Demographic patterns of human antibody levels to Simulium damnosum s.l. saliva in onchocerciasis-endemic areas: An indicator of exposure to vector bites.

BACKGROUND: In onchocerciasis endemic areas in Africa, heterogenous biting rates by blackfly vectors on humans are assumed to partially explain age- and sex-dependent infection patterns with Onchocerca volvulus. To underpin these assumptions and further improve predictions made by onchocerciasis transmission models, demographic patterns in antibody responses to salivary antigens of Simulium damnosum s.l. are evaluated as a measure of blackfly exposure. METHODOLOGY/PRINCIPAL FINDINGS: Recently developed IgG and IgM anti-saliva immunoassays for S. damnosum s.l. were applied to blood samples collected from residents in four onchocerciasis endemic villages in Ghana. Demographic patterns in antibody levels according to village, sex and age were explored by fitting generalized linear models. Antibody levels varied between villages but showed consistent patterns with age and sex. Both IgG and IgM responses declined with increasing age. IgG responses were generally lower in males than in females and exhibited a steeper decline in adult males than in adult females. No sex-specific difference was observed in IgM responses. CONCLUSIONS/SIGNIFICANCE: The decline in age-specific antibody patterns suggested development of immunotolerance or desensitization to blackfly saliva antigen in response to persistent exposure. The variation between sexes, and between adults and youngsters may reflect differences in behaviour influencing cumulative exposure. These measures of antibody acquisition and decay could be incorporated into onchocerciasis transmission models towards informing onchocerciasis control, elimination, and surveillance.

A Reevaluation of the Tolerability and Effects of Single-Dose Ivermectin Treatment on Onchocerca volvulus Microfilariae in the Skin and Eyes in Eastern Ghana.

Mass administration of ivermectin (IVM) has significantly reduced onchocerciasis prevalence, intensity, and morbidity in most endemic areas. Most IVM clinical trials were performed long ago in persons with high-intensity infections that are uncommon in West Africa today. This cohort treatment study recruited participants from a hypoendemic area in eastern Ghana to reevaluate the efficacy and tolerability of IVM with a special focus on the kinetics of microfilaria (Mf) clearance. Mf in the skin and anterior chambers (AC) were assessed by skin snip and slit lamp examinations at baseline and at 3 and 6 months after treatment with IVM 150 µg/kg. Most participants (184-231, 79.7%) enrolled were treatment-naïve. The baseline geometric mean skin Mf count was 12.67/mg (range 3-86). Although persons with MfAC at baseline (64/231, 27%) had significantly higher skin Mf counts than people without MfAC, 7 of 39 (15%) of persons with skin Mf counts in the range of 3-5 Mf/mg had MfAC. Skin Mf were detected in 14% (31/218) and 45% (96/216) of participants 3 and 6 months after IVM treatment, respectively. MfAC were detected in 12 of 212 (5.7%) study participants at 6 months. 81% (187 of 231) of participants experienced 439 adverse events within 7 days after treatment; all adverse events were mild (96.1%) or moderate. This study has provided new data on the kinetics of Mf in the skin and eyes after IVM treatment of persons with light to moderate intensity Onchocerca volvulus infections that are common in Africa at this time.

Drugs that target early stages of Onchocerca volvulus: A revisited means to facilitate the elimination goals for onchocerciasis.

Several issues have been identified with the current programs for the elimination of onchocerciasis that target only transmission by using mass drug administration (MDA) of the drug ivermectin. Alternative and/or complementary treatment regimens as part of a more comprehensive strategy to eliminate onchocerciasis are needed. We posit that the addition of "prophylactic" drugs or therapeutic drugs that can be utilized in a prophylactic strategy to the toolbox of present microfilaricidal drugs and/or future macrofilaricidal treatment regimens will not only improve the chances of meeting the elimination goals but may hasten the time to elimination and also will support achieving a sustained elimination of onchocerciasis. These "prophylactic" drugs will target the infective third- (L3) and fourth-stage (L4) larvae of Onchocerca volvulus and consequently prevent the establishment of new infections not only in uninfected individuals but also in already infected individuals and thus reduce the overall adult worm burden and transmission. Importantly, an effective prophylactic treatment regimen can utilize drugs that are already part of the onchocerciasis elimination program (ivermectin), those being considered for MDA (moxidectin), and/or the potential macrofilaricidal drugs (oxfendazole and emodepside) currently under clinical development. Prophylaxis of onchocerciasis is not a new concept. We present new data showing that these drugs can inhibit L3 molting and/or inhibit motility of L4 at IC50 and IC90 that are covered by the concentration of these drugs in plasma based on the corresponding pharmacological profiles obtained in human clinical trials when these drugs were tested using various doses for the therapeutic treatments of various helminth infections.

