Meba Banla

Ivermectin‐facilitated immunity in onchocerciasis; activation of parasite‐specific Th1‐type responses with subclinical Onchocerca volvulus infection

The present study examined the quantitative and qualitative changes registered in the parasite‐specific antibody response, cellular reactivity and cytokine production profile in onchocerciasis patients repeatedly treated with ivermectin over a period of 8 years. The densities of Onchocerca volvulus microfilariae (mf) in treated patients remained significantly reduced, whereas the number of permanently amicrofilaridermic patients (subclinical infection) increased with repeated treatments. In vitro cellular responses to O. volvulus antigen (OvAg) were highest (P<0.001) in untreated control individuals exposed to infection, but negative for mf of O. volvulus (endemic normals). Cellular reactivity in repeatedly treated patients was higher at 84 than at 36 months post initial treatment (p.i.t.); furthermore, the proliferative responses to OvAg, mycobacterial purified protein derivative (PPD) and streptococcal SL‐O were greater (P<0·05) at 84 months p.i.t. in amicrofilaridermic than in microfilaria‐positive onchocerciasis patients. In amicrofilaridermic patients such reactivity approached the magnitude observed in endemic normals. Peripheral blood mononuclear cells (PBMC) from patients and endemic normals produced equivalent amounts of IL‐2. IL‐4 and interferon‐gamma (IF N‐γ) in response to mitogenic stimulation with phytohaemagglutinin (PHA); in response to OvAg, however, significantly more IL‐2 and IFN‐γ were produced by PBMC from subclinical amicrofilaridermic patients or endemic normals than by mf‐positive patients OvAg‐specific production or IL‐4 by PBMC from treated patients was lower at 84 than at 36 months p.i.t. At three months p.i.t. the titres of circulating OvAg‐specific IgG1‐3 had increased (P<0·05), but they then continuously declined with repeated treatments. Only IgG1 and IgG4 bound to OvAg of mol. wt 2‐l2 kD at 1 month p.i.t., while recognition of OvAg of mol. wt 10–200 kD by IgG1, IgG2 and IgG4 reached a maximum intensity at 3–6 months p.i.t., with the overall intensity of binding to OvAg gradually weakening thereafter. These results suggest that onchocerciasis‐associated immuno‐suppression is reversible following ivermectin‐induced permanent clearance of microfilariae from the skin; and that a vigorous parasite‐specific cellular reactivity and a sustained production of IL‐2 and IFN‐γ in amicrofilaridermic individuals may contribute to controlling O. volvulus infection.

Ivermectin‐facilitated immunity in onchocerciasis. Reversal of lymphocytopenia, cellular anergy and deficient cytokine production after single treatment

A longitudinal investigation has been conducted into the cell‐mediated immune responses of onchocerciasis patients after a single‐dose treatment with ivermectin. Untreated patients tested for delayed cutaneous hypersensitivity (DCH) to seven recall antigens showed lower responses than infection‐free control individuals (P <0.01), but 6 and 14 months after treatment DCH reactions increased to similar levels to those seen in the controls. The in vitro cellular reactivity to Onchocerca volvulus‐derived antigen (OvAg) was reduced in untreated patients as compared with controls, and the lymphocyte blastogenic responses to OvAg and streptolysin‐O clearly improved up to 14 months after treatment. Peripheral blood mononuclear cells (PBMC) from untreated patients produced IL‐Iβ, tumour necrosis factor‐alpha (TNF‐α) and IL‐6 in response to mitogenic stimulation with phytohaemagglutinin (PHA), only low levels of IL‐Iβ, IL‐2 and TNF‐α in response to OvAg, but higher amounts of IL‐4 and interferon‐gamma (IFN‐γ) in response to OvAg than control individuals. After ivermectin treatment, the OvAg‐induced production of IL‐1β and TNF‐α increased significantly I and 14 months after treatment. The PHA‐induced production of IL‐2 and IL‐4 increased 1 month after treatment and remained significantly elevated until 14 months after treatment, whereas the OvAg‐specific secretion of IL‐2, IL‐4 and IFN‐γ did not change after ivermectin treatment. Flow cytometric analysis of lymphocyte‐subsets in the peripheral blood of untreated patients revealed a relative and absolute (P <0.01) diminution of CD4+ cells and a significantly smaller CD4+/CD8+ cell ratio as compared with controls. By 4 weeks after treatment and thereafter, CD4+ T cells increased relatively and absolutely (P <0.01); likewise there was an absolute increase in T‐helper‐inducer cells (CD4+CD45RO+) and a temporarily improved CD4+/ CD8+ cell ratio (P=0.001). The expression of the low‐affinity receptor for IgE (CD23) on total lymphocytes decreased from 14% to 7% by 14 months after treatment. The CD8+ cells and CD3+TCRγδ+ cells were higher in patients than in controls and both remained elevated until 14 months after treatment. These results suggest a distinctly improved cellular immunity in human onchocerciasis that was facilitated by ivermectin therapy.

