Treating Mansonella perstans is challenged by the low efficacy of registered antihelminthics. Wolbachia endobacteria provide an alternative treatment target because depletion results in amicrofilaremia in filarial infections with Wuchereria bancrofti and Onchocerca volvulus infections. This open-label, randomized study sought to confirm that i) Wolbachia are present in M. perstans in Ghana and ii) doxycycline treatment will deplete Wolbachia and cause a slow, sustained decline in microfilariae (MF). Two hundred and two Ghanaians with M. perstans infection were randomized into early (immediate) and delayed (6 months deferred) treatment groups, given doxycycline 200 mg/day for 6 weeks, and monitored for MF and Wolbachia levels at baseline, 4, 12, and 24 months after the study onset (= time of randomization and start of treatment for the early group). Per protocol analysis revealed that the median MF/mL in the early group declined from 138 at baseline to 64 at month 4 and further to 0 at month 12. In the delayed group, MF load did not change from a baseline median of 97 to 102 at month 4 but declined to 42 at month 12, that is, 6 months after receiving treatment, trailing the early group as expected. By month 24, both treatment groups had reached a median MF level of 0. After treatment, Wolbachia were depleted from MF by ≥ 1-log drop compared with baseline levels. We conclude that M. perstans in Ghana harbor Wolbachia that are effectively depleted by doxycycline with subsequent reduction in MF loads, most likely because of interruption of fertility of adult worms.

The filarial nematode Mansonella perstans is endemic throughout Africa, northern South America and the Caribbean. Interestingly, M. perstans-infected individuals present no distinct clinical picture associated with certain pathology. Due to its relatively silent nature, research on this tropical disease has been neglected, especially M. perstans-driven immune responses. A hindrance in obtaining data on M. perstans-specific responses has been the inability to obtain adult worms since their habitats in serous cavities are difficult to access. Thus, in this study, for the first time, we used Mansonella perstans worm antigen extract as stimulant to obtain filarial-specific recall and immunoglobulin responses from M. perstans microfilaremic individuals (Mp MF+) from Cameroon. Moreover, systemic immune profiles in sera and immune cell composition in peripheral blood from Mp MF+ and amicrofilaremic individuals (Mp MF-) were obtained. Our data reveal that Mp MF+ individuals showed significantly reduced cytokine (IL-4, IL-6 and IL-12p70) and chemokine levels (IL-8 and RANTES), but significantly higher MIP-1β as well as increased M. perstans-specific IgG4 levels compared to Mp MF- individuals. In contrast, upon re-stimulation with worm antigen extract, IFN-γ, IL-13, IL-10 and IL-17A secretion was enhanced in cell cultures from Mp MF+ individuals when compared to those from cultures of healthy European individuals. Moreover, analysis of immune cell composition in peripheral blood from Mp MF+ individuals revealed increased type 2 helper T (Th2), natural killer (NK), regulatory B and T cell (Breg and Treg) subsets but decreased type 1 regulatory T (Tr1) cells. In summary, this study deciphers for the first time, M. perstans-specific immune responses using worm antigen extract and shows that patent M. perstans infections have distinct Th2, Breg and Treg subsets accompanied with reduced systemic innate and adaptive immune responses and dominant filarial-specific IgG4 levels. Although there are approximately 114 million infected people with Mansonella perstans, there is a paucity of information about how this filarial nematode affects the host's immune system. M. perstans currently poses a potential risk to 581 million people throughout Africa and for many years infections were considered to be nonpathogenic because individuals presented undefined and non-distinct clinical symptoms. Due to lack of treatment and the potential consequences on co-infections, M. perstans infections are however, regarded as a public health problem in endemic areas. The long-lasting co-existence of the worm with its human host implies potential immune-modulatory properties that may also affect responses to other infections; an aspect that has not been well addressed. In this study, we were interested in determining whether M. perstans-infected patients from Cameroon show a distinct immunological profile. In addition, we applied for the first time M. perstans worm antigen extract in order to test its re-stimulation capacity on cells isolated from M. perstans-microfilaremic individuals. Overall this study shows that M. perstans-microfilaremic individuals have a distinct immune profile, which is characterized by increased Th2 and regulatory cell populations concomitant with reduced systemic cytokine/chemokine and increased filarial-specific IgG4 levels. In addition, the M. perstans-specific down-regulation of immune responses might be an explanation for the increased susceptibility and worsened disease course of HIV, tuberculosis and malaria in M. perstans endemic regions.

