Visceral leishmaniasis (VL or kala azar) is the most serious form of human leishmaniasis, responsible for over 20,000 deaths annually, and post kala azar dermal leishmaniasis (PKDL) is a stigmatizing skin condition that often occurs in patients after successful treatment for VL. Lack of effective or appropriately targeted cell mediated immunity, including CD8⁺ T cell responses, underlies the progression of VL and progression to PKDL, and can limit the therapeutic efficacy of anti-leishmanial drugs. Hence, in addition to the need for prophylactic vaccines against leishmaniasis, the development of therapeutic vaccines for use alone or in combined immuno-chemotherapy has been identified as an unmet clinical need. Here, we report the first clinical trial of a third-generation leishmaniasis vaccine, developed intentionally to induce Leishmania-specific CD8⁺ T cells. We conducted a first-in-human dose escalation Phase I trial in 20 healthy volunteers to assess the safety, tolerability and immunogenicity of a prime-only adenoviral vaccine for human VL and PKDL. ChAd63-KH is a replication defective simian adenovirus expressing a novel synthetic gene (KH) encoding two Leishmania proteins KMP-11 and HASPB. Uniquely, the latter was engineered to reflect repeat domain polymorphisms and arrangements identified from clinical isolates. We monitored innate immune responses by whole blood RNA-Seq and antigen specific CD8⁺ T cell responses by IFNγ ELISPOT and intracellular flow cytometry. ChAd63-KH was safe at intramuscular doses of 1x10¹⁰ and 7.5x10¹⁰ vp. Whole blood transcriptomic profiling indicated that ChAd63-KH induced innate immune responses characterized by an interferon signature and the presence of activated dendritic cells. Broad and quantitatively robust CD8⁺ T cell responses were induced by vaccination in 100% (20/20) of vaccinated subjects. The results of this study support the further development of ChAd63-KH as a novel third generation vaccine for VL and PKDL. This clinical trial (LEISH1) was registered at EudraCT (2012-005596-14) and ISRCTN (07766359). Leishmaniasis is a neglected disease of poverty with global public health impact. Caused by species of the Leishmania parasite, it may manifest itself as slow to heal skin ulcers, metastatic disease affecting the mouth and nose or systemic disease affecting internal organs (kala azar or visceral leishmaniasis). In common with other poverty-related neglected diseases, there have been few incentives for pharma to develop new drugs and vaccines. The few drugs currently available may cause serious side effects and/or only work well in some settings. No vaccines are available for prevention (prophylactic vaccines) or treatment (therapeutic vaccines). The clinical development of vaccines requires a series of clinical trials (Phases I–IV). Phase I studies assess primarily safety and are conducted in healthy volunteers. Later phase trials then involve either more healthy subjects (for prophylactic vaccines) or patients (for therapeutic vaccines) and provide additional safety data as well as preliminary data on efficacy. Here, we describe the results from a Phase I first-in-human trial of a new vaccine for leishmaniasis. Our vaccine is novel compared to those previously tested in that it is designed to target induction of a specific type of immune response (CD8⁺ T cells). We show here that the vaccine is safe in healthy volunteers and induces robust CD8⁺ T cell responses. The study represents an important step towards the development of a leishmaniasis vaccine.

Miltefosine has been used successfully to treat visceral leishmaniasis (VL) in India, but it was unsuccessful for VL in a clinical trial in Brazil. To identify molecular markers that predict VL treatment failure whole genome sequencing of 26 L. infantum isolates, from cured and relapsed patients allowed a GWAS analysis of SNPs, gene and chromosome copy number variations. A strong association was identified (p = 0·0005) between the presence of a genetically stable L. infantumMiltefosine Sensitivity Locus (MSL), and a positive response to miltefosine treatment. The risk of treatment failure increased 9·4-fold (95% CI 2·11-53·54) when an isolate did not have the MSL. The complete absence of the MSL predicted miltefosine failure with 0·92 (95% CI 0·65-0·996) sensitivity and 0·78 (95% CI 0·52-0·92) specificity. A genotyping survey of L. infantum (n = 157) showed that the frequency of MSL varies in a cline from 95% in North East Brazil to <5% in the South East. The MSL was found in the genomes of all L. infantum and L. donovani sequenced isolates from the Old World (n = 671), where miltefosine can have a cure rate higher than 93%. Knowledge on the presence or absence of the MSL in L. infantum will allow stratification of patients prior to treatment, helping to establish better therapeutic strategies for VL treatment. FUND: CNPq, FAPES, GCRF MRC and Wellcome Trust.

Anaerobic digestion (AD) as a waste management strategy for the organic fraction of municipal waste (OFMSW) has received attention in developed countries for several decades, leading to the development of large-scale plants. In contrast, AD of OFMSW has only recently drawn attention in developing countries. This systematic review was carried out to investigate the implementation of AD to treat the OFMSW in developing countries, focusing on assessing pilot and full-scale AD plants reported in the last ten years. Studies that met the selection criteria were analyzed and data regarding operating parameters, feedstock characteristics, and biogas, digestate, and energy production were extracted. As outlined in this systematic review, AD plants located in developing countries are mostly one-stage mesophilic systems that treat OFMSW via mono-digestion, almost exclusively with the aim of producing electrical energy. Based on the analysis done throughout this systematic review, it was noted that there is a large difference in the maturity level of AD systems between developing and developed countries, mainly due to the economic capacity of developed countries to invest in sustainable waste management systems. However, the number of AD plants reported in scientific papers is significantly lower than the number of installed AD systems. Research articles regarding large-scale implementation of AD to treat OFMSW in developed countries were analyzed and compared with developing countries. This comparison identified practices used in plants in developed countries that could be utilized in the large-scale implementation and success of AD in developing countries. These practices include exploiting potential products with high market-values, forming partnerships with local industries to use industrial wastes as co-substrates, and exploring different biological and physical pretreatment technologies. Additionally, the analysis of capital and operational costs of AD plants showed that costs tend to be higher for developing countries due to their need to import of materials and equipment from developed countries. Technical, economical, and political challenges for the implementation of AD at a large-scale in developing countries are highlighted.

