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Background The infestation with Echinococcus multilocularis larvae may persist in humans for up to decades without evident clinical symptoms. Longitudinal investigations are needed to understand the dynamic immunological processes in alveolar echinococcosis (AE) patients associated with an active and progressive, a stable or a regressive course of disease. Methodology/Principal findings This study evaluated the E. multilocularis specific antibody responses, systemic cytokine, and chemokine serum levels over a 10-year follow-up period, as well as cellular responsiveness in AE patients. Our results demonstrate a rapid decrease in antibodies against E. multilocularis specific antigen Em2+. Especially in cured patients, these antibodies remained negative, making them a significant predictor for cured AE. E. multilocularis specific IgG4, and indirect hemagglutination IHA decreased later in time, after around 5 years. While total IgE did not show significant dynamics over the course of disease, E. multilocularis specific IgE decreased after one to two years, and increasing levels were a significant predictor of progressive disease. There was no significant change in systemic IL-8, IL-9, CCL18 or CCL20 serum levels over time. Univariate analysis across groups indicated lower IL-8 levels in cured patients; however, this result could not be confirmed by multivariate analysis. Levels of CCL17 decreased during treatment, especially in cured patients, and thus might serve as a predictive or risk factor for progressive disease. Levels of IL-10 and CCL13 decreased during disease, especially after five and ten years of intervention. The E. multilocularis antigen (EmAg) inducible cellular productions of MCP1(CCL13), TARC(CCL17) and PARC(CCL18) were lowest in patients with cured AE and infection-free controls, while the EmAg inducible cellular production of IFN-γ increased after cure. Significant positive cytokine and chemokine correlations were observed in AE patients for IL-9, IL-10, CCL13(MCP-4), CCL17(TARC) and CCL20(LARC)(for all p, Author summary Alveolar echinococcosis (AE) is a severe disease caused by Echinococcus multilocularis, the fox tapeworm. Humans exposed to E. multilocularis may develop severe AE with progressive tissue and organ infiltrating growth of the larval stage. The E. multilocularis larvae appear to have developed effective immune evasion mechanisms which facilitate an asymptomatic incubation and an extended host and parasite coexistence for decades. Over a 10-year follow-up, this investigation aimed to gain a better understanding of the immunological process associated with an active and progressive, a stable or a regressive course of AE. In summary, the rapid decrease of antibodies against the E. multilocularis specific antigen Em2+, especially in cured patients, makes them a significant predictor for cured AE. The positive relation of E. multilocularis specific IgG4 responses and chemokine levels of TARC can indicate AE progression when both enhance over time. Enhanced levels of cytokines IL-8, IL-10, and chemokines MCP4 and LARC may predict AE regression. Repeated biomarker surveys are advisable to evaluate progression or regression of AE during longitudinal follow up, and such analyses can support imaging techniques and improve staging of AE patients.

Cache Valley virus (CVV) is a mosquito-borne virus in the genus Orthobunyavirus, family Peribunyaviridae. It was first isolated from a Culiseta inorata mosquito in Cache Valley, Utah in 1956 and is known to circulate widely in the Americas. While only a handful of human cases have been reported since its discovery, it is the causative agent of fetal death and severe malformations in livestock. CVV has recently emerged as a potential viral pathogen causing severe disease in humans. Currently, the only serological assay available for diagnostic testing is plaque reduction neutralization test which takes several days to perform and requires biocontainment. To expand diagnostic capacity to detect CVV infections by immunoassays, 12 hybridoma clones secreting anti-CVV murine monoclonal antibodies (MAbs) were developed. All MAbs developed were found to be non-neutralizing and specific to the nucleoprotein of CVV. Cross-reactivity experiments with related orthobunyaviruses revealed several of the MAbs reacted with Tensaw, Fort Sherman, Tlacotalpan, Maguari, Playas, and Potosi viruses. Our data shows that MAbs CVV14, CVV15, CVV17, and CVV18 have high specific reactivity as a detector in an IgM antibody capture test with human sera., Author summary Cache Valley virus is a mosquito-borne virus found throughout the Americas. It causes fetal death and severe malformations in livestock, and only a few cases of human viral disease have been identified. Currently, we do not fully understand the spectrum of disease in humans including its potential to cause fetal malformations. The only serological diagnostic assay available to detect recent viral infection is plaque reduction neutralization test which requires the use of live virus in biocontainment. In order to develop faster and safer serodiagnostics we generated 12 monoclonal antibodies for incorporation into new assays. These antibodies are specific to the nucleoprotein of the virus and cross-react with other closely related mosquito-borne viruses. Four of these antibodies were incorporated into an immunoassay for the detection of IgM from human sera demonstrating their utility in serodiagnosis. Rapid and higher throughput assays utilizing these antibodies will expand diagnostic capacity and facilitate research to increase our understanding of Cache Valley disease prevalence and the virus’s impact on at-risk populations.

Background Echinococcus multilocularis causes alveolar echinococcosis (AE), a rising zoonotic disease in the northern hemisphere. Treatment of this fatal disease is limited to chemotherapy using benzimidazoles and surgical intervention, with frequent disease recurrence in cases without radical surgery. Elucidating the molecular mechanisms underlying E. multilocularis infections and host-parasite interactions ultimately aids developing novel therapeutic options. This study explored an involvement of unfolded protein response (UPR) and endoplasmic reticulum-stress (ERS) during E. multilocularis infection in mice. Methods E. multilocularis- and mock-infected C57BL/6 mice were subdivided into vehicle, albendazole (ABZ) and anti-programmed death ligand 1 (αPD-L1) treated groups. To mimic a chronic infection, treatments of mice started six weeks post i.p. infection and continued for another eight weeks. Liver tissue was then collected to examine inflammatory cytokines and the expression of UPR- and ERS-related genes. Results E. multilocularis infection led to an upregulation of UPR- and ERS-related proteins in the liver, including ATF6, CHOP, GRP78, ERp72, H6PD and calreticulin, whilst PERK and its target eIF2α were not affected, and IRE1α and ATF4 were downregulated. ABZ treatment in E. multilocularis infected mice reversed, or at least tended to reverse, these protein expression changes to levels seen in mock-infected mice. Furthermore, ABZ treatment reversed the elevated levels of interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α and interferon (IFN)-γ in the liver of infected mice. Similar to ABZ, αPD-L1 immune-treatment tended to reverse the increased CHOP and decreased ATF4 and IRE1α expression levels. Conclusions and significance AE caused chronic inflammation, UPR activation and ERS in mice. The E. multilocularis-induced inflammation and consecutive ERS was ameliorated by ABZ and αPD-L1 treatment, indicating their effectiveness to inhibit parasite proliferation and downregulate its activity status. Neither ABZ nor αPD-L1 themselves affected UPR in control mice. Further research is needed to elucidate the link between inflammation, UPR and ERS, and if these pathways offer potential for improved therapies of patients with AE., Author summary Alveolar echinococcosis (AE) is a zoonotic disease, characterized by chronic progressive hepatic damage caused by the continuous tumor-like proliferation of the larval stage (metacestode) of the fox tapeworm Echinococcus multilocularis. Treatment of this fatal disease is limited to surgical intervention, preferably radical curative surgery if possible, and the use of parasitostatic benzimidazoles. It is not yet fully understood how the parasite can remain in the host’s tissue for prolonged periods, complicating the development of therapeutic applications. This work investigated an involvement of the unfolded protein response (UPR) and endoplasmic reticulum-stress (ERS) during E. multilocularis infection and upon treatment with either albendazole (ABZ) or anti-programmed death ligand-1 (αPD-L1) in mice. The results revealed increased expression levels of the ERS sensor ATF6 and of downstream target genes in liver tissue of E. multilocularis- compared to mock-infected mice. Additionally, hexose-6-phosphate dehydrogenase (H6PD), generating NADPH within the endoplasmic reticulum, and the lectin-chaperone calreticulin were increased in E. multilocularis infected liver tissue while the expression of the ERS associated genes ATF4 and IRE1α were decreased. The observed gene expression changes were at least partially reversed by ABZ treatment, which also reduced the AE-induced increase of the inflammatory cytokines IL-1β, IL-6, TNF-α and IFN-γ. PD-L1 blockade reversed the AE-induced changes of UPR and ERS associated proteins CHOP, ATF4 and IRE1α. Further investigation is needed to elucidate the link between inflammation and ERS in human patients with AE and whether modulation of these pathways may lead to improved therapy.

