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.

BACKGROUND: 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. METHODOLOGY/PRINCIPAL FINDINGS: Onchocerciasis patients were treated annually with ivermectin and their Onchocerca volvulus antigen (OvAg) specific IgG and cellular responses were investigated before and at 30 years post initial ivermectin treatment (30yPT). 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. CONCLUSIONS/SIGNIFICANCE: 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.

Sustainable control of onchocerciasis: ocular pathology in onchocerciasis patients treated annually with ivermectin for 23 years: a cohort study.

UNLABELLED: The evolution and persistence of ocular pathology was assessed in a cohort of Onchocerca volvulus infected patients treated annually with ivermectin for 23 years. Patients were resident in rural Central and Kara Region of Togo and ocular examinations included testing of visual acuity, slit lamp examination of the anterior eye segment and the eye fundus by ophthalmoscopy. Before ivermectin treatment, vivid O.volvulus microfilariae (MF) were observed in the right and left anterior eye chamber in 52% and 42% of patients (n = 82), and dead MF were seen in the right and left cornea in 24% and 15% of cases, respectively. At 23 years post initial treatment (PIT), none of the patients (n = 82) presented with MF in the anterior chamber and cornea. A complete resolution of punctate keratitis (PK) lesions without observable corneal scars was present at 23 years PIT (p<0.0001), and sclerosing keratitits (SK) lessened by half, but mainly in patients with lesions at early stage of evolution. Early-stage iridocyclitis diminished from 42%(rE) and 40%(lE) to 13% (rE+lE)(p<0.0001), but advanced iridocyclitis augmented (p<0.001) at 23 years PIT compared to before ivermectin. Advanced-stage papillitis and chorioretinitis did not regress, while early-stage papillitis present in 28%(rE) and 27%(lE) of patients at before ivermectin regressed to 17%(rE) and 18%(lE), and early-stage chorioretinitis present in 51%(rE+lE) of cases at before ivermectin was observed in 12%(rE) and 13%(lE) at 23 years PIT (p<0.0001). Thus, regular annual ivermectin treatment eliminated and prevented the migration of O. volvulus microfilariae into the anterior eye chamber and cornea; keratitis punctata lesions resolved completely and early-stage sclerosing keratitits and iridocyclitis regressed, whilst advanced lesions of the anterior and posterior eye segment remained progressive. In conclusion, annual ivermectin treatments may prevent the emergence of ocular pathology in those populations still exposed to O.volvulus infection. TRIAL REGISTRATION: www.pactr.org PACTR201303000464219).

Chemokines and cytokines in patients with an occult Onchocerca volvulus infection.

Repeated ivermectin treatment will clear microfilaria (Mf) of Onchocerca volvulus from skin and eyes of onchocerciasis patients while adult filaria remains alive and reproductive, and such occult O. volvulus infection may persist for years. To investigate the effect of residual adult filaria on the immune response profile, chemokines and cytokines were quantified 1) in onchocerciasis patients who developed an occult O. volvulus infection (Mf-negative) due to repeated ivermectin treatments, 2) patients who became Mf-negative without ivermectin treatments due to missing re-infection, and 3) endemic and non-endemic O. volvulus Mf-negative controls. With occult O. volvulus infection, serum levels of pro-inflammatory chemokines MCP-1/CCL2, MIP-1α/CCL3, MIP-1ß/CCL4, MPIF-1/CCL23 and CXCL8/IL-8 enhanced and approached higher concentrations as determined in infection-free controls, whilst regulatory and Th2-type cytokines and chemokines MCP-4/CCL13, MIP-1δ/CCL15, TARC/CCL17 and IL-13 lessened. Levels of Eotaxin-2/CCL24, MCP-3/CCL7 and BCA-1/CXCL13 remained unchanged. At 3 days post-initial ivermectin treatment, MCP-1/CCL2, MCP-4/CCL13, MPIF-1/CCL23 and Eotaxin-2/CCL24 were strongly enhanced, suggesting that monocytes and eosinophil granulocytes have mediated Mf clearance. In summary, with occult and expiring O. volvulus infections the serum levels of inflammatory chemokines enhanced over time while regulatory and Th2-type-promoting cytokines and chemokines lessened; these changes may reflect a decreasing effector cell activation against Mf of O. volvulus, and in parallel, an enhancing inflammatory immune responsiveness.