Human African trypanosomiasis is caused by two subspecies of Trypanosoma brucei . Trypanosoma brucei rhodesiense is found in East Africa and frequently causes acute disease, while Trypanosoma brucei gambiense is found in West Africa and is associated with chronic disease. Samples taken from a single focus of a Ugandan outbreak of T . b. rhodesiense in the 1980s were associated with either chronic or acute disease. We sequenced the whole genomes of two of these isolates, which showed that they are genetically distinct from each other. Analysis of single nucleotide polymorphism markers in a panel of 31 Ugandan isolates plus 32 controls revealed a mixture of East African and West African haplotypes, and some of these haplotypes were associated with the different virulence phenotypes. It has been shown recently that T . b. brucei and T . b. rhodesiense populations undergo genetic exchange in natural populations. Our analysis showed that these strains from the Ugandan epidemic were intermediate between the reference genome sequences of T . b. gambiense and T . b. brucei and contained haplotypes that were present in both subspecies. This suggests that the human-infective subspecies of T. brucei are not genetically isolated, and our data are consistent with genomic introgression between East African and West African T . b. brucei subspecies. This has implications for the control of the parasite, the spread of drug resistance, and understanding the variation in virulence and the emergence of human infectivity. IMPORTANCE We present a genetic study of the acute form of “sleeping sickness” caused by the protozoan parasite Trypanosoma brucei rhodesiense from a single outbreak in Uganda. This represents an advance in our understanding of the relationship between the T . b. rhodesiense and Trypanosoma brucei gambiense subspecies that have previously been considered geographically distinct. Our data suggest that introgression of West African-derived T. brucei haplotypes may be associated with differences in disease presentation in the East African disease. These findings are not only of scientific interest but also important for parasite control, as they suggest that the human-infective T. brucei subspecies are not genetically isolated.

Leishmania parasite infections, termed the leishmaniases, cause significant global infectious disease burden. The lifecycle of the parasite embodies three main stages that require precise coordination of gene regulation to survive environmental shifts between sandfly and mammalian hosts. Constitutive transcription in kinetoplastid parasites means that gene regulation is overwhelmingly reliant on post-transcriptional mechanisms, yet strikingly few Leishmania trans-regulators are known. Using optimized crosslinking and deep, quantified mass spectrometry, we present a comprehensive analysis of 1400 mRNA binding proteins (mRBPs) and whole cell proteomes from the three main Leishmania lifecycle stages. Supporting the validity, although the crosslinked RBPome is magnitudes more enriched, the protein identities of the crosslinked and non-crosslinked RBPomes were nearly identical. Moreover, multiple candidate RBPs were endogenously tagged and found to associate with discrete mRNA target pools in a stage-specific manner. Results indicate that in L. mexicana parasites, mRNA levels are not a strong predictor of the whole cell expression or RNA binding potential of encoded proteins. Evidence includes a low correlation between transcript and corresponding protein expression and stage-specific variation in protein expression versus RNA binding potential. Unsurprisingly, RNA binding protein enrichment correlates strongly with relative replication efficiency of the specific lifecycle stage. Our study is the first to quantitatively define and compare the mRBPome of multiple stages in kinetoplastid parasites. It provides novel, in-depth insight into the trans-regulatory mRNA:Protein (mRNP) complexes that drive Leishmania parasite lifecycle progression.

Background: Human visceral leishmaniasis, caused by infection with Leishmania donovani or L. infantum, is a potentially fatal disease affecting 50,000-90,000 people yearly in 75 disease endemic countries, with more than 20,000 deaths reported. Experimental models of infection play a major role in understanding parasite biology, host-pathogen interaction, disease pathogenesis, and parasite transmission. In addition, they have an essential role in the identification and pre-clinical evaluation of new drugs and vaccines. However, our understanding of these models remains fragmentary. Although the immune response to Leishmania donovani infection in mice has been extensively characterized, transcriptomic analysis capturing the tissue-specific evolution of disease has yet to be reported. Methods: We provide an analysis of the transcriptome of spleen, liver and peripheral blood of BALB/c mice infected with L. donovani. Where possible, we compare our data in murine experimental visceral leishmaniasis with transcriptomic data in the public domain obtained from the study of L. donovani-infected hamsters and patients with human visceral leishmaniasis. Digitised whole slide images showing the histopathology in spleen and liver are made available via a dedicated website, www.leishpathnet.org. Results: Our analysis confirms marked tissue-specific alterations in the transcriptome of infected mice over time and identifies previously unrecognized parallels and differences between murine, hamster and human responses to infection. We show commonality of interferon-regulated genes whilst confirming a greater activation of type 2 immune pathways in infected hamsters compared to mice. Cytokine genes and genes encoding immune checkpoints were markedly tissue specific and dynamic in their expression, and pathways focused on non-immune cells reflected tissue specific immunopathology. Our data also addresses the value of measuring peripheral blood transcriptomics as a potential window into underlying systemic disease. Conclusions: Our transcriptomic data, coupled with histopathologic analysis of the tissue response, provide an additional resource to underpin future mechanistic studies and to guide clinical research.