Despite worldwide mass drug administration, it is estimated that 68 million individuals are still infected with lymphatic filariasis with 19 million hydrocele and 17 million lymphedema reported cases. Despite the staggering number of pathology cases, the majority of LF-infected individuals do not develop clinical symptoms and present a tightly regulated immune system characterized by higher frequencies of regulatory T cells (Treg), suppressed proliferation and Th2 cytokine responses accompanied with increased secretion of IL-10, TGF-β and infection-specific IgG4. Nevertheless, the filarial-induced modulation of the host`s immune system and especially the role of regulatory immune cells like regulatory B (Breg) and Treg during an ongoing LF infection remains unknown. Thus, we analysed Breg and Treg frequencies in peripheral blood from Ghanaian uninfected endemic normals (EN), lymphedema (LE), asymptomatic patent (CFA+MF+) and latent (CFA+MF-) W. bancrofti-infected individuals as well as individuals who were previously infected with W. bancrofti (PI) but had cleared the infection due to the administration of ivermectin (IVM) and albendazole (ALB). In summary, we observed that IL-10-producing CD19<sup>+</sup>CD24<sup>high</sup>CD38d<sup>high</sup> Breg were specifically increased in patently infected (CFA+MF+) individuals. In addition, CD19<sup>+</sup>CD24<sup>high</sup>CD5<sup>+</sup>CD1d<sup>high</sup> and CD19<sup>+</sup>CD5<sup>+</sup>CD1d<sup>high</sup>IL-10<sup>+</sup> Breg as well as CD4<sup>+</sup>CD127<sup>-</sup>FOXP3<sup>+</sup> Treg frequencies were significantly increased in both W. bancrofti-infected cohorts (CFA+MF+ and CFA+MF-). Interestingly, the PI cohort presented frequency levels of all studied regulatory immune cell populations comparable with the EN group. In conclusion, the results from this study show that an ongoing W. bancrofti infection induces distinct Breg and Treg populations in peripheral blood from Ghanaian volunteers. Those regulatory immune cell populations might contribute to the regulated state of the host immune system and are probably important for the survival and fertility (microfilaria release) of the helminth. Regulation of the host`s immune system by filarial nematodes is crucial for the fertility and survival of the nematode. Indeed, the majority of W. bancrofti-infected individuals are characterized by a regulated state including increased regulatory T cells (Treg), IL-10, TGF-β and filarial-specific IgG4 and suppressed Th2 cytokine responses. However, the functional role of Treg populations and regulatory B cells (Breg) during filarial infection remains unknown. Thus, in this study we investigated whether W. bancrofti-infected individuals from Ghana harbored distinct Breg and Treg populations which might be important for filarial-specific immunomodulation. Overall, this study shows that W. bancrofti induces distinct Breg populations, especially in patently (microfilaremic) infected individuals who presented significantly increased frequencies of IL-10-producing CD19⁺CD24^highCD38d^high Breg. Furthermore, clearance of the infection, due to anti-filarial treatment, returned these regulatory immune cells to homeostatic levels showing that an ongoing filarial infection is important for the activation of distinct Breg and Treg subsets. Those regulatory immune cell subsets are a part of a complex system which are induced by filarial nematodes to modulate the host`s immune system and maintain long-term survival.

One of the most advantageous research aspects of the murine model of filariasis, Litomosoides sigmodontis, is the availability of mouse strains with varying susceptibility to the nematode infection. In C57BL/6 mice, L. sigmodontis worms are largely eliminated in this strain by day 40 post-infection and never produce their offspring, microfilariae (Mf). This provides a unique opportunity to decipher potential immune pathways that are required by filariae to achieve a successful infection. In this study we tracked worm development and patency, the production of microfilariae and thus the transmission life-stage, in Rag2IL-2Rγ−/− mice which are deficient in T, B and NK cell populations. Although worm burden was comparable between wildtype (WT) and Rag2IL-2Rγ−/− mice on d30, by day 72 post-infection, parasites in Rag2IL-2Rγ−/− mice were still in abundance, freely motile and all mice presented high quantities of Mf both at the site of infection, the thoracic cavity (TC), and in peripheral blood. Levels of cytokine (IL-4, IL-6, TNFα) and chemokine (MIP-2, RANTES, Eotaxin) parameters were generally low in the TC of infected Rag2IL-2Rγ−/−mice at both time-points. The frequency of neutrophils however was higher in Rag2IL-2Rγ−/−mice whereas eosinophils and macrophage populations, including alternatively activated macrophages, were elevated in WT controls. Our data highlight that adaptive immune responses prevent the development of patent L. sigmodontis infections in semi-susceptible C57BL/6 mice and suggest that induction of such responses may offer a strategy to prevent transmission of human filariasis.