Background Hundreds of millions of people in poor countries continue to suffer from disease caused by bloodfeeding hookworms. While mice and rats are not reliably permissive hosts for any human hookworm species, adult Golden Syrian hamsters are fully permissive for the human and animal pathogen Ancylostoma ceylanicum. Similar to humans, hamsters may be infected with A. ceylanicum third-stage larvae orally or percutaneously. Oral infection typically leads to consistent worm yields in hamsters but may not accurately reflect the clinical and immunological manifestations of human infection resulting from skin penetration. Methodology/Principal findings In this study we compared host responses following percutaneous infection to those utilizing an established oral infection protocol. Infected hamsters exhibited a dose-dependent pathology, with 1000 percutaneous larvae (L3) causing anemia and adult worm recovery comparable to that of 50 orally administered L3. A delayed arrival and maturity of worms in the intestine was observed, as was variation in measured cellular immune responses. A long-term study found that the decline in blood hemoglobin was more gradual and did not reach levels as low, with the nadir of disease coming later in percutaneously infected hamsters. Both groups exhibited moderate growth delay, an effect that was more persistent in the percutaneously infected group. Fecal egg output also peaked later and at lower levels in the percutaneously infected animals. In contrast to orally infected hamsters, antibody titers to larval antigens continued to increase throughout the course of the experiment in the percutaneous group. Conclusions/Significance These results demonstrate that the route of infection with A. ceylanicum impacts disease pathogenesis, as well as humoral and cellular immune responses in an experimental setting. These data further validate the utility of the Golden Syrian hamster as a model of both oral and percutaneous infection with human hookworms., Author summary Hookworms are bloodfeeding intestinal parasites that represent an important cause of anemia and growth delay in children from Low and Middle Income Countries (LMICs). The study of hookworm is limited by the limited availability of animal models that accurately reproduce the clinical features of human infection. We report here a detailed description of hookworm infection in the hamster, with comparison of two routes of infection: oral vs percutaneous. The results demonstrate differences in the time course of infection and primary immune responses based on whether infection occurs orally or via skin penetration. These results build on the current understanding of hookworm pathogenesis and extend the utility of this important animal model of human disease.

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., Author summary Onchocerciasis, a disease caused by the helminth parasite Onchocerca volvulus, is transmitted by the bites of female Simulium blackflies. The disease is still endemic in many African countries, and the World Health Organization has proposed elimination of its transmission in 12 countries by 2030. Understanding the heterogeneity in human exposure to vector bites can help discern which portion of the population is at higher risk of acquiring/ transmitting infection and is fundamental to identifying target groups for serological monitoring and transmission control. Traditionally, blackfly biting rates are estimated by performing human landing catches, a method that is often considered unethical and which can be unreliable as a representative measure. Therefore, we used our recently developed immunoassays to measure human antibody responses to antigens contained in the saliva of blackflies and deposited into human skin when they bloodfeed. In onchocerciasis endemic communities in Ghana, we measured antibody responses to understand age- and/or sex-related demographic patterns in vector exposure. We observed lower antibody responses in males compared to in females, and a substantial decline with increasing age, suggesting that high blackfly biting pressure induces desensitization in the human host.

Glanders is a highly contagious and potentially serious disease caused by Burkholderia mallei, a Tier 1 select agent. In this study, we raised a monoclonal antibody (mAb) against the lipopolysaccharide (LPS) of B. mallei and developed a competitive enzyme-linked immunosorbent assay (cELISA) for B. mallei infection. Using the titrated optimal conditions of B. mallei-LPS (2 ng) for microtiter plate coating, sample serum dilution at 1:20 and 3.5 ng/μL anti-LPS mAb B5, the cutoff value of the cELISA was determined using serum samples from 136 glanders-free seronegative horses in Hong Kong. All calculated percentage inhibition (PI) values from these seronegative samples were below 39.6% inhibition (1.5 standard deviations above mean PI) and was used as the cutoff value. The diagnostic sensitivity of the developed LPS-based cELISA was first evaluated using sera from donkeys and mice inoculated with B. mallei. An increasing trend of PI values above the defined cELISA cutoff observed in the donkey and mouse sera suggested positive detection of anti-LPS antibodies. The sensitivity and specificity of the LPS-based cELISA was further evaluated using 31 serologically positive horse sera from glanders outbreaks in Bahrain and Kuwait, of which 30 were tested positive by the cELISA; and 21 seronegative horse sera and 20 seronegative donkey sera from Dubai, of which all were tested negative by the cELISA. A cELISA with high sensitivity (97.2%) and specificity (100%) for the detection of B. mallei antibodies in different animals was developed., Author summary Glanders is a highly contagious and life-threatening disease caused by Burkholderia mallei, a Tier 1 select agent, with no available vaccine. The disease is endemic in the Middle East, Asia, Africa and South America with sporadic outbreaks and mainly occurs in horses, donkeys and mules, although it has also been reported in camels, tigers, lions, and even humans. As the bacterium is not easily isolated from clinical specimens and correct identification based on clinical signs is difficult, it is thus important to develop serological tests which can quickly diagnose B. mallei infection. In this study, we generated a monoclonal antibody against B. mallei lipopolysaccharide and used it to develop a competitive enzyme-linked immunosorbent assay (cELISA) for the serodiagnosis of B. mallei infection. The developed cELISA was optimized and evaluated using glanders-free and glanders-positive horses, donkeys and mice from Hong Kong and the Middle East, and was shown to be highly sensitive and specific for the detection of glanders in different animals. A simple and inexpensive test to allow for the early detection and diagnosis of suspected clinical cases as well as the screening of apparently asymptomatic animals will be helpful in controlling the spread and elimination of the disease.

Introduction: Chikungunya virus (CHIKV) is a mosquito-borne virus known to cause acute febrile illness associated with debilitating polyarthritis. In 2019, several institutions in Myanmar reported a CHIKV outbreak. There are no official reports of CHIKV cases between 2011 and 2018. Therefore, this study sought to determine the seroprevalence of CHIKV infection before the 2019 outbreak. Methods: A total of 1,544 serum samples were collected from healthy volunteers and patients with febrile illnesses in Yangon, Mandalay, and the Myeik district in 2013, 2015, and 2018. Participants ranged from one month to 65 years of age. Antibody screening was performed with in-house anti-CHIKV IgG and IgM ELISA. A neutralization assay was used as a confirmatory test. Results: The seroprevalence of anti-CHIKV IgM and anti-CHIKV IgG was 8.9% and 28.6%, respectively, with an overall seropositivity rate of 34.5%. A focus reduction neutralization assay confirmed 32.5% seroprevalence of CHIKV in the study population. Age, health status, and region were significantly associated with neutralizing antibodies (NAbs) and CHIKV seropositivity (p < 0.05), while gender was not (p = 0.9). Seroprevalence in 2013, 2015, and 2018 was 32.1%, 28.8%, and 37.3%, respectively. Of the clinical symptoms observed in participants with fevers, arthralgia was mainly noted in CHIKV-seropositive patients. Conclusion: The findings in this study reveal the circulation of CHIKV in Myanmar’s Mandalay, Yangon, and Myeik regions before the 2019 CHIKV outbreak. As no treatment or vaccine for CHIKV exists, the virus must be monitored through systematic surveillance in Myanmar., PLoS Neglected Tropical Diseases, 15(12), art. no. e0009961; 2021

In 2019, the World Health Organization declared 3 billion to be at risk of developing Crimean Congo Hemorrhagic Fever (CCHF). The causative agent of this deadly infection is CCHFV. The data related to the biology and immunology of CCHFV are rather scarce. Due to its indispensable roles in the viral life cycle, NP becomes a logical target for detailed viral immunology studies. In this study, humoral immunity to NP was investigated in CCHF survivors, as well as in immunized mice and rabbits. Abundant antibody response against NP was demonstrated both during natural infection in humans and following experimental immunizations in mice and rabbits. Also, cellular immune responses to recombinant NP (rNP) was detected in multispecies. This study represents the most comprehensive investigation on NP as an inducer of both humoral and cellular immunity in multiple hosts and proves that rNP is an excellent candidate warranting further immunological studies specifically on vaccine investigations., Author summary Crimean Congo Hemorrhagic Fever Virus (CCHFV) is the most lethal human pathogen of medical importance after the dengue virus among arboviruses. The increasing geographic spread of Hyalomma ticks, which are responsible for viral transmission widespread, threatens billions of people. WHO currently declares the field of research on CCHFV as the second most urgently needed areas of investigations on emerging pathogens. About 10 to 40% of those infected with the virus lose their life due to the rapidly developing severe clinical manifestations. Pandemic potential and the lack of any approved treatment or vaccine make raise the studies on CCHFV as critical. The studies on CCHFV are challenging due to the necessities of BSL-4 facilities and the immunological characterization of individual structural proteins will lay the groundwork for the steps to be taken to treat and prevent this emerging disease. As is known from other RNA viruses, nucleoprotein (NP) has crucial roles in the viral life cycle, both in viral replication and transcription and in the formation of the virion structure. So far, detailed and comprehensive immunological characterizations on NP in multiple are not undertaken. Our study was set out to embark such detailed investigation. The strong humoral and cellular immune response to NP demonstrated by this study indicates that NP might be an excellent candidate for future scrutinies on vaccines and diagnostic reagents.