Establishment of an in vitro culture system to study the developmental biology of Onchocerca volvulus with implications for anti-Onchocerca drug discovery and screening.

BACKGROUND: Infections with Onchocerca volvulus nematodes remain a threat in Sub-Saharan Africa after three decades of ivermectin mass drug administration. Despite this effort, there is still an urgent need for understanding the parasite biology especially the mating behaviour and nodule formation as well as the development of more potent drugs that can clear the developmental (L3, L4, L5) and adult stages of the parasite and inhibit parasite reproduction and behaviour. METHODOLOGY/PRINCIPAL FINDINGS: Prior to culture, freshly harvested O. volvulus L3 larvae from dissected Simulium damnosum flies were purified by centrifugation using a 30% Percoll solution to eliminate fly tissue debris and contaminants. Parasites were cultured in both cell-free and cell-based co-culture systems and monitored daily by microscopic visual inspection. Exhausted culture medium was replenished every 2-3 days. The cell-free culture system (DMEM supplemented with 10% NCS) supported the viability and motility of O. volvulus larvae for up to 84 days, while the co-culture system (DMEM supplemented with 10% FBS and seeded on LLC-MK2 feeder cells) extended worm survival for up to 315 days. Co-culture systems alone promoted two consecutive parasite moults (L3 to L4 and L4 to L5) with highest moulting rates (69.2±30%) observed in DMEM supplemented with 10% FBS and seeded on LLC-MK2 feeder cells, while no moult was observed in DMEM supplemented with 10% NCS and seeded on LEC feeder cells. In DMEM supplemented with 10% FBS and seeded on LLC-MK2 feeder cells, O. volvulus adult male worms attached to the vulva region of adult female worms and may have mated in vitro. Apparent early initiation of nodulogenesis was observed in both DMEM supplemented with 10% FBS and seeded on LLC-MK2 and DMEM supplemented with 10% NCS and seeded on LLC-MK2 systems. CONCLUSIONS/SIGNIFICANCE: The present study describes an in vitro system in which O. volvulus L3 larvae can be maintained in culture leading to the development of adult stages. Thus, this in vitro system may provide a platform to investigate mating behaviour and early stage of nodulogenesis of O. volvulus adult worms that can be used as additional targets for macrofilaricidal drug screening.

Preliminary evaluations of 3-dimensional human skin models for their ability to facilitate in vitro the long-term development of the debilitating obligatory human parasite Onchocerca volvulus.

Onchocerciasis also known as river blindness is a neglected tropical disease and the world's second-leading infectious cause of blindness in humans; it is caused by Onchocerca volvulus. Current treatment with ivermectin targets microfilariae and transmission and does not kill the adult parasites, which reside within subcutaneous nodules. To support the development of macrofilaricidal drugs that target the adult worm to further support the elimination of onchocerciasis, an in-depth understanding of O. volvulus biology especially the factors that support the longevity of these worms in the human host (>10 years) is required. However, research is hampered by a lack of access to adult worms. O. volvulus is an obligatory human parasite and no small animal models that can propagate this parasite were successfully developed. The current optimized 2-dimensional (2-D) in vitro culturing method starting with O. volvulus infective larvae does not yet support the development of mature adult worms. To overcome these limitations, we have developed and applied 3-dimensional (3-D) culture systems with O. volvulus larvae that simulate the human in vivo niche using in vitro engineered skin and adipose tissue. Our proof of concept studies have shown that an optimized indirect co-culture of in vitro skin tissue supported a significant increase in growth of the fourth-stage larvae to the pre-adult stage with a median length of 816-831 µm as compared to 767 µm of 2-D cultured larvae. Notably, when larvae were co-cultured directly with adipose tissue models, a significant improvement for larval motility and thus fitness was observed; 95% compared to 26% in the 2-D system. These promising co-culture concepts are a first step to further optimize the culturing conditions and improve the long-term development of adult worms in vitro. Ultimately, it could provide the filarial research community with a valuable source of O. volvulus worms at various developmental stages, which may accelerate innovative unsolved biomedical inquiries into the parasite's biology.