Mansonellosis remains one of the most neglected of tropical diseases and its current distribution in the entire forest block of southern Cameroon is unknown. In order to address this issue, we have surveyed the distribution of Mansonella perstans in different bioecological zones and in addition, elucidated the influence of multiple rounds of ivermectin (IVM) based mass drug administration (MDA). A mixed design was used. Between 2000 and 2014, both cross-sectional and longitudinal surveys were carried out in 137 communities selected from 12 health districts belonging to five main bioecological zones of the southern part of Cameroon. The zones comprised of grassland savanna (GS), mosaic forest savanna (MFS), forested savanna (FS), deciduous equatorial rainforest (DERF) and the dense humid equatorial rainforest (DHERF). The survey was carried out in some areas with no treatment history as well as those currently under IVM MDA. Individuals within the participatory communities were screened for the presence of M. perstans microfilariae (mf) in peripheral blood by the calibrated thick film method to determine both prevalence and geometric mean intensities at the community level. Apart from sporadic cases in savanna areas, distribution of M. perstans was strongly linked to the equatorial rainforest zones. Before CDTI, the highest mean prevalence (70.0 %) and intensity (17,382.2 mf/ml) were obtained in communities in Mamfes’ DHERF areas followed by communities in the DHERF zone of Lolodorf (53.8 % and 7,814.8 mf/ml, respectively). A longitudinal survey in Mamfe further showed that M. perstans infections had reduced by 34.5 % in DERF (P < 0.001) but not DHERF zones after ten years of IVM MDA. Further data from the cross-sectional study revealed that there was a decrease in prevalence in DHERF zones only after ten years of MDA. In DERF zones however, the infection was relatively lower after four years of MDA. The distribution of M. perstans in the southern part of Cameroon varies with bioecological zones and IVM MDA history. The zones with high prevalence and intensities lie in forested areas while those with low endemicity are in the savanna areas. MDA with ivermectin induced significant reduction in the endemicity of mansonellosis in the decidious equatorial rainforest. In contrast, the prevalence and intensity remained relatively high and stable in the dense humid equatorial rainforest zones even after a decade of mass drug administration with ivermectin. Since it is known that M. perstans down-regulates host's immune system, the findings from this work would be useful in designing studies to understand the impact of M. perstans on host immune response to vaccination and co-infection with other pathogens such as Mycobacterium spp. and Plasmodium spp. in areas of contrasting endemicities.

Approximately 114 million people are infected with Mansonella perstans in large proportions of Africa. In contrast to other filariae that infect humans, M. perstans-infected individuals show no distinct pathology or specific clinical picture, indicating a well-tuned adaptation to the host. In addition, since M. perstans adult worms reside in serous cavities which are difficult to access, research has been hindered and there is a paucity of knowledge about the biology of M. perstans, especially the development of the different life stages as well as M. perstans-driven immune responses. Thus in this study, an in vitro culture system was developed which allows an in-depth analysis of M. perstans. Culicoides species were caught in Ediki (Kumba), Southwest Region within Cameroon following a blood meal on a microfilaremic donor that had 1500 microfilariae/ml of peripheral blood and kept in captivity for 12 days at 23 °C. In a pilot experiment, 15 infective larvae were obtained from the midges and co-cultured with a confluent monolayer of monkey kidney epithelial cells (LLC-MK2) in DMEM medium supplemented with 10% FBS for up to 77 days. The resulting survival rates of 33% revealed that the cell-conditioned medium was suitable for long-term maintenance of M. perstans worms. To confirm these preliminary observations, 249 infective larvae were cultured for 50 days and their development was monitored daily and microscopically graded for motility. In total, 170 (68.3%) filariae survived and 124 (49.8%) larvae moulted between days 21–30 to become L5 stage larvae which were motile and showed continuous vigorous movement. We have established an in vitro culture system for the generation and long-term maintenance of viable M. perstans worms. This technique will be an important tool to study parasite biology and development, the role in host immunity, and might be helpful to discover novel treatment strategies against this filariae.