Proinflammatory and regulatory cytokines and chemokines in infants with uncomplicated and severe Plasmodium falciparum malaria

Cytokine and chemokine levels were studied in infants (<5 years) with uncomplicated (MM) and severe malaria tropica (SM), and in Plasmodium falciparum infection‐free controls (NEG). Cytokine plasma levels of interleukin (IL)‐10, IL‐13, IL‐31 and IL‐33 were strongly elevated in MM and SM compared to NEG (P < 0·0001). Inversely, plasma concentrations of IL‐27 were highest in NEG infants, lower in MM cases and lowest in those with SM (P < 0·0001, NEG compared to MM and SM). The levels of the chemokines macrophage inflammatory protein (MIP3)‐α/C–C ligand 20 (CCL20), monokine induced by gamma interferon (MIG)/CXCL9 and CXCL16 were enhanced in those with MM and SM (P < 0·0001 compared to NEG), and MIP3‐α/CCL20 and MIG/CXCL9 were correlated positively with parasite density, while that of IL‐27 were correlated negatively. The levels of 6Ckine/CCL21 were similar in NEG, MM and SM. At 48–60 h post‐anti‐malaria treatment, the plasma concentrations of IL‐10, IL‐13, MIG/CXCL9, CXCL16 and MIP3‐α/CCL20 were clearly diminished compared to before treatment, while IL‐17F, IL‐27, IL‐31 and IL‐33 remained unchanged. In summary, elevated levels of proinflammatory and regulatory cytokines and chemokines were generated in infants during and after acute malaria tropica. The proinflammatory type cytokines IL‐31 and IL‐33 were enhanced strongly while regulatory IL‐27 was diminished in those with severe malaria. Similarly, MIP3‐α/CCL20 and CXCL16, which may promote leucocyte migration into brain parenchyma, displayed increased levels, while CCL21, which mediates immune surveillance in central nervous system tissues, remained unchanged. The observed cytokine and chemokine production profiles and their dynamics may prove useful in evaluating either the progression or the regression of malarial disease.

Immunoregulation in onchocerciasis : predominance of Th1-type responsiveness to low molecular weight antigens of Onchocerca volvulus in exposed individuals without microfilaridermia and clinical disease

Chronic and generalized onchocerciasis is associated with suppression of the parasite‐specific cellular responsiveness, while exposed individuals without parasitological and clinical evidence of infection (endemic normals) display prominent cellular reactivity to Onchocerca volvulus antigens (OvAg). In order to identify those parasite antigens which may account for this differential cellular responsiveness, total adult worm‐derived OvAg were fractionated by means of preparative SDS–PAGE and blot‐elution into 22 antigen fractions of continuously decreasing molecular weight. Peripheral blood mononuclear cells (PBMC) from microfilariae (mf)‐positive onchocerciasis patients (n = 18) proliferated weakly in response to all OvAg fractions. In contrast, in vitro reactivity of PBMC from endemic normals (n = 9) was depressed in response to OvAg of mol. wt 200–30 kD only, while antigens of mol. wt <30 kD induced vigorous proliferation in these individuals compared with the microfilaridermic patients (P < 0.05). Highest proliferative reactivity of cells from endemic normals was observed in response to OvAg of mol. wt 15–11 kD. Furthermore, these low mol. wt antigen fractions induced substantial production of IL‐2 and interferon‐gamma (IFN‐γ) in PBMC from endemic normals, but not in those from onchocerciasis patients. Cells from individuals of both groups secreted similar amounts of IL‐5 in response to all OvAg fractions, with highest production again being induced by low mol. wt OvAg. In contrast, PBMC from onchocerciasis patients clearly produced more IL‐10 than did cells from endemic normals. This augmented IL‐10 production by PBMC from mf‐positive individuals was not only observed after stimulation with OvAg fractions, but was measured in unstimulated control cultures as well. IFN‐γ‐specific mRNA in antigen‐stimulated PBMC from endemic normals appeared to be more prominent than in cells from onchocerciasis patients. However, mRNA transcripts of IL‐10 and IL‐13 were clearly present in patients, but were absent or inconsistently observed in endemic normals. Our results suggest that vigorous Th1‐type cellular responsiveness encountered in endemic normals is restricted to low mol. wt antigens of O. volvulus, while such reactivity will not be present in mf‐positive individuals. Furthermore, spontaneous production of high levels of IL‐10 in onchocerciasis patients is likely to suppress Th1‐type immunity, and thus may favour manifestation of chronic onchocerciasis. These traits of cellular immunity may contribute to the differential outcome of O. volvulus infection, the manifestation of clinical disease, and may also regulate the build up of acquired immunity in humans.