Onchocerca volvulus is not detected in the cerebrospinal fluid of persons with onchocerciasis-associated epilepsy.

OBJECTIVES: Epidemiological evidence links onchocerciasis with the development of epilepsy. The aim of this study was to detect Onchocerca volvulus microfilariae or its bacterial endosymbiont, Wolbachia, in the cerebrospinal fluid (CSF) of persons with onchocerciasis-associated epilepsy (OAE). METHODS: Thirteen persons with OAE and O. volvulus skin snip densities of >80 microfilariae were recruited in Maridi County (South Sudan) and their CSF obtained. Cytospin centrifuged preparations of CSF were examined by light microscopy for the presence of O. volvulus microfilariae. DNA was extracted from CSF to detect O. volvulus (O-150 repeat) by quantitative real-time PCR, and Wolbachia (FtsZ gene) by standard PCR. To further investigate whether CSF from onchocerciasis-infected participants could induce seizures, 3- and 7-day old zebrafish larvae were injected with the CSF intracardially and intraperitoneally, respectively. For other zebrafish larvae, CSF was added directly to the larval medium. RESULTS: No microfilariae, parasite DNA, or Wolbachia DNA were detected in any of the CSF samples by light microscopy or PCR. All zebrafish survived the procedures and none developed seizures. CONCLUSIONS: The absence of O. volvulus in the CSF suggests that OAE is likely not caused by direct parasite invasion into the central nervous system, but by another phenomenon triggered by O. volvulus infection.

Antibody responses against the vaccine antigens Ov-103 and Ov-RAL-2 are associated with protective immunity to Onchocerca volvulus infection in both mice and humans.

BACKGROUND: The current strategy for the elimination of onchocerciasis is based on annual or bi-annual mass drug administration with ivermectin. However, due to several limiting factors there is a growing concern that elimination of onchocerciasis cannot be achieved solely through the current strategy. Additional tools are critically needed including a prophylactic vaccine. Presently Ov-103 and Ov-RAL-2 are the most promising vaccine candidates against an Onchocerca volvulus infection. METHODOLOGY/PRINCIPAL FINDINGS: Protection induced by immunization of mice with the alum-adjuvanted Ov-103 or Ov-RAL-2 vaccines appeared to be antibody dependent since AID-/- mice that could not mount antigen-specific IgG antibody responses were not protected from an Onchocerca volvulus challenge. To determine a possible association between antigen-specific antibody responses and anti-larvae protective immunity in humans, we analyzed the presence of anti-Ov-103 and anti-Ov-RAL-2 cytophilic antibody responses (IgG1 and IgG3) in individuals classified as putatively immune, and in infected individuals who developed concomitant immunity with age. It was determined that 86% of putatively immune individuals and 95% individuals with concomitant immunity had elevated IgG1 and IgG3 responses to Ov-103 and Ov-RAL-2. Based on the elevated chemokine levels associated with protection in the Ov-103 or Ov-RAL-2 immunized mice, the profile of these chemokines was also analyzed in putatively immune and infected individuals; both groups contained significantly higher levels of KC, IP-10, MCP-1 and MIP-1ß in comparison to normal human sera. Moreover, human monospecific anti-Ov-103 antibodies but not anti-Ov-RAL-2 significantly inhibited the molting of third-stage larvae (L3) in vitro by 46% in the presence of naïve human neutrophils, while both anti-Ov-103 and anti-Ov-RAL-2 antibodies significantly inhibited the molting by 70-80% when cultured in the presence of naive human monocytes. Interestingly, inhibition of molting by Ov-103 antibodies and monocytes was only in part dependent on contact with the cells, while inhibition of molting with Ov-RAL-2 antibodies was completely dependent on contact with the monocytes. In comparison, significant levels of parasite killing in Ov-103 and Ov-RAL-2 vaccinated mice only occurred when cells enter the parasite microenvironment. Taken together, antibodies to Ov-103 and Ov-RAL-2 and cells are required for protection in mice as well as for the development of immunity in humans. CONCLUSIONS/SIGNIFICANCE: Alum-adjuvanted Ov-103 and Ov-RAL-2 vaccines have the potential of reducing infection and thus morbidity associated with onchocerciasis in humans. The development of cytophilic antibodies, that function in antibody-dependent cellular cytotoxicity, is essential for a successful prophylactic vaccine against this infection.