Background Several infectious diseases are associated with hypothalamic-pituitary-adrenal (HPA) axis disorders by elevating circulating glucocorticoids (GCs), which are known to have an immunosuppressive potential. We conducted this study in golden hamsters, a suitable model for human visceral leishmaniasis (VL), to investigate the relationship of Leishmania (L.) infantum infection on cortisol production and VL severity. Methods L. infantum-infected (n = 42) and uninfected hamsters (n = 30) were followed-up at 30, 120, and 180 days post-infection (dpi). Plasma cortisol was analyzed by radioimmunoassay and cytokines, inducible nitric oxide synthase (iNOS), and arginase by RT-qPCR. Results All hamsters showed splenomegaly at 180 dpi. Increased parasite burden was associated with higher arginase expression and lower iNOS induction. Cortisol levels were elevated in infected animals in all-time points evaluated. Except for monocytes, all other leucocytes showed a strong negative correlation with cortisol, while transaminases were positively correlated. Immunological markers as interleukin (IL)-6, IL-1β, IL-10, and transforming growth-factor-β (TGF-β) were positively correlated to cortisol production, while interferon-γ (IFN-γ) presented a negative correlation. A network analysis showed cortisol as an important knot linking clinical status and immunological parameters. Conclusions These results suggest that L. infantum increases the systemic levels of cortisol, which showed to be associated with hematological, biochemical, and immunological parameters associated to VL severity., Author summary Visceral leishmaniasis (VL) is an infectious disease that is common in most tropical countries. VL has high morbidity and leads to death if not properly treated. In Brazil, Leishmania (Leishmania) infantum is the main causative agent of VL. Golden hamsters have proven to be a suitable model for VL. Despite the importance of hypothalamic-pituitary-adrenal (HPA) axis disturbances in infectious disease, few studies have addressed this issue in VL. In this study, we showed that L. infantum-infected hamsters present augmented levels of plasmatic cortisol in association with increased spleen parasite burden. Indeed, a strong positive correlation was observed between cortisol and biochemical parameters (AST/ALT/ALP) related to liver damage, as well as pro-inflammatory cytokines (IL-6 and IL-1β), anti-inflammatory cytokines (IL-10 and TGF-β), and the arginase enzyme that may favor the progression of infection. On the other side, cortisol was negatively correlated with leucocytes, except monocytes, and with IFN-γ and iNOS, which are involved in parasite-killing macrophage function. These results shed light on an unexplored aspect of VL pathogenesis, which is the importance of cortisol production in the disease-associated immune dysfunction.

Background Visceral leishmaniasis (VL) could progress to secondary hemophagocytic lymphohistiocytosis (HLH), which is a rare but life-threatening condition with poor prognosis. So far, the clinical and laboratory characteristics of VL associated HLH have not been well elucidated. Method and findings In this study, we retrospectively analyzed the clinical and laboratory profiles between 17 patients with VL associated HLH and 27 patients with VL alone admitted at the Beijing Friendship Hospital, Capital Medical University from May 2016 to March 2021. In addition to the identification of Leishmania infection, hemophagocytosis was identified in bone marrow in the most cases of VL associated HLH (15/17). The patients with VL associated HLH had higher chances of bleeding, hepatomegaly, thrombocytopenia, hypertriglyceridemia, hyperferritinemia, hypofibrinogenemia, elevated secretion of soluble IL-2 receptor or lower NK cell activity compared to patients with VL only. Furthermore, patients with VL associated HLH had higher inflammation status associated with higher levels of Th1 (TNF-α, IFN-γ, IL-1beta, IL-6, IL-8, IL-12p70), Th2 (IL-4) and Th17 cytokines (IL-17, IL-23) in the peripheral blood, and higher parasite load (qPCR and parasite culture). All 27 VL cases were totally recovered after being treated with Sodium Stibogluconate, five of the 17 patients with VL associated HLH died even after timely treatment with anti-parasite and immunosuppressive chemotherapy. Conclusion Without appropriate treatment, visceral leishmaniosis could develop to secondary HLH. The parasite culturing and qPCR detection of bone marrow samples facilitates the diagnosis of VL associated HLH in addition to other findings of HLH. Prompt treatment with anti-Leishmania and immunosuppressive chemotherapy is critical to reduce the mortality of VL associated HLH., Author summary Hemophagocytic lymphohistiocytosis (HLH) is a rare but life-threatening syndrome characterized by pro-inflammatory cytokine secretion, hyperinflammatory and multiple organ damages. Visceral leishmaniasis (VL) is a well-known cause of infection associated HLH and result in fatal consequence. However, it is not well characterized for the clinical and laboratory features of the visceral leishmaniasis associated HLH. In this study, we presented that, compared to the VL alone, patients with VL associated HLH had higher chances of bleeding, hepatomegaly, thrombocytopenia, hypertriglyceridemia, hyperferritinemia, hypofibrinogenemia, elevated secretion of soluble IL-2 receptor or lower NK cell activity. Moreover, patients with VL associated HLH also had higher levels of Th1, Th2 and Th17 cytokines in the sera and had higher parasite load in the bone marrow specimen. More cases with VL associated HLH had hepatosplenomegaly with iron overload in the magnetic resonance imaging. In the therapeutic strategy, besides the anti-Leishmania treatment, anti-inflammatory therapy to reduce cytokine storm and excessive immune responses facilitated the remission in the VL associated HLH cases.

Background Nifurtimox-eflornithine combination therapy (NECT) for the treatment of second stage gambiense human African trypanosomiasis (HAT) was added to the World Health Organization’s Essential Medicines List in 2009 after demonstration of its non-inferior efficacy compared to eflornithine therapy. A study of NECT use in the field showed acceptable safety and high efficacy until hospital discharge in a wide population, including children, pregnant and breastfeeding women, and patients with a HAT treatment history. We present here the effectiveness results after the 24-month follow-up visit. Methodology/Principal findings In a multicenter, open label, single arm phase IIIb study, second stage gambiense HAT patients were treated with NECT in the Democratic Republic of Congo. Clinical cure was defined 24 months after treatment as survival without clinical and/or parasitological signs of HAT. Of the 629 included patients, 619 (98.4%) were discharged alive after treatment and were examined for the presence of trypanosomes, white blood cell count in cerebro-spinal fluid, and disease symptoms. The clinical cure rate of 94.1% was comparable for all subpopulations analyzed at the 24-month follow-up visit. Self-reported adverse events during follow-up were few and concerned mainly nervous system disorders, infections, and gastro-intestinal disorders. Overall, 28 patients (4.3%) died during the course of the trial. The death of 16 of the 18 patients who died during the follow-up period was assessed as unlikely or not related to NECT. Within 24 months, eight patients (1.3%) relapsed and received rescue treatment. Sixteen patients were completely lost to follow-up. Conclusions/Significance NECT treatment administered under field conditions was effective and sufficiently well tolerated, no major concern arose for children or pregnant or breastfeeding women. Patients with a previous HAT treatment history had the same response as those who were naïve. In conclusion, NECT was confirmed as effective and appropriate for use in a broad population, including vulnerable subpopulations. Trial registration The trial is registered at ClinicalTrials.gov, number NCT00906880., Author summary The advanced stage of the neglected tropical disease human African trypanosomiasis was, until relatively recently, treated with an old toxic arsenical drug and there was little investment in an improved treatment option. Eflornithine alone was efficacious, but difficult to administer as it required four two-hour infusions a day for 14 days. Nifurtimox-eflornithine combination therapy (NECT) was developed as a simplified and easier to use treatment and was shown to be effective and sufficiently well tolerated in a randomized clinical trial. The present study was conducted to assess the overall effectiveness, including the feasibility of implementation of NECT under field conditions in a wider population than in the randomized clinical trial. We found that NECT can be implemented under field conditions and in remote areas, with the necessary logistical support and staff training for treatment administration. Adverse events, although very frequent, were considered acceptable given the severity of the disease. Less than 10% of patients showed severe adverse events. Over 24 months, the case fatality rate was 4.5% and relapses were rare (1.3%). The effectiveness of NECT was confirmed in a broad spectrum of second stage gambiense HAT patients, including children, pregnant and breastfeeding women, and patients who had been previously treated for HAT.