Applied field research for comprehensive helminth infection control.

The Institute for Tropical Medicine at University of Tübingen has established 30 years ago in Togo a Research Centre and Onchocerciasis Reference Laboratory (ORL). Onchocerca volvulus infection control and of other neglected tropical diseases has been the focus of activities, and those were performed together with the National Institute of Hygiene in Togo, the Medical Faculty at University of Lomé, national disease control programs and district and regional hospitals. The ORL contributed significantly to the assessment of ivermectin as the prime choice for onchocerciasis treatment, and 24 years of repeated annual treatment with ivermectin has progressively reduced disease prevalence and notably the level of ocular and dermal manifestations of onchocerciasis in the endemic population. The ORL has shown that large parts of the rural population in Togo is concurrently infected with intestinal and intravascular protozoan and helminth parasites, notably school children. The application of repeated treatments with albendazole and praziquantel against Schistosoma spp. and instestinal helminthes for several years has reduced infection intensities by more than 80%. Longitudinal investigations of the cellular immune responses in adults and children have found that parasite co-infections will generate prominent pro-inflammatory responses, and a single or few interventions will not suffice to eliminate co-infections and not establish an appropriately balanced immunity.

Of mice, cattle, and humans: the immunology and treatment of river blindness.

River blindness is a seriously debilitating disease caused by the filarial parasite Onchocerca volvulus, which infects millions in Africa as well as in South and Central America. Research has been hampered by a lack of good animal models, as the parasite can only develop fully in humans and some primates. This review highlights the development of two animal model systems that have allowed significant advances in recent years and hold promise for the future. Experimental findings with Litomosoides sigmodontis in mice and Onchocerca ochengi in cattle are placed in the context of how these models can advance our ability to control the human disease.

Microfilariae of the filarial nematode Litomosoides sigmodontis exacerbate the course of lipopolysaccharide-induced sepsis in mice.

Helminths facilitate their own survival by actively modulating the immune systems of their hosts. We investigated the impacts that different life cycle stages of the rodent filaria Litomosoides sigmodontis have on the inflammatory responses of mice injected with sublethal doses of lipopolysaccharide (LPS). Mice infected with female adult worms from prepatent infections, worms which have not yet started to release microfilariae, developed lower levels of proinflammatory cytokines in the peripheral blood after LPS challenge than sham-treated controls, demonstrating that female adult worms can mitigate the innate immune response. The presence of microfilariae in mice, however, through either direct injection or implantation of microfilaria-releasing adult female worms, turned the LPS challenge fatal. This lethal outcome was characterized by increased plasma levels of gamma interferon (IFN-gamma), tumor necrosis factor alpha (TNF-alpha), interleukin 12 (IL-12), and IL-6, greater numbers of macrophages and granulocytes in the peripheral blood, and decreased body temperatures in microfilaria-infected mice. Microfilaria-infected mice deficient in IFN-gamma receptor and TNF receptor 1 had increased survival rates after LPS challenge compared to immune-competent mice, suggesting that microfilariae worsen LPS-induced sepsis through actions of IFN-gamma and TNF-alpha. In summary, we have demonstrated that infection of mice with L. sigmodontis female adult worms from prepatent infections protects mice injected with LPS whereas microfilariae worsen LPS-induced sepsis through the induction of proinflammatory cytokines and upregulation of granulocytes, NK cells, and monocytes in the peripheral blood.

Antibody and cytokine responses in Dracunculus medinensis patients at distinct states of infection.