Perspectives on the utility of moxidectin for the control of parasitic nematodes in the face of developing anthelmintic resistance.

Macrocyclic lactone (ML) anthelmintics are the most important class of anthelmintics because of our high dependence on them for the control of nematode parasites and some ectoparasites in livestock, companion animals and in humans. However, resistance to MLs is of increasing concern. Resistance is commonplace throughout the world in nematode parasites of small ruminants and is of increasing concern in horses, cattle, dogs and other animals. It is suspected in Onchocerca volvulus in humans. In most animals, resistance first arose to the avermectins, such as ivermectin (IVM), and subsequently to moxidectin (MOX). Usually when parasite populations are ML-resistant, MOX is more effective than avermectins. MOX may have higher intrinsic potency against some parasites, especially filarial nematodes, than the avermectins. However, it clearly has a significantly different pharmacokinetic profile. It is highly distributed to lipid tissues, less likely to be removed by ABC efflux transporters, is poorly metabolized and has a long half-life. This results in effective concentrations persisting for longer in target hosts. It also has a high safety index. Limited data suggest that anthelmintic resistance may be overcome, at least temporarily, if a high concentration can be maintained at the site of the parasites for a prolonged period of time. Because of the properties of MOX, there are reasonable prospects that strains of parasites that are resistant to avermectins at currently recommended doses will be controlled by MOX if it can be administered at sufficiently high doses and in formulations that enhance its persistence in the host. This review examines the properties of MOX that support this contention and compares them with the properties of other MLs. The case for using MOX to better control ML-resistant parasites is summarised and some outstanding research questions are presented.

Development of a preliminary in vitro drug screening assay based on a newly established culturing system for pre-adult fifth-stage Onchocerca volvulus worms.

BACKGROUND: The human filarial parasite Onchocerca volvulus is the causative agent of onchocerciasis (river blindness). It causes blindness in 270,000 individuals with an additional 6.5 million suffering from severe skin pathologies. Current international control programs focus on the reduction of microfilaridermia by annually administering ivermectin for more than 20 years with the ultimate goal of blocking of transmission. The adult worms of O. volvulus can live within nodules for over 15 years and actively release microfilariae for the majority of their lifespan. Therefore, protracted treatment courses of ivermectin are required to block transmission and eventually eliminate the disease. To shorten the time to elimination of this disease, drugs that successfully target macrofilariae (adult parasites) are needed. Unfortunately, there is no small animal model for the infection that could be used for discovery and screening of drugs against adult O. volvulus parasites. Here, we present an in vitro culturing system that supports the growth and development of O. volvulus young adult worms from the third-stage (L3) infective stage. METHODOLOGY/PRINCIPAL FINDINGS: In this study we optimized the culturing system by testing several monolayer cell lines to support worm growth and development. We have shown that the optimized culturing system allows for the growth of the L3 worms to L5 and that the L5 mature into young adult worms. Moreover, these young O. volvulus worms were used in preliminary assays to test putative macrofilaricidal drugs and FDA-approved repurposed drugs. CONCLUSION: The culture system we have established for O. volvulus young adult worms offers a promising new platform to advance drug discovery against the human filarial parasite, O. volvulus and thus supports the continuous pursuit for effective macrofilaricidal drugs. However, this in vitro culturing system will have to be further validated for reproducibility before it can be rolled out as a drug screen for decision making in macrofilaricide drug development programs.

Development of Onchocerca volvulus in humanized NSG mice and detection of parasite biomarkers in urine and serum.

BACKGROUND: The study of Onchocerca volvulus has been limited by its host range, with only humans and non-human primates shown to be susceptible to the full life cycle infection. Small animal models that support the development of adult parasites have not been identified. METHODOLOGY/PRINCIPAL FINDINGS: We hypothesized that highly immunodeficient NSG mice would support the survival and maturation of O. volvulus and alteration of the host microenvironment through the addition of various human cells and tissues would further enhance the level of parasite maturation. NSG mice were humanized with: (1) umbilical cord derived CD34+ stem cells, (2) fetal derived liver, thymus and CD34+ stem cells or (3) primary human skeletal muscle cells. NSG and humanized NSG mice were infected with 100 O. volvulus infective larvae (L3) for 4 to 12 weeks. When necropsies of infected animals were performed, it was observed that parasites survived and developed throughout the infection time course. In each of the different humanized mouse models, worms matured from L3 to advanced fourth stage larvae, with both male and female organ development. In addition, worms increased in length by up to 4-fold. Serum and urine, collected from humanized mice for identification of potential biomarkers of infection, allowed for the identification of 10 O. volvulus-derived proteins found specifically in either the urine or the serum of the humanized O. volvulus-infected NSG mice. CONCLUSIONS/SIGNIFICANCE: The newly identified mouse models for onchocerciasis will enable the development of O. volvulus specific biomarkers, screening for new therapeutic approaches and potentially studying the human immune response to infection with O. volvulus.