Coinfections with Schistosoma haematobium, Necator americanus and Entamoeba histolytica/Entamoeba dispar in Children: Chemokine and Cytokine Responses and Changes after Antiparasite Treatment

The effect of polyparasite infections on cytokine and chemokine responses as well as the effect of antiparasite treatment was studied in children without parasite infection (the G0 group), in children singly infected with Schistosoma haematobium (the G1 group), and in children multiply infected with S. haematobium/Schistosoma mansoni, Entamoeba histolytica/Entamoeba dispar, and Necator americanus (the G3+ group). Linear regression analysis disclosed a significant risk for coinfection with hookworm and Schistosoma species. Polyparasite infections detected in 23% of children before treatment were present in 5% at 15 months after treatment. Chemokine responses to S. mansoni adult worm antigen (SmAg) diminished after treatment for macrophage inflammatory chemokine (MIP)-1alpha/chemokine (C-C motif) ligand (CCL)-3 (among G3+ children, by a factor of 200 [95% confidence interval {CI}, 33-1111]) and for MIP-1beta/CCL-4 (among G3+ children, by a factor of 26 [95% CI, 6-117]) but were enhanced for thymus- and activation-regulated chemokine/CCL-17 (among G3+ children, by a factor of 10 [95% CI, 3-32]) (P < .001 for all). In response to E. histolytica antigen, interleukin (IL)-13 levels increased after treatment among G1 children by a factor of 138 (95% CI, 12-1569) and among G3+ children by a factor of 21 (95% CI, 7-64) (P < .001 for both). Cellular production of interferon (IFN)-gamma in response to SmAg decreased 4 weeks after treatment among G3+ children, whereas T helper cell type 2 (Th2) IL-13 production was enhanced among G1 and G3+ children. In summary, polyparasite infections with S. haematobium/S. mansoni, E. histolytica/E. dispar, and N. americanus generated prominent proinflammatory cytokine and chemokine responses, and, after antihelminth treatment, the inflammatory chemokine response lessened as the Th2 responsiveness in coinfected children increased.

Parasite‐specific antibody and cytokine profiles in newborns from Plasmodium falciparum and Entamoeba histolytica/ dispar‐infected mothers