Dracunculiasis is a promising candidate for eradication, but transmission of Dracunculus medinensis and recrudescence of the disease have been observed repeatedly. In the present investigation, the D. medinensis-specific cellular cytokine response profiles and the parasite-specific antibody subclass reactivity were evaluated in dracunculiasis patients at distinct states of infection. Analysis of the cellular cytokine response in dracunculiasis patients disclosed a D. medinensis antigen-specific depression of IFN-gamma production with patent D. medinensis infection, while the T helper type 2 cytokine IL-10 was similar in patent, post-patent and control individuals, and IL-5 production was always the highest in controls. In parallel, diminished IFN-gamma and IL-12 responses to antigens from Ascaris lumbricoides, Entamoeba histolytica and mycobacteria were observed in patent and post-patent dracunculiasis cases. The parasite-specific IgG(1) and IgG(4) subclass reactivity profiles corresponded with the D. medinensis infection state, and a clear distinction between patent and post-patent patients and controls was found. Overall a depressed cytokine release was observed with patent D. medinensis, which extended beyond the parasite-specific immune responsiveness. The detection of D. medinensis-specific IgG(1) and IgG(4) isotypes may help to distinguish newly exposed, patent and post-patent D. medinensis infections.

Cytokine and chemokine responses in patients co-infected with Entamoeba histolytica/dispar, Necator americanus and Mansonella perstans and changes after anti-parasite treatment.

This study examined the impact of concurrent parasite infections (amoebiasis, filariasis, necatoriasis) and the effect of anti-parasite treatment on cytokine and chemokine responses in singly and poly-parasitized patients. Cellular reactivity and parasite-specific Th1- and Th2-type cytokine and chemokine profiles were investigated before and six weeks after treatment. In those patients infected with three parasite species, cellular secretion of interleukin 5 (IL-5) and IL-12p40 by PBMC was strongly diminished (p<0.005) but IL-10 was elevated in parasite-infected patients (p<0.0001) in response to protozoa- and helminth-specific as well as bacteria-specific antigens. Macrophage inflammatory chemokines (MIP-1alpha/CCL3 and MIP-1beta/CCL4), macrophage-derived chemokine (MDC/CCL22) and neutrophil activating chemokine (IL-8/CXCL8) were produced by PBMC in similar amounts in endemic controls and singly and poly-parasitized patients, but thymus and activation-regulated chemokine (TARC/CCL17) was produced the highest by PBMC from patients with triple parasite infections (p<0.0001). Following anti-parasite therapy, secretion of IL-12p40 and IL-5 augmented significantly in treated patients while IL-10, MDC, MIP-1alpha, TARC and IL-8 substantially diminished (all p<10(-5)) when their PBMC were activated with parasite- and bacteria-specific antigens. In summary, PBMC from poly-parasitized patients responded to protozoa- and helminth-specific antigens with a compromised IL-5 and IL-12p40 but high IL-10 and a substantial chemokine release. Chemokines may attract and activate effector cells in peri-parasitic tissues to limit parasite proliferation and dissemination, while depressed IL-5 and IL-12p40 but prominent IL-10 may prevent eosinophil and cytotoxic cell-mediated inflammatory processes and pathogenesis to the host. The changes in this profile following anti-parasite therapy disclosed the dynamics of an immune adaptation associated with parasite accumulation and also with clearance of parasite infections.

Longitudinal clinical and serological survey of abdominal angiostrongyliasis in Guaporé, southern Brazil, from 1995 to 1999.

Abdominal angiostrongyliasis is a zoonotic infection caused by Angiostrongylus costaricensis, a nematode with an intra-vascular location in the mesentery. Our objective was to address several aspects of the natural history of this parasitosis, in a longitudinal clinical and seroepidemiological study. A total of 179 individuals living in a rural area with active transmission in southern Brazil were followed for five years (1995-1999) resulting in yearly prevalence of 28.2%, 4.2%, 10%, 20.2% and 2.8% and incidences of 0%, 5.9%, 8% and 1.5%, respectively. Both men and woman were affected with higher frequencies at age 30-49 years. In 32 individuals serum samples were collected at all time points and IgG antibody reactivity detected by ELISA was variable and usually persisting not longer than one year. Some individual antibody patterns were suggestive of re-infection. There was no association with occurrence of abdominal pain or of other enteroparasites and there was no individual with a confirmed (histopathologic) diagnosis. Mollusks were found with infective third-stage larvae in some houses with an overall prevalence of 16% and a low parasitic burden. In conclusion, abdominal angiostrongyliasis in southern Brazil may be a frequent infection with low morbidity and a gradually decreasing serological reactivity.