Prediction and validation of the structural features of Ov58GPCR, an immunogenic determinant of Onchocerca volvulus.

Onchocerciasis is a severely debilitating yet neglected tropical disease (NTD) that creates social stigma, generates and perpetuates poverty, and leads ultimately in some cases to irreversible unilateral or bilateral blindness if untreated. Consequently, the disease is a major impediment to socioeconomic development. Many control programs have been launched for the disease with moderate successes achieved. This mitigated hit is partially due to the lingering need for reliable, non-invasive and easily applicable tools for mapping endemic regions and post-elimination surveillance. In this work, bioinformatics analyses combined with immunological assays were applied in a bid to develop potential tools for diagnosis and assessing the success of drug treatment programs. We report that (i) the O. volvulus antigen, Ov58GPCR is a G-protein coupled receptor (GPCR) conserved in related nematodes, (ii) synthetic peptides predicted to be in the extracellular domain (ECD) of Ov58GPCR are indeed immunogenic epitopes in actively-infected individuals, (iii) synthetic peptide cocktails discriminate between actively-infected individuals, treated individuals and healthy African controls, (iv) polyclonal antibodies against one of the peptides or against the bacterially-expressed ECD reacted specifically with the native antigen of O. volvulus total and surface extracts, (v) Ov58GPCR is transcribed in both larvae and adult parasite stages, (vi) IgG and IgE responses to the recombinant ECD decline with ivermectin treatment. All these findings suggest that the extracellular domain and synthetic peptides of Ov58GPCR, as well as the specific immune response generated could be harnessed in the context of disease diagnosis and surveillance.

Characterization of Divalent Metal Transporter 1 (DMT1) in Brugia malayi suggests an intestinal-associated pathway for iron absorption.

Lymphatic filariasis and onchocerciasis are neglected parasitic diseases which pose a threat to public health in tropical and sub-tropical regions. Strategies for control and elimination of these diseases by mass drug administration (MDA) campaigns are designed to reduce symptoms of onchocerciasis and transmission of both parasites to eventually eliminate the burden on public health. Drugs used for MDA are predominantly microfilaricidal, and prolonged rounds of treatment are required for eradication. Understanding parasite biology is crucial to unravelling the complex processes involved in host-parasite interactions, disease transmission, parasite immune evasion, and the emergence of drug resistance. In nematode biology, large gaps still exist in our understanding of iron metabolism, iron-dependent processes and their regulation. The acquisition of iron from the host is a crucial determinant of the success of a parasitic infection. Here we identify a filarial ortholog of Divalent Metal Transporter 1 (DMT1), a member of a highly conserved family of NRAMP proteins that play an essential role in the transport of ferrous iron in many species. We cloned and expressed the B. malayi NRAMP ortholog in the iron-deficient fet3fet4 strain of Saccharomyces cerevisiae, performed qPCR to estimate stage-specific expression, and localized expression of this gene by immunohistochemistry. Results from functional iron uptake assays showed that expression of this gene in the iron transport-deficient yeast strain significantly rescued growth in low-iron medium. DMT1 was highly expressed in adult female and male B. malayi and Onchocerca volvulus. Immunolocalization revealed that DMT1 is expressed in the intestinal brush border, lateral chords, and reproductive tissues of males and females, areas also inhabited by Wolbachia. We hypothesize based on our results that DMT1 in B. malayi functions as an iron transporter. The presence of this transporter in the intestine supports the hypothesis that iron acquisition by adult females requires oral ingestion and suggests that the intestine plays a functional role in at least some aspects of nutrient uptake.

Impact of five annual rounds of mass drug administration with ivermectin on onchocerciasis in Sierra Leone.

