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Onchocerca volvulus-specific antibody and cellular responses in onchocerciasis patients treated annually with ivermectin for 30 years and exposed to parasite transmission in central Togo

['Saskia I. Johanns', 'University Clinics Tübingen', 'Institute For Tropical Medicine', 'Eberhard-Karls University', 'Tübingen', 'Richard G. Gantin', 'Onchocerciasis Reference Laboratory', 'Institut National D Hygiene', 'Centre Hospitalier Regional', 'Sokode']

Date: 2022-05

Repeated annual ivermectin treatment of onchocerciasis patients durably inhibited their patent O. volvulus infections despite ongoing low-level parasite transmission in the study area. Repeated MDA with ivermectin affects the expression of immunity in patients. O. volvulus parasite-specific antibody levels diminished to levels seen in infection-free endemic controls. With low antibody levels, antibody-dependent cellular cytotoxic responses against tissue-dwelling O. volvulus larvae will weaken. O. volvulus antigen inducible cytokine and chemokine production increased in treated mf-negative patients, while their innate responsiveness to mitogen declined. Such lower innate responsiveness in elderly patients could contribute to reduced adaptive immune responses to parasite infections and vaccines. On the other hand, increased specific cellular chemokine responses in mf-negative onchocerciasis patients could reflect effector cell activation against tissue invasive larval stages of O. volvulus. The annual Simulium damnosum s.l. biting rate observed in the Mô river basin was similar to levels prior to initiation of MDA with ivermectin, and the positive rtPCR results reported here confirm ongoing O. volvulus transmission.

Repeated annual ivermectin treatments eliminated persisting O. volvulus microfilariae (mf) from the skin of patients and abrogated patent infections. The OvAg-specific IgG1 and IgG4 responses were diminished at 30yPT to the levels observed in endemic controls. Prior to starting ivermectin treatment, OvAg-induced cellular productions of IL-10, IFN-γ, CCL13, CCL17 and CCL18 were low in patients, and at 30yPT, cellular cytokine and chemokine responses increased to the levels observed in endemic controls. In contrast, mitogen(PHA)- induced IL-10, IFN-γ, CCL17 and CCL18 cellular production was diminished. This divergent response profile thus revealed increased parasite antigen-specific but reduced polyclonal cellular responsiveness in patients. The transmission of O. volvulus continued at the patients’ location in the Mô river basin in central Togo 2018 and 2019 when 0.58% and 0.45%, respectively, of Simulium damnosum s.l. vector blackflies carried O. volvulus infections.

Annual mass drug administrations (MDA) of ivermectin will strongly reduce Onchocerca volvulus microfilariae (mf) in the skin and in the onchocerciasis patients’ eyes. Ivermectin treatment will also affect the expression of immunity in patients, such that activated immune defenses may help control and contribute to clearance of mf of O. volvulus. Longitudinal surveys are a prerequisite to determining the impact of ivermectin on the status of anti-parasite immunity, notably in risk zones where parasite transmission and active O. volvulus infections persist.

Repeated MDA with ivermectin affects immune responses, such that activated immune defenses may enhance clearance of mf of O. volvulus. Longitudinal surveys are required to determine the impact of ivermectin on the status of immunity, notably in risk zones where parasite transmission and active O. volvulus infections persist. We examined the changes of O. volvulus parasite-specific antibody and cellular immune responsiveness in patients treated annually with ivermectin for 30 years. Treatment prevented patent O. volvulus infections, whilst parasite antigen-specific cytokine and chemokine responses increased but O.volvulus-specific antibody responses declined. Such decreased antibody levels could weaken antibody-dependent cellular cytotoxic responses to infective and tissue-dwelling O. volvulus larvae. Strengthened monocyte attracting and activation regulated chemokine responses could enhance effector cell migration and activation against larval stages of O.volvulus, possibly also eliciting resistance to further parasite infections.