Background Chagas disease, caused by the protozoan Trypanosoma cruzi, is endemic in Latin America and is widely distributed worldwide because of migration. In 30% of cases, after years of infection and in the absence of treatment, the disease progresses from an acute asymptomatic phase to a chronic inflammatory cardiomyopathy, leading to heart failure and death. An inadequate balance in the inflammatory response is involved in the progression of chronic Chagas cardiomyopathy. Current therapeutic strategies cannot prevent or reverse the heart damage caused by the parasite. Aspirin-triggered resolvin D1 (AT-RvD1) is a pro-resolving mediator of inflammation that acts through N-formyl peptide receptor 2 (FPR2). AT-RvD1 participates in the modification of cytokine production, inhibition of leukocyte recruitment and efferocytosis, macrophage switching to a nonphlogistic phenotype, and the promotion of healing, thus restoring organ function. In the present study, AT-RvD1 is proposed as a potential therapeutic agent to regulate the pro-inflammatory state during the early chronic phase of Chagas disease. Methodology/Principal findings C57BL/6 wild-type and FPR2 knock-out mice chronically infected with T. cruzi were treated for 20 days with 5 μg/kg/day AT-RvD1, 30 mg/kg/day benznidazole, or the combination of 5 μg/kg/day AT-RvD1 and 5 mg/kg/day benznidazole. At the end of treatment, changes in immune response, cardiac tissue damage, and parasite load were evaluated. The administration of AT-RvD1 in the early chronic phase of T. cruzi infection regulated the inflammatory response both at the systemic level and in the cardiac tissue, and it reduced cellular infiltrates, cardiomyocyte hypertrophy, fibrosis, and the parasite load in the heart tissue. Conclusions/Significance AT-RvD1 was shown to be an attractive therapeutic due to its regulatory effect on the inflammatory response at the cardiac level and its ability to reduce the parasite load during early chronic T. cruzi infection, thereby preventing the chronic cardiac damage induced by the parasite., Author summary Chagas disease is prevalent in Latin America and is widely distributed worldwide due to migration. In 30% of patients, if the parasite is left untreated, the disease may progress from an acute symptomless phase to chronic myocardial inflammation, which can cause heart failure and death, years after the infection. Imbalances in the inflammatory response are related to this progression. Current treatments cannot prevent or reverse the cardiac damage inflicted by the parasite. Aspirin-triggered resolvin D1, also named AT-RvD1, can modify cellular and humoral inflammatory responses leading to the resolution of inflammation, thus promoting healing and restoring organ function. In this study, AT-RvD1, in an N-formyl peptide receptor 2 (FPR2)-dependent manner, was shown to regulate local and systemic inflammation and decrease cellular infiltration in the heart tissue of mice chronically infected with the parasite and reduce cardiac hypertrophy and fibrosis in the early stages of the chronic phase of the disease. Importantly, AT-RvD1 was able to decrease parasite load in the infected hearts. Thus, this research indicates that At-RvD1 treatment is a potential therapeutic strategy that offers an improvement on current drug therapies.

Funder: IBERS, Aberystwyth University PhD studentship, Funder: Higher Education Funding Council for Wales (HEFCW) - Global Challenges Research Fund (GCRF), Extracellular Vesicles (EVs) are an integral component of cellular/organismal communication and have been found in the excreted/secreted (ES) products of both protozoan and metazoan parasites. Within the blood fluke schistosomes, EVs have been isolated from egg, schistosomula, and adult lifecycle stages. However, the role(s) that EVs have in shaping aspects of parasite biology and/or manipulating host interactions is poorly defined. Herein, we characterise the most abundant EV-enriched protein in Schistosoma mansoni tissue-migrating schistosomula (Schistosoma mansoni Larval Extracellular Vesicle protein 1 (SmLEV1)). Comparative sequence analysis demonstrates that lev1 orthologs are found in all published Schistosoma genomes, yet homologs are not found outside of the Schistosomatidae. Lifecycle expression analyses collectively reveal that smlev1 transcription peaks in cercariae, is male biased in adults, and is processed by alternative splicing in intra-mammalian lifecycle stages. Immunohistochemistry of cercariae using a polyclonal anti-recombinant SmLEV1 antiserum localises this protein to the pre-acetabular gland, with some disperse localisation to the surface of the parasite. S. mansoni-infected Ugandan fishermen exhibit a strong IgG1 response against SmLEV1 (dropping significantly after praziquantel treatment), with 11% of the cohort exhibiting an IgE response and minimal levels of detectable antigen-specific IgG4. Furthermore, mice vaccinated with rSmLEV1 show a slightly reduced parasite burden upon challenge infection and significantly reduced granuloma volumes, compared with control animals. Collectively, these results describe SmLEV1 as a Schistosomatidae-specific, EV-enriched immunogen. Further investigations are now necessary to uncover the full extent of SmLEV1's role in shaping schistosome EV function and definitive host relationships.

The metacestode stage of Echinococcus granulosus can cause cystic echinococcosis (CE), which still widely occurs around the world. Since the early 1970s, benzimidazoles have been shown to inhibit the growth of cysts and used to treat CE. However, benzimidazoles are still ineffective in 20%-40% of cases. In order to explore the new agents against CE, we have investigated the therapeutic effect of the recombinant adenoviral vector expressing mouse IL-28B (rAd-mIL-28B) on protoscoleces-infected mice. In our study, we successfully established the model mice which infected with protoscoleces intraperitoneally. At 18 weeks post-infection, the mice received rAd-mIL-28B (1×107 PFU) weekly by intramuscular injection for 6 weeks. Compared with the untreated control (13.1 ± 2.2 g), there was a significant reduction in cysts wet weight in rAd-mIL-28B group (8.3 ± 3.5 g) (P, Author summary Echinococcosis is a chronic zoonotic parasitic disease, which is caused by the larval stage of Echinococcus granulosus (E. granulosus) and Echinococcus multiocularis (E. multiocularis). This disease is still widely prevalent in the world and seriously endangers human health and life, causing heavy burdens and economic losses to agriculture and animal husbandry. China is also one of the high incidence areas of the disease. At present, the preferred treatment is surgical excision of the parasitic mass, but patients with multiple cysts appear in multiple organs have to receive drug therapy. The approved chemotherapeutic drugs in clinic, such as albendazole (ABZ) and mebendazole, often do not work expectedly with a high rate of recurrence. Therefore, it is urgent to develop the new anti-echinococcal drug. IL-28B is an important member of type-III IFNs, which is equipped with the capacity of anti-viral activity and anti-tumor. In our previous study, we found that IL-28B could inhibit the proliferation of cervical cancer cells via down-regulating Treg cells in mice. This study mainly studied the therapeutic effect of rAd-mIL-28B on E. granulosus-infected mice, and the results showed that rAd-mIL-28B could relieve the parasitic burden and inhibit Treg cells meanwhile improve the Th1 and Th17 immune responses. It may contribute to another choice for the anti-echinococcal treatment.

The Human T-cell Leukemia Virus-1 (HTLV-1)-Associated Myelopathy/Tropical Spastic Paraparesis (HAM/TSP) is a devastating neurodegenerative disease with no effective treatment, which affects an increasing number of people in Brazil. Immune cells from the adaptive compartment are involved in disease manifestation but whether innate cell functions participate in disease occurrence has not been evaluated. In this study, we analyzed innate cell responses at steady state and after blood cell stimulation using an agonist of the toll-like receptor (TLR)7/8-signaling pathway in blood samples from HTLV-1-infected volunteers, including asymptomatic carriers and HAM/TSP patients. We observed a lower response of IFNα+ DCs and monocytes in HAM/TSP compared to asymptomatic carriers, as a potential consequence of corticosteroid treatments. In contrast, a higher frequency of monocytes producing MIP-1α and pDC producing IL-12 was detected in HAM/TSP blood samples, together with higher IFNγ responsiveness of NK cells, suggesting an increased sensitivity to inflammatory response in HAM/TSP patients compared to asymptomatic carriers. This sustained inflammatory responsiveness could be linked or be at the origin of the neuroinflammatory status in HAM/TSP patients. Therefore, the mechanism underlying this dysregulations could shed light onto the origins of HAM/TSP disease., Author summary The infection by the Human T-cell Leukemia Virus-1 (HTLV-1) is quite frequent in Brazil. Between 1–5% of infected individuals develop a devastating neurodegenerative disease (HAM/TSP) as a result of a sustained inflammation in the central nervous system, with no effective treatment. So far, inflammation has been linked to the deregulated activation of T-cells, but the role of innate cells has not been investigated yet. In this work, we aimed to characterize the responsiveness of innate cells, as this immune population is cornerstone of efficient immune response, but also might participate in disease exacerbation found in chronic infection. Our findings suggest an impaired antiviral response and increased inflammatory responsiveness by dendritic cells and monocytes in HAM/TSP patients compared to asymptomatic carriers. This sustained inflammatory responsiveness upon innate cell activation could participate in the establishment of the HAM/TSP disease.

