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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 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.

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.

Beside diagnostic uncertainties due to the lack of a perfect gold standard test for Helicobacter pylori infection, the diagnosis and the prevalence estimation for this infection encounter particular challenges in Africa including limited diagnostic tools and specific genetic background. We developed and evaluated the accuracy of an enzyme-linked immunosorbent assay (ELISA) system tailored for H. pylori genetics in Africa (HpAfr-ELISA). Strains belonging to main genetic populations infecting Africans were exploited as sources for whole-cell antigens to establish in-house the ELISA system. A phase II unmatched case-control study explored the diagnostic accuracy of the HpAfr-ELISA using a training set of samples collected from dyspeptic patients from Kinshasa, the Democratic Republic of Congo (DRC) who had been tested with invasive standard tests (i.e., histology, culture, and rapid urease test) in 2017. Then the assay was cross-validated through a community-based survey assessing the prevalence of H. pylori and associated factors in 425 adults from Mbujimayi, DRC in 2018. Bayesian inferences were used to deal with statistical uncertainties of estimates (true prevalence, sensitivity, and specificity) in the study population. At its optimal cut-off-value 20.2 U/mL, the assay achieved an estimated sensitivity of 97.6% (95% credible interval [95%CrI]: 89.2; 99.9%) and specificity of 90.5% (95%CrI: 78.6; 98.5). Consistent outcomes obtained at repeated tests attested the robustness of the assay (negative and positive agreements always > 70%). The true prevalence of H. pylori was estimated 53.8% [95%CrI: 42.8; 62.7%]. Increasing age (adjusted odds ratio [aOR] > 1.0 [95% confidence interval (CI): > 1.0; 1.1]; p, Author summary Enzyme-linked immunosorbent assay (ELISA) systems are useful tools for mass screening of Helicobacter pylori infection but their use encounters important challenges in Africa. This is mainly due to the specific genetic background of African H. pylori that may jeopardize the accuracy of currently available ELISA kits. In this study, we developed a new ELISA system, i.e. HpAfr-ELISA, based on pooled antigens of H. pylori that represent main genetic populations colonizing Africa. This new assay could be validated analytically using training samples collected in H. pylori positive and negative patients from the Democratic Republic of Congo (DRC). Then, the tool was successfully applied to a real-world community-based mass survey aiming at assessing the epidemiology of H. pylori in a rural area of the DRC. The characteristics of the HpAfr-ELISA assay indicated good robustness and high performances for detection of H. pylori. Statistical modeling enabled estimating the true latent prevalence of H. pylori infection and identifying factors predicting the exposition to the infection in the study population. This outcome will be useful for drawing efficient health policies and optimally allocating available resources to fight the H. pylori infection and related complications (e.g., gastric cancer) in the study area and globally in Africa.

Anemia caused by trypanosome infection is poorly understood. Autoimmunity during Trypanosoma brucei infection was proposed to have a role during anemia, but the mechanisms involved during this pathology have not been elucidated. In mouse models and human patients infected with malaria parasites, atypical B-cells promote anemia through the secretion of autoimmune anti-phosphatidylserine (anti-PS) antibodies that bind to uninfected erythrocytes and facilitate their clearance. Using mouse models of two trypanosome infections, Trypanosoma brucei and Trypanosoma cruzi, we assessed levels of autoantibodies and anemia. Our results indicate that acute T. brucei infection, but not T. cruzi, leads to early increased levels of plasma autoantibodies against different auto antigens tested (PS, DNA and erythrocyte lysate) and expansion of atypical B cells (ABCs) that secrete these autoantibodies. In vitro studies confirmed that a lysate of T. brucei, but not T. cruzi, could directly promote the expansion of these ABCs. PS exposure on erythrocyte plasma membrane seems to be an important contributor to anemia by delaying erythrocyte recovery since treatment with an agent that prevents binding to it (Annexin V) ameliorated anemia in T. brucei-infected mice. Analysis of the plasma of patients with human African trypanosomiasis (HAT) revealed high levels of anti-PS antibodies that correlated with anemia. Altogether these results suggest a relation between autoimmunity against PS and anemia in both mice and patients infected with T. brucei., Author summary African trypanosomes are relevant pathogens and a cause of great morbidity and economic burden in endemic countries. Anemia during infection by African Trypanosomes is one of the most common pathologies associated with the disease, but the mechanisms leading to it are poorly understood. Here, we report evidence of autoimmunity, particularly autoantibodies against the lipid phosphatidylserine that are secreted by autoimmune/atypical B-cells, in Trypanosoma brucei infected mice and that are also found in Human African Trypanosomiasis patients and correlate with levels of anemia. These findings point to a relation between autoimmunity and anemia during infection with African Trypanosomes, possibly through the secretion of autoantibodies against phosphatidylserine.