Onchocerciasis is a neglected tropical disease, and a major cause of debilitating skin disease and ocular damage that can lead to irreversible blindness. Annual mass drug administrations (MDA) of ivermectin strongly reduces the load of Onchocerca volvulus microfilaria (mf) in the skin and in the patients’ eyes. Evolution of onchocerciasis as a disease is prevented by MDA, but recent studies have shown that O. volvulus transmission has not been completely interrupted.

Funding: The study was supported by the German Agency for International Cooperation (gtz/giz) (PN77.2210), the UNDP/WB/WHO/TDR Programme Grant ID890067 and Grant ID930543, the Edna McConnell Clark Foundation (Grant 13694), the Commission of the European Community (STD 3/0178, Contract No TS3-CT92-0057, INCO DEV SCOOTT Contract No 032321, FP7 Project Grant Acronym E-PIAF #242131), the DFG Grant Kn244/3-1, Di308/12-1 and So367/1-2 and the Federal Ministry for Education and Research (BMBF Grant 01KA1008 and Grant 01DG20021). The Medizinische Fakultät, Eberhard-Karls Universität Tübingen, the University Clinics Molecular Medicine Programme supported SIJ. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2022 Johanns et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Introduction

In the past 3 decades repeated annual treatments with ivermectin have largely eliminated Onchocerca volvulus microfilariae (mf) from the skin and eyes of onchocerciasis patients, thereby preventing the emergence of ocular pathologies [1] and profoundly improving dermal health in affected individuals [2]. By targeting high coverage of ivermectin mass drug administration (MDA) to eligible populations, onchocerciasis intervention programs aim to suppress the transmission of O. volvulus. Although the symptoms of the disease have largely disappeared, O. volvulus adult worms survive because single annual doses of ivermectin do not kill adult female O. volvulus and do not exert embryotoxic or embryostatic effects. Such medication does suppress the release of mf from gravid female O. volvulus for several months, and this measure must be applied repeatedly until fertile female O. volvulus either stop reproducing mf or die [3–6]. Following the ivermectin-facilitated reduction of mf load in onchocerciasis patients their cellular anergy reversed, parasite-specific Th1-type responses and serum chemokine levels increased [7–12], and such activated immune responses can lead to clearance of mf of O. volvulus. After 15 years of repeated ivermectin treatments, parasite antigen-mediated cellular production of Th1-, Th2-type and Treg-type cytokines declined in onchocerciasis patients [13], while their O.volvulus-specific antibody responses persisted at levels higher than those observed in infection-free endemic controls [7,13,14].

In onchocerciasis and lymphatic filariasis IL-10 is a prominent cytokine mediating immune suppression and cellular hypo-responsiveness to filarial and bystander antigens [15–17]. O. volvulus antigen-induced IL-10 production will dampen the activity of Th1-type, pro-inflammatory IFN-γ, whilst neutralisation of IL-10 enhances IFN-γ production in patients [18,19]. Stronger IFN-γ responses were observed in O. volvulus microfilariae negative individuals considered putatively immune to onchocerciasis [20]. In addition, peripheral blood cells from O. volvulus microfilariae positive patients produced less monocyte and T-cell activating and chemoattracting chemokines MCP-4 (CCL13), TARC (CCL17) and PARC (CCL18), suggesting that persistent O. volvulus can suppress these chemokines. Following ivermectin treatment in onchocerciasis patients, serum chemokine levels increased, and this may contribute to dermal immune responses along with killing and clearance- of O. volvulus microfilariae. However, to which extent parasite-specific and innate immune responses may change in onchocerciasis patients with a reduced parasite load and ultimate elimination remains an unexplored issue.

The present study investigated the strength and expression of O. volvulus-specific immune responses in onchocerciasis patients after 30 years of repeated ivermectin treatments. Cellular production of cytokines, of activation-regulated chemokines, and parasite-specific antibody reactivity were examined, and exposure of the study population to O. volvulus parasite transmission by infected Simulium damnosum s.l. vector blackflies was also determined.

