone. Despite the fact that most trypanosomes cannot be considered natural pathogens for rodents, experimental infections in mice have shed a tremendous amount of light on the general biology of these parasites and their interaction with and evasion of the mammalian immune system. Different aspects including inflammation, vaccine failure, antigenic variation, resistance/sensitivity to normal human serum and the influence of tsetse compounds on parasite transmission have all been addressed using mouse models. In more recent years, the introduction of various 'knock-out' mouse strains has allowed to analyse the implication of various cytokines, particularly TNF, IFNγ and IL-10, in the regulation of parasitaemia and induction of pathological conditions during infection.

Immunosuppression of Syrian golden hamsters accelerates relapse but not the emergence of resistance in Leishmania infantum following recurrent miltefosine pressure.

Although miltefosine (MIL) has only been approved for the treatment of visceral leishmaniasis (VL) in 2002, its application in monotherapy already led to the development of two confirmed MIL-resistant isolates by 2009. Although liposomal amphotericin B is recommended as first-line treatment in Europe, MIL is still occasionally used in HIV co-infected patients. Since their immune system is incapable of controlling the infection, high parasite burdens and post-treatment relapses are common. Linked to the particular pharmacokinetic profile of MIL, successive treatment of recurrent relapses could in principle facilitate the emergence of drug resistance. This study evaluated the effect of immunosuppression (cyclophosphamide 150 mg/kg once weekly) on the development of MIL-resistance in Syrian golden hamsters infected with Leishmania infantum. The hamsters were treated with MIL (20 mg/kg orally for 5 days) whenever clinical signs of infection or relapse were observed. The immunosuppression resulted in a significant depletion of CD4+ lymphocytes and MHCII-expressing cells in peripheral blood, and a concomitant increase in tissue parasite burdens and shorter time to relapse, but the strain's susceptibility upon repeated MIL exposure remained unaltered. This study demonstrates that immunosuppression accelerates the occurrence of relapse without expediting MIL resistance development.

In vitro and in vivo pharmacodynamics of three novel antileishmanial lead series.

OBJECTIVES: Three new chemical series (bicyclic nitroimidazoles, aminopyrazoles and oxaboroles) were selected by Drugs for Neglected Diseases initiative as potential new drug leads for leishmaniasis. Pharmacodynamics studies included both in vitro and in vivo efficacy, cross-resistance profiling against the current antileishmanial reference drugs and evaluation of their cidal activity potential. METHODS: Efficacy against the reference laboratory strains of Leishmania infantum (MHOM/MA(BE)/67/ITMAP263) and L. donovani (MHOM/ET/67/L82) was evaluated in vitro on intracellular amastigotes and in vivo in the early curative hamster model. Cidal activity was assessed over a period of 15 days in an in vitro 'time-to-kill' assay. Cross-resistance was assessed in vitro on a panel of L. infantum strains with different degrees of resistance to either antimony, miltefosine or paromomycin. RESULTS: All lead compounds showed potent and selective in vitro activity against the Leishmania strains tested and no cross-resistance could be demonstrated against any of the current antileishmanial drugs. Cidal activity was obtained in vitro for all series within 15 days of exposure with some differences noted between L. donovani and L. infantum. When evaluated in vivo, all lead compounds showed high efficacy and no adverse effects were observed. CONCLUSIONS: The new lead series were shown to have cidal pharmacodynamic activity. The absence of cross-resistance with any of the current antileishmanial drugs opens possibilities for combination treatment to reduce the likelihood of treatment failures and drug resistance.

Streptococcus pneumoniae galU gene mutation has a direct effect on biofilm growth, adherence and phagocytosis in vitro and pathogenicity in vivo.

Streptococcus pneumoniae, the most common cause of bacterial pneumonia, has developed a wide range of virulence factors to evade the immune system of which the polysaccharide capsule is the most important one. Formation of this capsule is dependent on the cps gene locus, but also involves other genes-like galU. The pyrophosphorylase encoded by galU plays a role in the UDP-glucose metabolism of prokaryotes and is required for the biosynthesis of capsular polysaccharides. In this paper, the effect of a galU mutation leading to a dysfunctional UDP-glucose pyrophosphorylase (UDPG:PP) on in vitro biofilm biomass, adherence to lung epithelial cells and macrophage phagocytosis is studied. Last, in vivo virulence using a Galleria mellonella model has been studied. We show that the mutation improves streptococcal adherence to epithelial cells and macrophage phagocytosis in vitro, while there is no definitive correlation on biofilm formation between parent and mutant strains. Moreover, in vivo virulence is attenuated for all mutated strains. Together, these results demonstrate that a galU mutation in S. pneumoniae influences host cell interactions in vitro and in vivo and can strongly influence the outcome of a streptococcal infection. As such, UDPG:PP is worth investigating further as a potential drug target.