Background The assessment of chemotherapeutic responses in Post Kala-azar Dermal Leishmaniasis (PKDL), especially its macular form is challenging, emphasizing the necessity for ‘test of cure’ tools. This study explored the diagnostic and prognostic potential of IgG subclasses and associated cytokines for monitoring the effectiveness of chemotherapy in PKDL. Methods Participants included PKDL cases at (a) disease presentation, (b) immediately at the end of treatment (12 weeks for Miltefosine or 3 weeks for Liposomal Amphotericin B, LAmB and (c) at any time point 6 months later, for estimating anti-leishmanial immunoglobulin (Ig, IgG, IgM, IgG1, IgG2 and IgG3) and cytokines (IL-10, IL-6). Results In PKDL, Ig levels were elevated, with IgG3 and IL-10 being the major contributors. Miltefosine decreased both markers substantially and this decrease was sustained for at least six months. In contrast, LAmB failed to decrease IgG3 and IL-10, as even after six months, their levels remained unchanged or even increased. Conclusions In PKDL, IgG3 and IL-10 proved to be effective predictors of responsiveness to chemotherapy and may be considered as a non invasive alternative for longitudinal monitoring., Author summary Post Kala-azar Dermal Leishmaniasis (PKDL) is a dermal condition that occurs in East Africa and South Asia, the latter in 5–10% of patients after apparent cure from Visceral Leishmaniasis (VL). Till date, conventional knowledge in South Asia was that the polymorphic form of PKDL comprising of macules, papules and nodules was the predominant disease form, constituting 85–90%. However, since 2014, implementation of active-case surveillance led to unearthing of a large number of macular, hypopigmented cases, and was reported to contribute to nearly 50% of the disease burden. In particular, the macular form poses a diagnostic dilemma as microscopically parasites are difficult to identify in their lesions, and repigmentation occurs months after parasite clearance, emphasizing the need for developing non-invasive approaches for measurement of parasite burden. Till date, no formal clinical trial for treatment of PKDL has been undertaken where the parasite load was quantified and treatment remains empirical. This is primarily due to PKDL cases being unwilling to provide a repeat skin biopsy once their lesions have declined. Therefore, in cases where treatment failure occurs, it cannot be precisely identified, and could potentially lead to these cases becoming mobile disease reservoirs, thereby adversely impacting on the ongoing VL elimination programme. This study addressed this critical lacuna, where it was established that in both clinical types of PKDL, circulating levels of IgG3 and IL-10 can be considered as effective markers for monitoring treatment outcome. At disease presentation, the raised levels of IgG subclasses and associated cytokines (IL-10 and IL-6) declined following therapy with Miltefosine, the maximum decrease being with IgG3 along with IL-10; importantly, this decrease was sustained for at least six months. In contrast, LAmB failed to decrease the levels of immunoglobulins and associated cytokines even six months after completion of treatment; in fact the antibody levels either increased or remained unchanged. Taken together, this study has established the potential of IgG3 and IL10 as a non-invasive alternative for monitoring of chemotherapeutic responses in PKDL.

The neglected tropical disease onchocerciasis, or river blindness, is caused by infection with the filarial nematode Onchocerca volvulus. Current estimates indicate that 17 million people are infected worldwide, the majority of them living in Africa. Today there are no non-invasive tests available that can detect ongoing infection, and that can be used for effective monitoring of elimination programs. In addition, to enable pharmacodynamic studies with novel macrofilaricide drug candidates, surrogate endpoints and efficacy biomarkers are needed but are non-existent. We describe the use of a multimodal untargeted mass spectrometry-based approach (metabolomics and lipidomics) to identify onchocerciasis-associated metabolites in urine and plasma, and of specific lipid features in plasma of infected individuals (O. volvulus infected cases: 68 individuals with palpable nodules; lymphatic filariasis cases: 8 individuals; non-endemic controls: 20 individuals). This work resulted in the identification of elevated concentrations of the plasma metabolites inosine and hypoxanthine as biomarkers for filarial infection, and of the urine metabolite cis-cinnamoylglycine (CCG) as biomarker for O. volvulus. During the targeted validation study, metabolite-specific cutoffs were determined (inosine: 34.2 ng/ml; hypoxanthine: 1380 ng/ml; CCG: 29.7 ng/ml) and sensitivity and specificity profiles were established. Subsequent evaluation of these biomarkers in a non-endemic population from a different geographical region invalidated the urine metabolite CCG as biomarker for O. volvulus. The plasma metabolites inosine and hypoxanthine were confirmed as biomarkers for filarial infection. With the availability of targeted LC-MS procedures, the full potential of these 2 biomarkers in macrofilaricide clinical trials, MDA efficacy surveys, and epidemiological transmission studies can be investigated., Author summary Today’s diagnosis of infection with the filarial parasite Onchocerca volvulus mainly depends on the microscopic analysis of skin biopsies and serological testing. The work presented here describes the use of multiple mass spectrometry-based screening methods (metabolomics and lipidomics) to search for biomarkers indicative of infection with Onchocerca volvulus. This resulted in the identification of elevated concentrations of the plasma metabolites inosine and hypoxanthine as biomarkers for filarial infection, and of the urine metabolite cis-cinnamoylglycine as biomarker for O. volvulus. Further evaluation of these biomarkers in a geographically distinct non-endemic population however invalidated the use of urine cis-cinnamoylglycine. These findings are of utmost importance as it not only opens new avenues in the development of non-invasive diagnostic tools for filarial infections, but also emphasizes the need for evaluation and validation of newly discovered biomarkers in different populations from different geographies.

Nodding syndrome (NS) is a catastrophic and enigmatic childhood epilepsy, accompanied by multiple neurological impairments and neuroinflammation. Of all the infectious, environmental and psychological factors associated with NS, the major culprit is Onchocerca Volvulus (Ov)–a parasitic worm transmitted to human by blackflies. NS seems to be an ’Autoimmune Epilepsy’ in light of the recent findings of deleterious autoimmune antibodies to Glutamate receptors and to Leiomodin-I in NS patients. Moreover, we recently found immunogenetic fingerprints in HLA peptide-binding grooves associate with protection or susceptibility to NS. Macrophage migration inhibitory factor (MIF) is an immune-regulatory cytokine playing a central role in modulating innate and adaptive immunity. MIF is also involved in various pathologies: infectious, autoimmune and neurodegenerative diseases, epilepsy and others. Herein, two functional polymorphisms in the MIF gene, a −794 CATT5–8 microsatellite repeat and a −173 G/C single-nucleotide polymorphism, were assessed in 49 NS patients and 51 healthy controls from South Sudan. We also measured MIF plasma levels in established NS patients and healthy controls. We discovered that the frequency of the high-expression MIF -173C containing genotype was significantly lower in NS patients compared to healthy controls. Interestingly however, MIF plasma levels were significantly elevated in NS patients than in healthy controls. We further demonstrated that the HLA protective and susceptibility associations are dominant over the MIF association with NS. Our findings suggest that MIF might have a dual role in NS. Genetically controlled high-expression MIF genotype is associated with disease protection. However, elevated MIF in the plasma may contribute to the detrimental autoimmunity, neuroinflammation and epilepsy., Author summary Nodding syndrome (NS) is a devastating and enigmatic childhood disease, accompanied by multiple neurological impairments and neuroinflammation. NS is associated with the parasite Onchocerca volvulus (Ov) and other environmental factors. It was shown that NS seems to be an "Autoimmune-Epilepsy". However, it is still not clear why only some children, within the same family, tribe and district, develop this malady in South-Sudan. Recently, we hypothesized that immunogenetic factors might address this question, and indeed demonstrated that few amino acids in human leukocyte antigen (HLA) peptide-binding grooves associate with either protection or susceptibility to NS. In the current study, we evaluate the contribution of macrophage migration inhibitory factor (MIF) to NS pathology. MIF is an immune-regulatory cytokine that is involved in numerous pathologies, which vary from infectious diseases, autoimmunity to neurodegenerative diseases. Immunogenetic analysis of MIF in South-Sudanese NS patients and healthy controls shed new light on the epidemiology of this disease. Our findings suggest that MIF promoter genotypes that are linked with high-protein expression are associated with disease protection. However, in affected patients, with established NS, MIF plasma levels are elevated compared to control subjects and might be involved with autoimmunity and neuroinflammation.