Patients and methodology Ethics statement. At each follow up the aims, procedures and risks of the surveys were explained to all participants in the local language by the medical staff at the Centre Hospitalier Regional (CHR) de Sokodé. Skin snip specimen used in this study were collected from participants who gave oral informed consent. Blood samples used in this study were collected from participants (years 1989 until 2005) who gave oral informed consent, from year 2005 until year 2019 participants provided written informed consent. Formal consent was obtained from the parent or guardian of participants under the age of 18 years. The investigations were authorized by the Ministry of Health in Togo and re-approved every 5–10 years (No.2824/87/MSP-ASCF; No.1999/292/MS/CAB; No.0407/2007/MS/CAB/DGS; No.0060/2013/MS/CAB/DGS) and the Comite de Bioethique pour la Recherche en Santé (No.013/2015/CBRS).

Survey sites and study participants Female and male study participants were permanent residents in rural villages in central and northern Togo in West Africa. Surveys were conducted in 1989, 1994, 2004 and 2019 in the villages of Bouzalo (09°06’05"N; 01°02’37"E), Kéméni (09°14’03"N; 01°14’36"E) and Sagbadai (09°04’06"N; 01°04’17"E) in the Région Centrale. All villages are sentinel villages of the National Onchocerciasis Control Program (NOCP) and have received annual mass drug administration (MDA) since 1989 via community-directed distribution of ivermectin (150 μg/kg body weight). All onchocerciasis patients participated in regular surveys conducted by the NOCP. Patients were apparently healthy males and non-pregnant women with a documented history of microfilaria of O. volvulus in skin biopsies. In 1989 at the first surveys all patients were positive for mf of O. volvulus. The density of O. volvulus mf was determined in skin biopsies (mf/mg skin) taken from the right and left hip. Endemic controls from the above sentinel villages who joined the surveys in 1989 and 2019 were never diagnosed as being O. volvulus mf-positive, and they also received annual ivermectin under the community-directed mass drug administration (MDA).

Serum collection from ivermectin treated onchocerciasis patients and endemic controls Blood samples were collected from onchocerciasis patients who participated in regular surveys at baseline (before first ivermectin treatment), at 5 years, 12 years, and 30 years post initial treatment with ivermectin (PT). From the same onchocerciasis patients, sera were collected (paired samples) at baseline from n = 98, at 5yPT from n = 22, at 12yPT from n = 42 and at 30yPT from n = 86 participants. The endemic control (endmCTRL) group participants comprised n = 11 at baseline and n = 22 in 2019 at 30yPT, but were not the same individuals at the two timepoints. They were all permanent residents in the OCP sentinel villages, treated annually with ivermectin at 150 μg/kg, but were never found positive for microfilariae of O. volvulus in any survey. The density of O. volvulus microfilariae (mf) was determined in skin biopsies (mf/mg skin) taken from the right and left hip by means of cornea scleral punch (Holth- or Walser-type). Skin biopsies were immediately weighed and then immersed in 100μl physiological saline in separate wells of flatbottomed microtiter plates and stored at room temperature in a high-humidity atmosphere. Microfilariae were counted by microscopical examination after overnight incubation. Patients were treated annually with ivermectin 150 μg/kg and all patients were followed individually until 30 years post initiation of treatment (PT). At each timepoint of examination all participants gave their informed consent, and for correct and complete understanding explanations were always given in the local language.