Longitudinal clinical and serological survey of abdominal angiostrongyliasis in Guaporé, southern Brazil, from 1995 to 1999

Angiostrongilíase abdominal é uma zoonose causada pelo Angiostrongylus costaricensis, nematódeo que se localiza no interior de vasos mesentéricos. Nosso objetivo foi de abordar vários aspectos da história natural da parasitose, num estudo longitudinal clínico-sorológico. Um total de 179 indivíduos residentes em área rural no sul do Brasil, com transmissão ativa, foram seguidos por cinco anos. Neste período foram registradas prevalências de 28,2%, 4,2%, 10%, 20,2% e 2,8% e incidências de 0%, 5,9%, 8% e 1,5%. Tanto o sexo masculino quanto o feminino foram afetados com maiores frequências na faixa etária dos 30 aos 49 anos. Em 32 indivíduos, amostras de soro foram coletadas em todas as etapas e a reatividade de IgG detectada por ELISA foi variável e geralmente não persistindo mais do que um ano. Alguns padrões individuais foram sugestivos de re-infecção. Não houve associação com a ocorrência nem de dor abdominal nem com outras enteroparasitoses e não houve nenhum caso com diagnóstico confirmado (histopatológico) da infecção. Moluscos foram encontrados portando larvas infectantes de terceiro estadio, em algumas moradias, com uma prevalência geral de 16% e baixas cargas parasitárias. Em conclusão, a angiostrongilíase abdominal no sul do Brasil pode ser uma infecção frequente, porém com baixa morbidade e reatividade sorológica de gradual declínio.

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)-gamma, interleukin (IL)-10 and tumor necrosis factor (TNF)-alpha 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-1beta, TNF-alpha, IFN-gamma and IL-5, but PBMC from mothers secreted significantly more IL-10. Parasite-specific production of inflammatory and Th(1)- and Th(2)-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 Th(1)- and Th(2)-type cytokine responses, and such broad and mixed cytokine spectrum may be of advantage upon secondary parasite challenge in later life.

Caracterización de antígenos de bajo peso molecular de angiostrongylus costaricensis, reconocidos durante una infección experimental en roedores / Characterization of low molecular weight antigens of angiostrongylus costaricensis recognized during an experimental infection in mice

La respuesta inmune hacia componentes de bajo peso molecular del antígeno somático de A. costaricensis fue investigada en el modelo animal experimental. Durante la fase crónica de la infección y mediante el Western blot, son detectados por la IgG e IgG1 tres antígenos inmunodominantes: uno de 20 otro de 15 y uno de 2 kDa respectivamente. Mediante el método de digestión de Edmann se obtuvo la secuencia de aminoácidos inicial de cada componente: para la banda de 15 kDa una secuencia con un 100 por ciento de homología con la enzima glutation-S-transferasa de Brugia malayi y Onchocerca volvulus, para el componete de 2kDa una secuencia con 95 por ciento de homología con la proteína Ubiquitina, y para el compontente de 20 kDa una secuencia de trece aminoácidos, que no presenta homología con alguna proteína hasta ahora descrita. En general la reacción cruzada contra estos componentes es muy débil y en el caso particular del componente de 20 kDa no se detecta ninguna reacción cruzada. Se proponen futuros estudios con sueros humanos, para verificar la utilidad de dichos antígenos en el desarrollo de una prueba diagnóstica específica para la angiostrongiliosis abdominal.

Regulatory effects of IL-12 and IL-18 on Onchocerca volvulus- and Entamoeba histolytica-specific cellular reactivity and cytokine profiles.