No vaccine to protect against an estimated 238,000 shigellosis deaths per year is widely available. S. sonnei is the most prevalent Shigella, and multiple serotypes of S. flexneri, which change regionally and globally, also cause significant disease. The leading Shigella vaccine strategies are based on the delivery of serotype specific O-antigens. A strategy to minimize the complexity of a broadly-protective Shigella vaccine is to combine components from S. sonnei with S. flexneri serotypes that induce antibodies with maximum cross-reactivity between different serotypes. We used the GMMA-technology to immunize animal models and generate antisera against 14 S. flexneri subtypes from 8 different serotypes that were tested for binding to and bactericidal activity against a panel of 11 S. flexneri bacteria lines. Some immunogens induced broadly cross-reactive antibodies that interacted with most of the S. flexneri in the panel, while others induced antibodies with narrower specificity. Most cross-reactivity could not be assigned to modifications of the O-antigen, by glucose, acetate or phosphoethanolamine, common to several of the S. flexneri serotypes. This allowed us to revisit the current dogma of cross-reactivity among S. flexneri serotypes suggesting that a broadly protective vaccine is feasible with limited number of appropriately selected components. Thus, we rationally designed a 4-component vaccine selecting GMMA from S. sonnei and S. flexneri 1b, 2a and 3a. The resulting formulation was broadly cross-reactive in mice and rabbits, inducing antibodies that killed all S. flexneri serotypes tested. This study provides the framework for a broadly-protective Shigella vaccine which needs to be verified in human trials., Author summary A strategy to optimize the composition for a broadly-protective Shigella vaccine is to combine components directed against S. sonnei with S. flexneri serotypes to induce antibody responses with the maximum cross-reactivity between different serotypes. Based on mouse and rabbit immunogenicity, we selected 4 GMMA-immunogens, derived from S. sonnei and S. flexneri 1b, 2a and 3a, able to induce antibodies that were broadly bactericidal against most epidemiologically significant S. flexneri strains in mice and rabbits. This was not predicted on the basis of O-antigen modifications conferring serotype or group specificities and allowed revisiting the dogma of cross-protection among S. flexneri serotypes. Overall, this study provides a framework for the rational design of a broadly-protective vaccine that will be evaluated in upcoming human vaccine trials. It also tackles a key issue regarding Shigella vaccine development that is balancing a sufficient number of antigenic components in the vaccine to provide adequate coverage of serotype diversity while minimizing complexity.

Extracellular vesicles (EVs) are protein-loaded nano-scaled particles that are extracellularly released by eukaryotes and prokaryotes. Parasite’s EVs manipulate the immune system, making them probable next-generation vaccines. Schistosomal EVs carry different proteins of promising immunizing potentials. For evaluating the immune-protective role of Schistosoma mansoni (S. mansoni) egg-derived EVs against murine schistosomiasis, EVs were isolated from cultured S. mansoni eggs by progressive sequential cooling ultra-centrifugation technique. Isolated EVs were structurally identified using transmission electron microscope and their protein was quantified by Lowry’s technique. Experimental mice were subcutaneously immunized with three doses of 20 μg EVs (with or without alum adjuvant); every two weeks, then were challenged with S. mansoni cercariae two weeks after the last immunizing dose. Six weeks post infection, mice were sacrificed for vaccine candidate assessment. EVs protective efficacy was evaluated through parasitological, histopathological, and immunological parameters. Results showed significant reduction of tegumentally deranged adult worms, hepatic and intestinal egg counts reduction by 46.58%, 93.14% and 93.17% respectively, accompanied by remarkable amelioration of sizes, numbers and histopathology of hepatic granulomata mediated by high interferon gamma (IFN γ) and antibody level. Using sera from vaccinated mice, the molecular weight of EVs’ protein components targeted by the antibody produced was recognized by western immunoblot. Results revealed two bands of ~ 14 KDa and ~ 21 KDa, proving that EVs are able to stimulate specific antibodies response. In conclusion, the present study highlighted the role of S. mansoni-egg derived EVs as a potential vaccine candidate against murine schistosomiasis mansoni., Author summary All types of cells were proved to secrete nano-spheres called extracellular vesicles (EVs). Parasites derived-EVs have the ability to manipulate the immune system, and carry many proteins that have been used as a vaccine candidate. In this study, the author used EVs derived from cultured Schistosome mansoni eggs as an immunizing agent against experimental schistosomiasis. Mice were immunized with three subcutaneous doses of EVs. Two weeks after the last vaccination dose mice were exposed to cercarial challenge. Six weeks later mice were sacrificed and vaccine efficacy was evaluated using different parasitological, histopathological and immunological parameters. Results showed significant reduction in the parasite burden especially the hepatic and intestinal egg count. Adults obtained from the vaccinated mice showed sever derangement in their protective tegumental layer with loss of muscle tone when examined by electron microscope. EVs induced high level of protective IFN γ and antibody. This was accompanied by amelioration of the hepatic histopathology which is the main cause of the clinical manifestation.

Two hookworm vaccine candidates, Na-GST-1 and Na-APR-1, formulated with Glucopyranosyl Lipid A (GLA-AF) adjuvant, have been shown to be safe, well tolerated, and to induce antibody responses in a Phase 1 clinical trial (Clinicaltrials.gov NCT02126462) conducted in Gabon. Here, we characterized T cell responses in 24 Gabonese volunteers randomized to get vaccinated three times with Na-GST-1 and Na-APR-1 at doses of 30μg (n = 8) or 100μg (n = 10) and as control Hepatitis B (n = 6). Blood was collected pre- and post-vaccination on days 0, 28, and 180 as well as 2-weeks after each vaccine dose on days 14, 42, and 194 for PBMCs isolation. PBMCs were stimulated with recombinant Na-GST-1 or Na-APR-1, before (days 0, 28 and 180) and two weeks after (days 14, 42 and 194) each vaccination and used to characterize T cell responses by flow and mass cytometry. A significant increase in Na-GST-1 -specific CD4+ T cells producing IL-2 and TNF, correlated with specific IgG antibody levels, after the third vaccination (day 194) was observed. In contrast, no increase in Na-APR-1 specific T cell responses were induced by the vaccine. Mass cytometry revealed that, Na-GST-1 cytokine producing CD4+ T cells were CD161+ memory cells expressing CTLA-4 and CD40-L. Blocking CTLA-4 enhanced the cytokine response to Na-GST-1. In Gabonese volunteers, hookworm vaccine candidate, Na-GST-1, induces detectable CD4+ T cell responses that correlate with specific antibody levels. As these CD4+ T cells express CTLA-4, and blocking this inhibitory molecules resulted in enhanced cytokine production, the question arises whether this pathway can be targeted to enhance vaccine immunogenicity., Author summary Two hookworm vaccine candidate (Na-GST-1 and Na-APR-1) have been tested in Gabonese and found to be safe and to induce antibody response. We aimed to study the cellular immune responses among vaccinated and unvaccinated volunteers. We found that Na-GST-1 induced CD4+ T cell responses (IL-2, TNF) among the vaccinated volunteers that received the high vaccine dose (100 ug). Furthermore Na-GST-1 specific memory T cells were found to express the inhibitory molecule CTLA-4. These responses was not observed in those who received the low dose of the Na-GST-1 vaccine, or those who received Na-APR-1 or HBV. By blocking CTLA-4, we observed an increase in TNF production. Our data suggest that an intervention involving blockage of the CTLA-4 molecule in the vaccinated could be beneficial in endemic settings where vaccine responses have been shown to be lower compared to non-endemic settings.