Cellular cytokine and chemokine responses in patients and controls Blood samples (Table 1) were collected, peripheral mononuclear cells (PBMC) isolated and cells stimulated in vitro with O. volvulus antigen (OvAg) or with mitogen (PHA) for 48 hours. In onchocerciasis patients (OnchoPAT, n = 32) and O. volvulus mf-negative endemic controls (endmCTR, n = 33) PBMC responses to stimulation were analysed at baseline (beforeIVM) and at 30 years post initial treatment (30yPT). The OnchoPAT group participants examined beforeIVM and 30yPT were the same individuals while the endmCTRL group participants were not the same individuals. PPT PowerPoint slide

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TIFF original image Download: Table 1. The leukocyte blood cells, erythrocyte (RBC) and thrombocyte counts, haemoglobin concentration and haematocrit measured in peripheral blood samples from O. volvulus microfilariae positive onchocerciasis patients’ (OnchoPAT) patients and O. volvulus microfilariae-negative endemic control (endmCTRL) were surveyed in 1989 prior to initiation of community directed treatment intervention (CDTI) with ivermectin (beforeIVM) and at 30 years post annually repeated ivermectin treatments (30yPT) in year 2019. https://doi.org/10.1371/journal.pntd.0010340.t001

Isolation and culture of peripheral blood mononuclear cells (PBMC) In 1989 and 2019 venous blood samples (10 ml) were collected from the same n = 32 participants for in vitro cell culture purposes. A complete blood count including a white blood cell differential was conducted for each participant. Haematological examinations were performed in the central laboratory at CHR. PBMC were isolated from whole blood by Biocoll (Biochrom, Germany) density gradient centrifugation. For this, 9 ml of venous blood was diluted with 9 ml of RPMI (Gibco, UK) supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin and 0.25 μg/ml amphotericin B (Gibco, USA). Diluted blood was layered on Biocoll separation solution and samples were centrifuged for 30 min at 1,600 rpm. The PBMC layer was extracted and transferred into RPMI (as above) and centrifuged for 15 min at 1,600 rpm. This step was repeated twice, and thereafter cell pellets were resuspended in RPMI as described above containing 10% foetal bovine serum (FBS). These freshly isolated PBMCs were cultured in vitro at a concentration of 2.5 x 106 cells/ml in 0.5 ml of RPMI (as above) with 10% FBS in 48-well microtiter plates (COSTAR No3548). PBMC were stimulated with the mitogen Phytohemagglutinin (PHA, 5 μg/ml), the O. volvulus adult worm antigen (OvAg, 35μg/ml), or left unstimulated (negative control). Cell cultures were incubated at 37°C with 5% CO 2 for 48 hours, and thereafter, cell culture supernatants were harvested and stored at -20°C until further use. Cell culture supernatants from the years 1989 and 1995 were stored at -80°C.

Cytokine- and chemokine-specific enzyme-linked immunosorbent assay (ELISA) The cytokine and chemokine concentrations in cell culture supernatants of PBMC were quantified using specific sandwich ELISA (R&D Systems). High binding 96-well microtiter plates (CORNING No3690) were coated with capture antibody diluted in PBS according to the manufacturer’s specifications. The plates were sealed, incubated at room temperature overnight, and subsequently washed three times with washing buffer (PBS with 0.05% Tween20, pH 7.4; Sigma-Aldrich P3563). Blocking reagent diluent (RD; R&D Systems) was added to each well and incubated for 1h at room temperature. The solution was then discarded, and plates washed as above. Next, cell culture supernatants were added to wells and a seven-point standard sample row, prepared as recommended by the manufacturer, was applied. The plates were then sealed and incubated at room temperature for 2h, washed as above, and the detection antibody, diluted in RD according to the manufacturer’s specifications, was added to each well. Plates were incubated at room temperature for 2h, then washed as above, and streptavidin-horseradish peroxidase (Streptavidin-HRP, R&D Systems) diluted in RD was added to each well and incubated for 20 min. After 3 washing steps, the TMB substrate solution (Thermo Fisher Scientific #34021) was added to each well and the colouration was stopped with sulfuric acid. The optical density (OD) was determined at 450 nm in each well using a microplate photometer (BIOTEK EL808). The concentration of cytokines (IL-10, IFN-γ) and chemokines (CCL13, CCL17, CCL18) in cell culture supernatants were calculated in relation to the respective standard curves.