BACKGROUND: Onchocerciasis is endemic in 12 of the 14 health districts of Sierra Leone. Good treatment coverage of community-directed treatment with ivermectin was achieved between 2005 and 2009 after the 11-year civil conflict. Sentinel site surveys were conducted in 2010 to evaluate the impact of five annual rounds of ivermectin distribution. METHODS: In total, 39 sentinel villages from hyper- and meso-endemic areas across the 12 endemic districts were surveyed using skin snips in 2010. Results were analyzed and compared with the baseline data from the same 39 villages. RESULTS: The average microfilaridermia (MF) prevalence across 39 sentinel villages was 53.10% at baseline. The MF prevalence was higher in older age groups, with the lowest in the age group of 1-9 years (11.00%) and the highest in the age group of 40-49 years (82.31%). Overall mean MF density among the positives was 28.87 microfilariae (mf)/snip, increasing with age with the lowest in the age group of 1-9 years and the highest in the age group of 40-49 years. Males had higher MF prevalence and density than females. In 2010 after five rounds of mass drug administration, the overall MF prevalence decreased by 60.26% from 53.10% to 21.10%; the overall mean MF density among the positives decreased by 71.29% from 28.87 mf/snip to 8.29 mf/snip; and the overall mean MF density among all persons examined decreased by 88.58% from 15.33 mf/snip to 1.75 mf/snip. Ten of 12 endemic districts had > 50% reduction in MF prevalence. Eleven of 12 districts had ≥50% reduction in mean MF density among the positives. CONCLUSIONS: A significant reduction of onchocerciasis MF prevalence and mean density was recorded in all 12 districts of Sierra Leone after five annual MDAs with effective treatment coverage. The results suggested that the onchocerciasis elimination programme in Sierra Leone was on course to reach the objective of eliminating onchocerciasis in the country by the year 2025. Annual MDA with ivermectin should continue in all 12 districts and further evaluations are needed across the country to assist the NTDP with programme decision making.

Factors affecting onchocerciasis transmission: lessons for infection control.

INTRODUCTION: Onchocerca volvulus infects in excess of 15 million people. The vectors are Simulium blackflies, varieties of which differ in their ecologies, behavior and vectorial abilities. Control of the vectors and mass administrations of ivermectin have succeeded in reducing prevalences with elimination achieved in some foci, particularly in Central and southern America. In Africa, progress towards elimination has been less successful. Areas covered: Even with community directed treatment with ivermectin (CDTI), control has been difficult in African areas with initial prevalences in excess of 55%, especially if only annual treatments are dispensed. This is partly attributable to insufficient coverage, but the appearance of incipiently resistant non-responding parasites and lack of attention to vector biology in modeling and planning outcomes of intervention programmes have also played their parts, with recrudescence now appearing in some treated areas. Expert commentary: The biology of onchocerciasis is complex involving different vectors with differing abilities to transmit parasites, diverse pathologies related to geographical and parasite variations and endosymbionts in both parasite and vector. Modeling to predict epidemiological and control outcomes is addressing this complexity but more attention needs to be given to the vectors' roles to further understanding of where and when control measures will succeed.

Stage-Specific Transcriptome and Proteome Analyses of the Filarial Parasite Onchocerca volvulus and Its Wolbachia Endosymbiont.

Onchocerciasis (river blindness) is a neglected tropical disease that has been successfully targeted by mass drug treatment programs in the Americas and small parts of Africa. Achieving the long-term goal of elimination of onchocerciasis, however, requires additional tools, including drugs, vaccines, and biomarkers of infection. Here, we describe the transcriptome and proteome profiles of the major vector and the human host stages (L1, L2, L3, molting L3, L4, adult male, and adult female) of Onchocerca volvulus along with the proteome of each parasitic stage and of its Wolbachia endosymbiont (wOv). In so doing, we have identified stage-specific pathways important to the parasite's adaptation to its human host during its early development. Further, we generated a protein array that, when screened with well-characterized human samples, identified novel diagnostic biomarkers of O. volvulus infection and new potential vaccine candidates. This immunomic approach not only demonstrates the power of this postgenomic discovery platform but also provides additional tools for onchocerciasis control programs. IMPORTANCE: The global onchocerciasis (river blindness) elimination program will have to rely on the development of new tools (drugs, vaccines, biomarkers) to achieve its goals by 2025. As an adjunct to the completed genomic sequencing of O. volvulus, we used a comprehensive proteomic and transcriptomic profiling strategy to gain a comprehensive understanding of both the vector-derived and human host-derived parasite stages. In so doing, we have identified proteins and pathways that enable novel drug targeting studies and the discovery of novel vaccine candidates, as well as useful biomarkers of active infection.