In the present study, the cytokines interleukin (IL)-12 and IL-18 were evaluated for their capacity to modulate and to re-direct in vitro parasite antigen-specific cellular responsiveness in patients exposed to Onchocerca volvulus and Entamoeba histolytica infection. We found that IL-18 was highly capable of reducing parasite antigen-induced IL-10 production by PBMC. In contrast, addition or neutralization of IL-12, also in combination with IL-18 and the interferon-gamma-inducible chemokine IP-10 did not affect IL-10 production. Interestingly, the highest IL-10 levels were measured when IL-18 and IP-10 were both neutralized. Although having no effect on IL-10, IL-12 strongly promoted spontaneous and parasite antigen-driven IFN-gamma production by PBMC, whereas IL-18 was only moderately affecting IFN-gamma release by PBMC re-stimulated with E. histolytica- or O. volvulus-specific antigens. Both IL-12 and IL-18 diminished the cellular production of IL-13, and a synergistic effect was observed when the cytokines were combined. Likewise, neutralization of IL-12 enhanced Entamoeba and Onchocerca antigen-driven IL-13 production, but no further increase of IL-13 was observed, when anti-IL-12 and anti-IL-18 were used together. This study disclosed that IL-18 will significantly down-regulate parasite-specific IL-10 production, whereas IL-12 induced IFN-gamma and inhibited IL-13 production by PBMC from humans exposed to O. volvulus and E. histolytica. Such selective immune-regulatory capacity of IL-12 and IL-18 may comprise an important tool to re-direct polarized cytokine responses towards a balanced Th1/Th2 cytokine profile, which may prevent pathology and promote immunity against helminth and protozoan parasite infections.

Litomosoides sigmodontis cystatin acts as an immunomodulator during experimental filariasis.

During chronic filariasis, parasite-specific cellular responsiveness is profoundly down-regulated. Cystatins, a group of cysteine protease inhibitors, have been implicated in this suppressive activity. In an attempt to investigate the effects of cystatins in vivo, we isolated and expressed a 14 kDa protein of the rodent filaria Litomosoides sigmodontis with substantial homologies to cystatins from human pathogenic filariae. Cystatin was detected in antigen preparations of several developmental stages of L. sigmodontis, as well as in the supernatants of in vitro cultured adult worms. On closer examination, L. sigmodontis cystatin (Ls-Cystatin) migrated as two separate bands at 14 and 15 kDa. When cystatin was introduced into the peritoneal cavity of C57BL/6 mice via micro-osmotic pumps, the production of nitric oxide was profoundly reduced upon microfilarial challenge and, at the same time, synthesis of TNF-alpha mRNA became up-regulated. Furthermore, antigen-specific proliferative response of spleen cells to circulating L. sigmodontis microfilariae was significantly diminished in the presence of cystatin, whereas the antibody production was not suppressed. In vaccination trials, using the L. sigmodontis/BALB/c mouse model of filariasis, L. sigmodontis cystatin did not generate protective effects in terms of adult worm recovery, however, lower numbers of patent infections, i.e. less infections with microfilaraemia were observed in vaccinated animals. These results suggested that cystatin acts as an immunomodulatory molecule during the course of a filarial infection, and its neutralisation might contribute to generate protective immune responses.

Detection of the acute phase of abdominal angiostrongyliasis with a parasite-specific IgG enzyme linked immunosorbent assay

Angiostrongylus costaricensis may cause intestinal lesions of varied severity when it accidentally infects man in Central and South America. First-stage larvae have never been detected in stools. Therefore, a parasite-specific IgG ELISA was evaluated for the determination of the acute phase of infection. The specificity and the sensitivity of the immunoassay was shown to be 76.2 percent and 91.1 percent, respectively. Eight serum samples taken from patients with histopathological diagnosis, at different time points (3 to 15 months) after surgical treatment, showed a sharp and early decline in antibody reactivity. The titration of anti-A. costaricensis antibodies has proved to be a useful method for the diagnosis of acute abdominal angiostrongyliasis