The Glossina morsitans tsetse fly saliva: general characteristics and identification of novel salivary proteins.

The tsetse fly (Glossina spp.) is an obligate blood-sucking insect that transmits different human-pathogenic and livestock threatening trypanosome species in Africa. To obtain more insight in the tsetse salivary function, some general aspects of the tsetse fly saliva and its composition were studied. Direct pH and protein content measurements revealed a moderately alkaline (pH approximately 8.0) salivary environment with approximately 4.3 microg soluble proteins per gland and a constant representation of the major saliva proteins throughout the blood-feeding cycle. Although major salivary genes are constitutively expressed, upregulation of salivary protein synthesis within 48 h after the blood meal ensures complete protein replenishment from day 3 onwards. Screening of a non-normalised Glossina morsitans morsitans lambdagt11 salivary gland expression library with serum from a saliva-immunized rabbit identified three full-length cDNAs encoding for novel salivary proteins with yet unknown functions: a 8.3 kDa glycine/glutamate-rich protein (G. morsitans morsitans salivary gland protein Gmmsgp1), a 12.0 kDa proline-rich protein (Gmmsgp2), and a 97.4 kDa protein composed of a metallophosphoesterase/5'nucleotidase region with a glutamate/aspartate/asparagines-rich region (Gmmsgp3).

Using microdialysis to analyse the passage of monovalent nanobodies through the blood-brain barrier.

BACKGROUND AND PURPOSE: Nanobodies are promising antigen-binding moieties for molecular imaging and therapeutic purposes because of their favourable pharmacological and pharmacokinetic properties. However, the capability of monovalent nanobodies to reach targets in the CNS remains to be demonstrated. EXPERIMENTAL APPROACH: We have assessed the blood-brain barrier permeability of Nb_An33, a nanobody against the Trypanosoma brucei brucei variant-specific surface glycoprotein (VSG). This analysis was performed in healthy rats and in rats that were in the encephalitic stage of African trypanosomiasis using intracerebral microdialysis, single photon emission computed tomography (SPECT) or a combination of both methodologies. This enabled the quantification of unlabelled and (99m) Tc-labelled nanobodies using, respectively, a sensitive VSG-based nanobody-detection elisa, radioactivity measurement in collected microdialysates and SPECT image analysis. KEY RESULTS: The combined read-out methodologies showed that Nb_An33 was detected in the brain of healthy rats following i.v. injection, inflammation-induced damage to the blood-brain barrier, as in the late encephalitic stage of trypanosomiasis, significantly increased the efficiency of passage of the nanobody through this barrier. Complementing SPECT analyses with intracerebral microdialysis improved analysis of brain disposition. There is clear value in assessing penetration of the blood-brain barrier by monovalent nanobodies in models of CNS inflammation. Our data also suggest that rapid clearance from blood might hamper efficient targeting of specific nanobodies to the CNS. CONCLUSIONS AND IMPLICATIONS: Nanobodies can enter the brain parenchyma from the systemic circulation, especially in pathological conditions where the blood-brain barrier integrity is compromised.

Control of experimental Trypanosoma brucei infections occurs independently of lymphotoxin-alpha induction.

Trypanosome infections are marked by severe pathological features, including anemia, splenomegaly, and suppression of T-cell proliferation. We have used lymphotoxin-alpha-deficient (LT-alpha(-/-)) mice, as well as LT-alpha-tumor necrosis factor-double-deficient (LT-alpha(-/-) TNF(-/-)) mice, to analyze the contributions of these related cytokines in both induction of trypanosomosis-associated immunopathology and infection control. Moreover, as the cytokine-deficient mice used have no detectable lymph nodes and lack germinal-center formation upon immune stimulation, we have analyzed the functional importance of both the lymph nodes and spleen during experimental Trypanosoma brucei infections. First, we show that the absence of LT-alpha does not significantly alter early trypanosomosis development or pathology but does result in better control of late-stage parasitemia levels and slightly prolonged survival. This increased survival of infected LT-alpha(-/-) mice coincides with the appearance of increased chronic-stage anti-trypanosome immunoglobulin M (IgM)-IgG2a serum titers that are generated in the absence of functional peripheral lymphoid tissue and do not require germinal-center formation. Second, we show that splenectomized mice control their parasitemia to the same extent as fully immune-competent littermates. Finally, using LT-alpha(-/-) TNF(-/-) double-deficient mice, we show that in these mice T. brucei infections are very well controlled during the chronic infection stage and that infection-induced pathology is minimized. Together, these findings indicate that while increased IgM-IgG2a anti-trypanosome antibody titers (generated in the absence of LT-alpha, peripheral lymph nodes, and germinal-center formation) coincide with improved parasitemia control, it is TNF that has a major impact on trypanosomosis-associated immunopathology.