Dual protonophore-chitinase inhibitors dramatically affect O. volvulus molting.

The L3-stage-specific chitinase OvCHT1 has been implicated in the development of Onchocerca volvulus, the causative agent of onchocerciasis. Closantel, a known anthelmintic drug, was previously discovered as a potent and specific OvCHT1 inhibitor. As closantel is also a known protonophore, we performed a simple scaffold modulation to map out the structural features that are relevant for its individual or dual biochemical roles. Furthermore, we present that either OvCHT1 inhibition or protonophoric activity was capable of affecting O. volvulus L3 molting and that the presence of both activities in a single molecule yielded more potent inhibition of the nematode's developmental process.

Molecular characterization and evaluation of Onchocerca volvulus-secreted larval acidic protein 1 (SLAP1) as a putative vaccine candidate on endemic population of lymphatic filariasis.

Filarial parasites infected nearly 160 million of the global population with onchocerciasis and lymphatic filariasis, and further, a billion of people are estimated to be at risk of infection, rendering them among the most prevalent infectious agents in the world today. Given the complexity of their life cycle and the immune evasion mechanisms of these organisms, development of a vaccine remains to be a long-term challenge. Though a number of immunodominant antigens have been characterized, the presence of homologous proteins in humans or the allelic variants are some of the major drawbacks. One of the extensively studied vaccine candidates is abundant larval transcripts (ALT) family of proteins for the following properties: highly regulated expression, abundance, excreted-secreted product of infective stage larvae, and essentially for parasite establishment and survival in the host. In the present study, stage-specific expression of secreted larval acidic protein 1 (SLAP1) was identified; an ALT orthologue from Onchocerca volvulus was cloned, expressed, and purified as a recombinant protein. Immunogenicity of OvSLAP1 was demonstrated with sera and peripheral blood mononuclear cells from endemic regions of Brugia malayi and Wuchereria bancrofti. OvSLAP1 antibodies were predominated by IgG1 and IgG2 in endemic normal (EN) and chronic pathology (CP) subjects. It has also induced marked cellular response as observed by lymphoproliferation assay. The study revealed that OvSLAP1 can segregate humoral (EN mean optical density (OD) = 0.87 ± 0.035, CP mean OD = 0.59 ± 0.029) and cellular (EN mean stimulation index (SI) = 5.87 ± 0.167, CP mean SI = 3.5 ± 0.134) immune responses between EN and CP individuals (P < 0.001), signifying its prophylactic ability and vitality for protection from filarial infections in endemic population.

Retarded Onchocerca volvulus L1 to L3 larval development in the Simulium damnosum vector after anti-wolbachial treatment of the human host.

BACKGROUND: The human parasite Onchocerca volvulus harbours Wolbachia endosymbionts essential for worm embryogenesis, larval development and adult survival. In this study, the development of Wolbachia-depleted microfilariae (first stage larvae) to infective third stage larvae (L3) in the insect vector Simulium damnosum was analysed. METHODS: Infected volunteers in Cameroon were randomly and blindly allocated into doxycycline (200 mg/day for 6 weeks) or placebo treatment groups. After treatment, blackflies were allowed to take a blood meal on the volunteers, captured and dissected for larval counting and DNA extraction for quantitative real-time PCR analysis. RESULTS: PCR results showed a clear reduction in Wolbachia DNA after doxycycline treatment in microfilariae from human skin biopsies with > 50% reduction at one month post-treatment, eventually reaching a reduction of > 80%. Larval stages recovered from the insect vector had similar levels of reduction of endosymbiotic bacteria. Larval recoveries were analysed longitudinally after treatment to follow the kinetics of larval development. Beginning at three months post-treatment, significantly fewer L3 were seen in the blackflies that had fed on doxycycline treated volunteers. Concomitant with this, the proportion of second stage larvae (L2) was significantly increased in this group. CONCLUSIONS: Doxycycline treatment and the resulting decline of Wolbachia endobacteria from the microfilaria resulted in retarded development of larvae in the insect vector. Thus, anti-wolbachial treatment could have an additive effect for interrupting transmission by reducing the number of L3 that can be transmitted by blackflies.