O. volvulus (OvAg) antigen-specific antibody ELISA The IgG1 and IgG4 responses of the study participants were investigated using an antigen extract of adult O. volvulus (OvAg). Isolation of O. volvulus and preparation OF adult worm-derived antigen (OvAg) was performed as described previously [21,22]. Nodules containing adult O. volvulus were surgically removed from patients and adult male and female worms were isolated by collagenase digestion. Freshly isolated worms were extensively washed in sterile phosphate-buffered saline (PBS), transferred into a Ten Broek tissue grinder, and then homogenized extensively on ice. The homogenate was then sonicated twice (30% intensity) for 10 min on ice and centrifuged at 16,000 g for 30 min at 4°C. The supernatants were collected then sterile filtered (0.22 mm) and stored in aliquots at -70C° until use. The protein concentration of the antigen preparation was determined with the BCA protein assay (Pierce, Rockford, USA). 96-well microtiter plates (CORNING No3690) were coated with 50 μl/well of OvAg diluted in PBS (pH 7.4) to a concentration of 5 μg/ml and incubated at 4°C overnight. The OvAg solution was discarded, and plates blocked using 50 μl/well of PBS/0.05%Tween20 (pH 7.4) (Sigma-Aldrich P3563) supplemented with 5% foetal bovine serum (FBS, Therma Fisher Scientific No10270106) for 1.5 h at room temperature. Plates were washed three times using PBS-Tween20 and then 50 μl of serum samples diluted 1:4 in PBS were applied to each well. The samples were incubated for 2 h at 37°C, washed as above and 50 μl of anti-human IgG4 HRP-conjugated monoclonal antibody (Thermo Fisher Scientific A10654) diluted 1:500 in PBS-Tween20 with 5% FBS was added, and plates were incubated for 1.5 h at room temperature. Then plates were washed as described above and TMB substrate solution (Thermo Fisher Scientific #34021) was added to each well, colouration was stopped with sulfuric acid and optical density (OD) was determined at 450 nm in each well using a microplate photometer (BIOTEK EL808).

Skin biopsy collection Before ivermectin MDA (delivered by community-directed drug distributors, CDDs), participants gave their informed consent for the collection of skin biopsies to detect O. volvulus microfilariae (mf). From each participant, a skin biopsy was taken from the left and right iliac crest (for a total of two snips) with a sterile 2-mm Holth corneo-scleral punch biopsy tool. Immediately, skin snips were incubated with physiological saline solution for 30 minutes, and each biopsy was microscopically examined for emerging O. volvulus mf and their number counted. After this first examination, the two biopsies were transferred separately into a round-bottom well of a 96-well plate containing saline solution, and after 24-hour incubation biopsies were re-examined as before. The use of two incubation steps for skin biopsies is the standard procedure applied by the National Programme for Onchocerciasis Control (PNLO/APOC), and this approach makes it possible to detect O. volvulus mf which may emerge slowly from skin.

Blood, stool, and urine sample examination for parasite (co-)infections For the determination of intestinal helminth and protozoan parasites, fresh stool samples (0.5 g) were mixed with saline and dispersed on 2 microscope slides covered with a 24×48-mm slide; samples were examined by laboratory technicians at the Centre Hospitalier Regional (CHR) de Sokode. All stool samples were examined using the Kato-Katz technique for quantification of helminth eggs per gram stool (helm-TEST; Labmaster). To detect Schistosoma haematobium infestation, 10 ml of urine from each participant was filtered (polycarbonate membrane; pore size, 12 μm; Whatman); the filters were then examined under a microscope, and S. haematobium eggs were quantified. For detection of Plasmodium spp. parasites, a thick blood film and a rapid detection test kit was applied (Malaria P.f HRP-II Antigen Rapid Test, Standard Diagnostics Inc., Korea).