Passage of parasites and their antigens across the placenta occurs with metazoan as well as protozoan parasites, and this study addressed to which extent exposure to and infection of mothers with Plasmodium spp. and Entamoeba histolytica/dispar has sensitized their offspring for parasite‐specific immune responses. While at delivery none of the mothers presented with an acute malaria attack, 42% were seropositive for P. falciparum. In half of the mothers cysts of E. histolytica/dispar were detected in stool specimen, 51% of them were found seropositive for E. histolytica, and E. histolytica‐specific immunoglobulin A (IgA) responses were detected in neonates of seropositive mothers as well. Umbilical cord blood cells (UCBC) from neonates, when activated with the mitogen phytohaemagglutinine (PHA) and bacterial streptolysin O (SL‐O), released significantly less interferon (IFN)‐γ, interleukin (IL)‐10 and tumor necrosis factor (TNF)‐α into cell culture supernatants than peripheral blood cells (PBMC) of mothers. In response to Plasmodium‐ and Entamoeba‐specific antigens UCBC and PBMC produced equal amounts of IL‐1β, TNF‐α, IFN‐γ and IL‐5, but PBMC from mothers secreted significantly more IL‐10. Parasite‐specific production of inflammatory and Th1‐ and Th2‐type cytokines was similar in newborns of Plasmodium and Entamoeba seropositive and seronegative mothers. In summary, repeated exposure and subclinical infection of mothers with E. histolytica or P. falciparum will suffice to prime in utero their children for inflammatory and both Th1‐ and Th2‐type cytokine responses, and such broad and mixed cytokine spectrum may be of advantage upon secondary parasite challenge in later life.

Efficacy of ivermectin in the treatment of concomitant Mansonella perstans infections in onchocerciasis patients

As part of an ivermectin dose-ranging study of onchocerciasis patients in Togo, 55 onchocerciasis patients with concomitant mansonelliasis received single oral doses either of ivermectin (100 to 200 micrograms/kg body weight) or placebo. As expected, Onchocerca volvulus microfilariae in the skin were greatly reduced in number soon after drug treatment, but microfilariae of Mansonella perstans reacted differently. Microfilarial densities of M. perstans were assessed with a filtration technique both before, and 4 times after, treatment. In untreated patients microfilarial densities were stable until the end of the study at 6 months. In patients receiving ivermectin, microfilarial densities dropped on average to less than 60% of the pre-treatment level and remained there until the final post-treatment examination. This partial reduction was probably not caused by a microfilaricidal effect of ivermectin, but rather by an altered distribution of microfilariae in the peripheral blood and in a suspected microfilarial reservoir.

Onchocerca volvulus-specific antibody and cytokine responses in onchocerciasis patients after 16 years of repeated ivermectin therapy.

The recommended control option against onchocerciasis is repeated ivermectin treatment, which will need to be implemented for decades, and it remains unknown how repeated ivermectin therapy might affect immunity against Onchocerca volvulus in the long term. O. volvulus‐specific antibody reactivity and cellular cytokine production were investigated in onchocerciasis patients receiving ivermectin (150 µg/kg) annually for 16 years. In treated patients, the T helper type 2 (Th2) cytokine interleukin (IL)‐5 and T regulatory IL‐10 in response to O. volvulus antigen (OvAg) and bacteria‐derived Streptolysin O (SL‐O) diminished to levels found in infection‐free endemic controls; also, cellular release of Th1‐type interferon (IFN)‐γ at 16 years post initial ivermectin treatment (p.i.t.) approached control levels. In ivermectin‐treated onchocerciasis patients, IL‐5 production in responses to the mitogen phytohaemagglutinin (PHA) decreased, but IL‐10 in response PHA increased, and neither attained the cytokine production levels of endemic controls. At 16 years p.i.t., O. volvulus‐specific IgG1 and IgG4 subclass reactivity still persisted at higher levels in onchocerciasis patients than in O. volvulus exposed but microfilariae‐free endemic controls. In addition, cytokine responses remained depressed in onchocerciasis patients infected concurrently with Mansonella perstans and Necator americanus or Entamoeba histolytica/dispar. Thus, long‐term ivermectin therapy of onchocerciasis may not suffice to re‐establish fully a balanced Th1 and Th2 immune responsiveness in O. volvulus microfilariae‐negative individuals. Such deficient reconstitution of immune competence may be due to an as yet continuing and uncontrolled reinfection with O. volvulus, but parasite co‐infections can also bias and may prevent the development of such immunity.