Schistosoma haematobium is the leading cause of urogenital schistosomiasis and it is recognised as a class 1 carcinogen due to the robust association of infection with bladder cancer. In schistosomes, tetraspanins (TSPs) are abundantly present in different parasite proteomes and could be potential diagnostic candidates due to their accessibility to the host immune system. The large extracellular loops of six TSPs from the secretome (including the soluble excretory/secretory products, tegument and extracellular vesicles) of S. haematobium (Sh-TSP-2, Sh-TSP-4, Sh-TSP-5, Sh-TSP-6, Sh-TSP-18 and Sh-TSP-23) were expressed in a bacterial expression system and polyclonal antibodies were raised to the recombinant proteins to confirm the anatomical sites of expression within the parasite. Sh-TSP-2, and Sh-TSP-18 were identified on the tegument, whereas Sh-TSP-4, Sh-TSP-5, Sh-TSP-6 and Sh-TSP-23 were identified both on the tegument and internal tissues of adult parasites. The mRNAs encoding these TSPs were differentially expressed throughout all schistosome developmental stages tested. The potential diagnostic value of three of these Sh-TSPs was assessed using the urine of individuals (stratified by infection intensity) from an endemic area of Zimbabwe. The three Sh-TSPs were the targets of urine IgG responses in all cohorts, including individuals with very low levels of infection (those positive for circulating anodic antigen but negative for eggs by microscopy). This study provides new antigen candidates to immunologically diagnose S. haematobium infection, and the work presented here provides compelling evidence for the use of a biomarker signature to enhance the diagnostic capability of these tetraspanins., Author summary Schistosoma haematobium, the leading cause of urogenital schistosomiasis, affects millions of people worldwide. Infection with this parasite is associated with different clinical complications such as squamous cell carcinoma and genital malignancy in women. Despite its importance, there is a lack of sensitive and specific diagnostics that support control and elimination initiatives against this devastating disease. Herein, we have characterised six molecules belonging to the tetraspanin family of membrane proteins, providing details about their relative expression during parasite’s development and their localization in adult forms of S. haematobium. Furthermore, we have characterised the antibody responses against three of these molecules in urine from infected human subjects from an endemic area, providing compelling evidence for the use of these molecules to diagnose urogenital schistosomiasis.

Background To determine the seroprevalence and transmission dynamics of dengue virus (DENV), age-stratified longitudinal serological surveys were conducted in Bangphae district, Ratchaburi province, Thailand, for 3 years between April 2012 and April 2015. Methodology The surveys enrolled 2012 healthy children and adults between 1 and 55 years-of-age, and a longitudinal serosurvey of six repeated bleeds of the same cohort of individuals was conducted every 8 months for the first 2 years (M0, M8, M16) and every half a year (M24, M30, M36) for the rest of the study period. All samples were tested using in-house indirect sandwich dengue IgG ELISA to determine DENV antibody titer, and 640 paired samples which showed rising of DENV IgG titers in paired serum were further tested using in-house neutralization assay, Plaque Reduction Neutralization Test (PRNT50). Principal findings When compared against the gold standard based on the results of PRNT50, sensitivity and specificity of indirect ELISA were found to be both about 85%. The overall DENV IgG positivity determined by ELISA was 74.3% in 2012 and increased to 79.4% by the final sample collection in 2015. In our study sample, more than 98% of subjects older than 25 years were found to be seropositive. Among 518 IgG negative subjects at enrollment, the seroconversion rates were measured in paired bleeds; the rates (between successive visits, approximately 6 months) ranged between 4.8% (between M16 and M24) and 14.7% (between M0 and M8). The dominant serotype of primary DENV infection cases based on seroconversion was identified from the PRNT results and it was DENV-2. Conclusions Our study documented high levels of seroprevalence and rate of transmission. Given the importance of the serostatus and disease burden in consideration for dengue vaccine introduction, our data could be used in decision-making on implementation of various dengue control and preventive measures., Author summary To estimate the proportion of individuals with past exposure to dengue virus (DENV), we conducted repeated serological surveys in Bangphae district, Ratchaburi province, Thailand, between April 2012 and April 2015. About 45% of the subjects were under 15 years-of-age. The surveys enrolled 2012 healthy individuals between 1 and 55 years-of-age, and a longitudinal serosurvey of six repeated bleeds of the same cohort of individuals was conducted approximately every 6 months. All samples were tested using an indirect dengue IgG ELISA and a subset of paired samples which showed rising of IgG values in pairs were tested using neutralization assay, used as the gold standard. When compared against the gold standard, sensitivity and specificity of indirect ELISA were found to be both 85%. The proportion of the study population with past DENV infection measured by IgG ELISA was 74.3% in 2012 and increased to 79.4% by 2015. More than 98% of subjects older than 25 years showed to have past exposure to dengue. Among 518 subjects without past exposure, the rate of getting infection was between 4.8–14.7% (between successive visits, approximately 6 months). The dominant serotype was DENV-2. The level of proportion of individuals with past DENV infection is an important consideration, and our data could be used in decision-making for dengue vaccine introduction.

Infection with the human T cell leukaemia virus type 1 (HTLV-1) subtype C is endemic among Aboriginal people in central Australia. To provide insights into the risk factors for transmission, we conducted the first large-scale, community-based prevalence study in seven remote Aboriginal communities. Residents >2 years old were invited to participate in the study between August 2014 and June 2018. HTLV-1 infection was defined as a positive western blot (WB) test or a positive HTLV-1 PCR. 720 community residents participated in the study (children, Author summary In the first large scale community study of HTLV-1 prevalence in central Australia we found an adult prevalence of 36.8%, the highest reported worldwide. Prevalence increased with age suggesting that sexual contact may be the predominant mode of transmission. Although a wide range of HTLV-1 proviral loads was found, in nearly 40% of participants this exceeded 1000 copies per 105 peripheral blood leukocytes, a level that has been associated with an increased risk of chronic lung disease and death in this population. The median PVL for participants who were symptomatic with HTLV-1 associated inflammatory diseases was 1.34 log10 higher than that of those who were asymptomatic. These data demand a public health initiative to reduce HTLV-1 transmission among Aboriginal Australians.

Background Brazil faced a yellow fever(YF) outbreak in 2016–2018 and vaccination was considered for autoimmune rheumatic disease patients(ARD) with low immunosuppression due to YF high mortality. Objective This study aimed to evaluate, prospectively for the first time, the short-term immunogenicity of the fractional YF vaccine(YFV) immunization in ARD patients with low immunossupression. Methods and Results A total of 318 participants(159 ARD and 159 age- and sex-matched healthy controls) were vaccinated with the fractional-dose(one fifth) of 17DD-YFV. All subjects were evaluated at entry(D0), D5, D10, and D30 post-vaccination for clinical/laboratory and disease activity parameters for ARD patients. Post-vaccination seroconversion rate(83.7%vs.96.6%, p = 0.0006) and geometric mean titers(GMT) of neutralizing antibodies[1143.7 (95%CI 1012.3–1292.2) vs.731 (95%CI 593.6–900.2), p0.05). Conclusion Fractional-dose 17DD-YF vaccine in ARD patients resulted in a high rate of seroconversion rate(>80%) but lower than controls, with a longer but less intense viremia. This vaccine was immunogenic, safe and did not induce flares in ARD under low immunosuppression and may be indicated in YF outbreak situations and for patients who live or travel to endemic areas. Trial registration This clinical trial was registered with Clinicaltrials.gov (#NCT03430388)., Author summary Yellow fever is a viral hemorragic fever with high mortality rate and the vaccine is a remarkably successful way of preventing it. As a live attenuated virus vaccine, it is not recommended for rheumatic and other immunossupressed patients in general. However, in an outbreak scenario, the risk of dying of the disease can be higher than the risk of a vaccine serious adverse event. In 2018, the fractional-dose yellow fever vaccine was offered to the hospital employees and to the rheumatic patients without or with low immunossupression therapy in Hospital das Clinicas of University of São Paulo, during the yellow fever outbreak in São Paulo, Brazil. In order to optimize the yellow fever vaccine (YFV) supply, the fractional-dose (corresponding to one fifth) was adopted in the public vaccine campaign. This is the first study evaluating the primary vaccination with fractional-dose YFV in autoimmune rheumatic diseases(ARD) patients (n = 159) under low immunosuppression. Most vaccinated participants were able to produce enough neutralizing antibodies to be protected against yellow fever (seroconversion rate of 84% versus 96% in healthy controls). Neither activity of the rheumatic disease or serious adverse event was identified during the 30 days of followup after the vaccination.

B cells played an important role in Schistosoma infection-induced diseases. TLR7 is an intracellular member of the innate immune receptor. The role of TLR7 on B cells mediated immune response is still unclear. Here, C57BL/6 mice were percutaneously infected by S. japonicum for 5–6 weeks. The percentages and numbers of B cells increased in the infected mice (p < 0.05), and many activation and function associated molecules were also changed on B cells. More splenic cells of the infected mice expressed TLR7, and B cells were served as the main cell population. Moreover, a lower level of soluble egg antigen (SEA) specific antibody and less activation associated molecules were found on the surface of splenic B cells from S. japonicum infected TLR7 gene knockout (TLR7 KO) mice compared to infected wild type (WT) mice (p < 0.05). Additionally, SEA showed a little higher ability in inducing the activation of B cells from naive WT mice than TLR7 KO mice (p < 0.05). Finally, the effects of TLR7 on B cells are dependent on the activation of NF-κB p65. Altogether, TLR7 was found modulating the splenic B cell responses in S. japonicum infected C57BL/6 mice., Author summary Schistosomiasis seriously jeopardizes public health and social development in tropical and subtropical regions. B cells play an important role in Schistosoma infection-induced diseases. TLR7 is an intracellular member of the innate immune receptor. The role of TLR7 on B cells mediated immune response is still unclear. Here, we found the percentage and numbers of B cells increased in the infected mice (p < 0.05), and the expression of many activation and function associated molecules were also changed on B cells. B cells were served as the main cell population expressed TLR7. When TLR7 gene was knockout, the soluble egg antigen (SEA) specific antibody and activation associated molecules were decreased in S. japonicum infected mice. Additionally, SEA could induce the activation of B cells by TLR7. Finally, we found the effects of TLR7 on B cells are dependent on the activation of NF-κB p65. In this study, the characteristic of B cells in the spleen of S. japonicum infected C57BL/6 mice was explored, and the role of TLR7 on the progress of B cell activation and differentiation was investigated.