Simulium damnosum s.l. collection From year 1976 until 2014 the collection of S. damnosum s.l. was conducted at specific catch points at river Mô by trained fly catchers in proximity to the sentinel villages Bouzalo, Sagbadai and Kemeni/Aleheride during the rainy season on five consecutive days in August and September. Collections took place from 7am to 6pm alternating the fly catchers every two hours. Approaching blackflies were caught upon landing and before biting. In addition, from April 2018 until February 2020, throughout the years weekly collections of S. damnosum s.l. were continued at the river Mô site, in proximity to the villages of Bouzalo and Sagbadai (Region Centrale). The blackflies caught daily were first frozen, then suspended in 70% alcohol, and 25 individual S. damnosum s.l. were pooled into a single tube in ethanol and stored below -20°C until DNA extraction and real-time-PCR (rtPCR). The sampling procedure was the same as above, and this long-term collection was used to determine the annual biting rate (ABR), which was calculated by multiplying the average number of blackflies caught daily by the number of days per week in the month to add up to 12 months.

Dissection of Simulium damnosum s.l. The captured S.damnosum s..l. were inactivated at 4°C, dissected using a stereo microscope and classified as parous or nulliparous. The parous flies were divided into head, thorax, and abdomen. Each part was dissected by teasing it apart in a normal saline solution, using dissecting needles and a stereo dissecting microscope, and examined for O. volvulus larvae. L3 larvae were defined as infective stages present in the head. Dissections were carried out by experienced technicians.

DNA-Extraction from captured Simulium damnosum For DNA extraction the S. damnosum samples in ethanol were evaporated overnight, ground in 80μl PBS and the DNA was extracted using the QIAmp DNA Mini Kit (QIAGEN) following the protocol for DNA extraction from tissues. The DNA concentration of each sample was measured by absorbance at 260 nm using the NanoDrop 1000.

Real-time polymerase chain reaction for Ov33 and Ov150 In blackfly samples O. volvulus DNA was detected by real-time polymerase chain reaction (PCR) using the MIC qPCR system (BIOZYM, Germany). The O. volvulus specific gene sequences of Ov33 and Ov150 were selected for qPCR and respective primers and probes were applied as previously described [23,24]. All samples were measured in duplicates. The threshold and Ct values were determined using the MIC qPCR software. The prevalence of Ov33 and/or Ov150 in blackflies pools was calculated as described by Katholi et al. [24]. The following primers and probes were used for the qPCR reaction: Sequence 5’-3’ OV33fwd: GCA AGC TCC AGT TGA AGC AC; OV33rev:CGG CAT TTT CAC GTC CAA GT; OV33probe: FAM-AGA ACC ACC ACA TTT CTG CGT CGC A-TAM; OV150fw: TGT GGA AAT TCA CCT AAA TAT G; OV150rev: AAT AAC TGA CCT ATG ACC; OV150probe: FAM-TAG GAC CCA ATT CGA ATG TAT GTA CCC-TAM The Ov33 and Ov150 PCR reaction mix was at a total volume 25ul per tube. For Ov33 PCR: Primer Fwd (1.2ul; 10uM); Primer Rev (1.2ul; 10uM); Probe (0.7ul; 10uM); For Ov150 PCR: Primer Fwd (1.2ul; 20uM); Primer Rev (1.2ul; 20uM); Probe (0.7ul; 10uM); for both qPCR: TaqMan 2x Universal MasterMix: (12.4ul); DNA: 5ul; H2O: 4.5ul The Cycle conditions were for Ov33: Activation (50°C; 2min)(1x); Hold (95°C;10min)(1x); Cycles 60x Denaturation (95°C;10s); Annealing (54°C;30s); and for Ov150: Activation (50°C;2min)(1x); Hold (95°C;10min)(1x); Cycles 60x Denaturation (95°C;15s); Annealing (49°C;30s); Elongation (60°C;2min).

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