Chemokines in onchocerciasis patients after a single dose of ivermectin

Ivermectin treatment will effectively diminish microfilariae (Mf) of Onchocerca volvulus in the skin of patients, but therapy is associated with adverse host inflammatory responses. To investigate the association of proinflammatory chemokines with the intensity of infection and clinical adverse reactions, chemokine serum levels were measured in patients following ivermectin treatment (100 µg/kg, 150 µg/kg or 200 µg/kg) or placebo. The density of O. volvulus Mf per mg skin decreased by 85%, 97%, 97% and 90% at day 3, at month 3, month 6 and at 1 year post‐ivermectin. The cutaneous T cell‐attracting chemokine (CTACK/CCL27) was found highly elevated in onchocerciasis patients compared to infection‐free European controls (P = 0·0004) and it did not change following ivermectin or placebo to 1 year post‐therapy. The chemokine RANTES/CCL5 (regulated on activated and normally T cell‐expressed) was similarly high in onchocerciasis patients and infection‐free European controls; the RANTES/CCL5 levels did not change following treatment until 6 months post‐therapy but were slightly elevated at 1 year post‐therapy (P < 0·02). In contrast, the Th2‐type chemoattractants, thymus and activation regulated chemokine (TARC/CCL17) and macrophage‐derived chemokine (MDC/CCL22), were activated at 3 days post‐ivermectin (P < 0·0001) to return to pretreatment or lower levels thereafter. The Th1‐type chemoattractants, macrophage inflammatory protein (MIP)‐1α/CCL3 and MIP‐1β/CCL4 were low before ivermectin treatment, but following clearance of microfilariae of O. volvulus their levels increased from 6 months post‐therapy onwards (for both at 12 months post‐therapy, P < 0·0001). The adverse reaction scores (RS) in treated patients increased significantly on day 3 (P < 0·02) while it remained unchanged in those who received placebo (P = 0·22); RS interacted with the microfilarial density (P = 0·01), but not with the dose of ivermectin or with the serum levels of MIP‐1α/CCL3, MIP‐1β/CCL4, TARC/CCL17, MDC/CCL22 and CTACK/CCL27. Our observations suggest that following ivermectin, macrophages as well as memory Th2‐type lymphocytes and B cells, attracted and activated by MDC/CCL22, TARC/CCL17 and CTACK/CCL27, may contribute to dermal immune responses and O. volvulus Mf killing and clearance. The transient changes of TARC/CCL17 and MDC/CCL22 were not associated with clinical adverse responses, and the later rise of MIP‐1α/CCL3 and MIP‐1β/CCL4 showed a reactivation of Type 1 immune responses associated with persistent low levels of O. volvulus microfilariae and an expiring O. volvulus infection.

A Recombinant Positive Control for Serology Diagnostic Tests Supporting Elimination of Onchocerca volvulus

Background Serological assays for human IgG4 to the Onchocerca volvulus antigen Ov16 have been used to confirm elimination of onchocerciasis in much of the Americas and parts of Africa. A standardized source of positive control antibody (human anti-Ov16 IgG4) will ensure the quality of surveillance data using these tests. Methodology/Principal Findings A recombinant human IgG4 antibody to Ov16 was identified by screening against a synthetic human Fab phage display library and converted into human IgG4. This antibody was developed into different positive control formulations for enzyme-linked immunosorbent assay (ELISA) and rapid diagnostic test (RDT) platforms. Variation in ELISA results and utility as a positive control of the antibody were assessed from multiple laboratories. Temperature and humidity conditions were collected across seven surveillance activities from 2011–2014 to inform stability requirements for RDTs and positive controls. The feasibility of the dried positive control for RDT was evaluated during onchocerciasis surveillance activity in Togo, in 2014. When the anti-Ov16 IgG4 antibody was used as a standard dilution in horseradish peroxidase (HRP) and alkaline phosphatase (AP) ELISAs, the detection limits were approximately 1ng/mL by HRP ELISA and 10ng/mL by AP ELISA. Positive control dilutions and spiked dried blood spots (DBS) produced similar ELISA results. Used as a simple plate normalization control, the positive control antibody may improve ELISA data comparison in the context of inter-laboratory variation. The aggregate temperature and humidity monitor data informed temperature parameters under which the dried positive control was tested and are applicable inputs for testing of diagnostics tools intended for sub-Saharan Africa. As a packaged positive control for Ov16 RDTs, stability of the antibody was demonstrated for over six months at relevant temperatures in the laboratory and for over 15 weeks under field conditions. Conclusions The recombinant human anti-Ov16 IgG4 antibody-based positive control will benefit inter-laboratory validation of ELISA assays and serve as quality control (QC) reagents for Ov16 RDTs at different points of the supply chain from manufacturer to field use.