Homeostatic perturbation caused by infection fosters two major defense strategies, resistance and tolerance, which promote the host’s survival. Resistance relates to the ability of the host to restrict the pathogen load. Tolerance minimizes collateral tissue damage without directly affecting pathogen fitness. These concepts have been explored mechanistically in murine models of malaria but only superficially in human disease. Indeed, individuals infected with Plasmodium vivax may present with asymptomatic malaria, only mild symptoms, or be severely ill. We and others have reported a diverse repertoire of immunopathological events that potentially underly susceptibility to disease severity in vivax malaria. Nevertheless, the combined epidemiologic, clinical, parasitological, and immunologic features associated with defining the disease outcomes are still not fully understood. In the present study, we perform an extensive outlining of cytokines and inflammatory proteins in plasma samples from a cohort of individuals from the Brazilian Amazon infected with P. vivax and presenting with asymptomatic (n = 108) or symptomatic (n = 134) disease (106 with mild presentation and 28 with severe malaria), as well as from uninfected endemic controls (n = 128) to elucidate these gaps further. We employ highly multidimensional Systems Immunology analyses using the molecular degree of perturbation to reveal nuances of a unique profile of systemic inflammation and imbalanced immune activation directly linked to disease severity as well as with other clinical and epidemiologic characteristics. Additionally, our findings reveal that the main factor associated with severe cases of P. vivax infection was the number of symptoms, despite of a lower global inflammatory perturbation and parasitemia. In these participants, the number of symptoms directly correlated with perturbation of markers of inflammation and tissue damage. On the other hand, the main factor associated with non-severe infections was the parasitemia values, that correlated only with perturbation of inflammatory markers, such as IL-4 and IL-1β, with a relatively lower number of symptoms. These observations suggest that some persons present severe vivax regardless of pathogen burden and global inflammatory perturbation. Such patients are thus little tolerant to P. vivax infection and show higher susceptibility to disrupt homeostasis and consequently exhibit more clinical manifestations. Other persons are capable to tolerate higher parasitemia with lower inflammatory perturbation and fewer symptoms, developing non-severe malaria. The analytical approach presented here has capability to define in more details the determinants of disease tolerance in vivax malaria., Author summary Plasmodium vivax infection can result in a broad spectrum of disease manifestations, ranging from asymptomatic malaria to severe life-threatening disease. Despite significant advances in the current understanding of the critical factors associated with the disease outcomes in vivax malaria, the immunopathological events responsible for the diversity of severe manifestations in the disease remain deeply unknown. Here, a large panel of cytokines/chemokines were assessed in plasma samples from a Brazilian cohort of P. vivax patients presenting with asymptomatic infection or symptomatic malaria at the time of diagnosis, as well as from uninfected endemic controls, to define the relationships between systemic inflammation, disease presentation, parasitemia, and epidemiologic characteristics. In-depth analyses using the molecular degree of perturbation were employed to reveal nuances of a unique profile of systemic inflammation and imbalanced immune activation directly linked to disease severity. Moreover, the discoveries diagrammed the occurrence of disease tolerance by narrowing down the interactions between the systemic degree of inflammatory perturbation and parasitemia values in vivax malaria patients.

Background Trichinellosis is a serious zoonotic disease distributed around the world. It is needed to develop a safe, effective and feasible anti-Trichinella vaccine for prevention and control of trichinellosis. The aim of this study was to construct a recombinant Lactobacillus plantarum encoding Trichinella spiralis inorganic pyrophosphatase (TsPPase) and investigate its immune protective effects against T. spiralis infection. Methodology/Principal findings The growth of recombinant L. plantarum was not affected by TsPPase/pSIP409-pgsA′ plasmid, and the recombinant plasmid was inherited stably in bacteria. Western blot and immunofluorescence assay (IFA) indicated that the rTsPPase was expressed on the surface of recombinant L. plantarum. Oral vaccination with rTsPPase induced higher levels of specific serum IgG, IgG1, IgG2a and mucosal secretory IgA (sIgA) in BALB/c mice. ELISA analysis revealed that the levels of IFN-γ and IL-4 released from spleen, mesenteric lymph nodes and Peyer’s patches were evidently increased at 2–4 weeks following vaccination, compared to MRS (De Man, Rogosa, Sharpe) medium control group (P < 0.05). Immunization of mice with rTsPPase exhibited a 67.18, 54.78 and 51.91% reduction of intestinal infective larvae, adult worms and muscle larvae at 24 hours post infection (hpi), 6 days post infection (dpi) and 35 dpi, respectively (P < 0.05), and the larval molting and development was significantly inhibited by 45.45% at 24 hpi, compared to the MRS group. Conclusions TsPPase plays a crucial role in T. spiralis molting and development, oral vaccination with rTsPPase induced a significant local mucosal sIgA response and systemic Th1/Th2 immune response, and immune protection against T. spiralis infection in BALB/c mice., Author summary In the previous study, a Trichinella spiralis inorganic pyrophosphatase (TsPPase) was expressed and its role in larval molting and development was observed. In this study, a recombinant TsPPase/pSIP409-pgsA′ plasmid was constructed and transferred into Lactobacillus plantarum NC8, the rTsPPase was expressed on the surface of recombinant L. plantarum NC8. Oral immunization of mice with rTsPPase DNA vaccine elicited a high level of specific serum IgG, IgG1, IgG2a and mucosal secretory IgA (sIgA). The levels of IFN-γ and IL-4 released from spleen, mesenteric lymph nodes and Peyer’s patches were evidently increased at 2–4 weeks following vaccination. Immunization of mice with rTsPPase showed a significant reduction of intestinal infective larvae, adult worms and muscle larvae, and intestinal larval molting and development was significantly suppressed. The results indicated that oral vaccination with rTsPPase elicited a significant local mucosal sIgA response and specific systemic Th1/Th2 immune response, and an obvious protective immunity against T. spiralis infection.

Development of a rapid, on-site detection tool for snakebite is highly sought after, owing to its clinically and forensically relevant medicolegal significance. Polyvalent antivenom therapy in the management of such envenomation cases is finite due to its poor venom neutralization capabilities as well as diagnostic ramifications manifested as untoward immunological reactions. For precise molecular diagnosis of elapid venoms of the big four snakes, we have developed a lateral flow kit using a monoclonal antibody (AB1; IgG1 – κ chain; Kd: 31 nM) generated against recombinant cytotoxin-7 (rCTX-7; 7.7 kDa) protein of the elapid venom. The monoclonal antibody specifically detected the venoms of Naja naja (p < 0.0001) and Bungarus caeruleus (p, Author summary Detection of the snake species responsible for the envenomation in a victim is crucial for clinical and forensic management of poisoning cases. Polyvalent antivenom therapy can seldom lead to immunological complications manifested in the form of symptoms ranging from serum sickness to myalgia. Use of monovalent antivenom therapy is being recommended for targeted venom neutralization in envenomed individuals with minimum side effects. Moreover, the development of field applicable venom detection devices is the need of the hour for enabling orderly detection of poisoning cases at the crime scene, besides testing for illegal trade of snake parts, including venom, protected under the Wildlife Act. The monovalent antivenom therapy and the field level detection of venom conducive to adequate crime scene management is limited by the tools available for species or family-specific identification of the venom under question. For differential detection of the Elapids of the big four snakes from the Viperidae, we have developed a monoclonal antibody-based lateral flow assay kit using recombinant Cytotoxin– 7 protein. The limit of quantitation for the detection of venoms of N. naja and B. caeruleus was ascertained to be 170 pg/μL and 2.1 ng/ μL in spiked buffer samples and 28.7 ng/ μL and 110 ng/ μL in spiked serum samples, respectively. Thus, the kit can effectively detect the venoms of elapids of the big four snakes in both simple and complex matrices of the samples and can be adapted for its use in differential diagnosis.