Leishmania are intracellular protozoan parasites that cause a wide spectrum of clinical manifestations in genetically susceptible individuals with an insufficient or balanced Th1 immune response to eliminate the parasite. MiRNAs play important regulatory role in numerous biological processes including essential cellular functions. miR146-a acts as an inhibitor of interleukin 1 receptor associated kinase 1 (IRAK1) and tumour necrosis factor (TNF) receptor associated factor 6 (TRAF6) present in the toll-like receptors pathway while miR499a modulates TGF-β and TNF signalling pathways. Here, we investigated whether MIRNA146A rs2910164 and MIRNA499 rs3746444 variants are associated with the development of L. guyanensis (Lg)-cutaneous leishmaniasis (CL). The variants MIR146A rs2910164 and MIR499A rs3746444 were assessed in 850 patients with Lg-CL and 891 healthy controls by polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP). Plasma cytokines were measured using the BioPlex assay. Carriers of rs2910164 CC genotype have 30% higher odds of developing CL (ORadjage/sex = 1.3 [95%CI 0.9–1.8]; Padjage/sex 0.14) compared to individuals with the genotype GG (ORadjage/sex = 0.77 [95%CI 0.56–1.0]; Padjage/sex 0.14) if exposed to Lg-infection. Heterozygous GC individuals also showed lower odds of developing CL (ORadjage/sex = 0.77 [95%CI 0.5–1.1]; Padjage/sex 0.09). Homozygosity for the allele C is suggestive of an association with the development of Lg-CL among exposed individuals to Lg-infection. However, the odds of developing CL associated with the CC genotype was evident only in male individuals (ORadjage = 1.3 [95% CI = 0.9–2.0]; Padjage = 0.06). Individuals homozygous for the G allele tend to have higher plasma IL-8 and CCL5. Similarly, for the MIR499A rs3746444, an association with the G allele was only observed among male individuals (OR = 1.4 [1.0–1.9]; P = 0.009). In a dominant model, individuals with the G allele (GG-GA) when compared to the AA genotype reveals that carriers of the G allele have 40% elevated odds of developing Lg-CL (ORadjage = 1.4 [1.1–1.9]). Individuals with the GG genotype have higher odds of developing Lg-CL (ORadjage/sex = 2.0 [95%CI 0.83–5.0]; Padjage = 0.01. Individuals homozygous for the G allele have higher plasma IL-8. Genetic combinations of both variants revealed that male individuals exposed to Lg bearing three or four susceptible alleles have higher odds of developing Lg-CL (OR = 2.3 [95% CI 1.0–4.7]; p = 0.017). Both MIR146A rs2910164 and MIR499A rs3746444 are associated with the development of Lg-CL and this association is prevalent in male individuals., Author summary Leishmaniasis is caused by infection with Leishmania parasites. In regions with the presence of Leishmania parasites, all people do not develop the disease despite similar exposure. Only a proportion of inhabitants progress to the development of disease. Clinical manifestations depend on the vector and Leishmania species, as well the host genetic background and genetically determined immune responses. miRNAs play important roles in regulating gene expression and many biological processes including immune pathways. miR-146a targets TRAF6 and IRAK1 genes, that encode key adaptor molecules downstream of toll-like receptors (TLRs). TLRs are critical in immune response to Leishmania-infection. miR499-a modulates inflammation-related signalling pathways such as TGFβ, TNFα and TLR pathways. In this study, we showed that MIR146A and MIR499A variants are risk factors to developing cutaneous leishmaniasis caused by L. guyanensis in Amazonas state of Brazil. Individuals with these variants are susceptible to the development of CL.

Immune evasion by Treponema pallidum subspecies pallidum (T. pallidum) has been attributed to antigenic variation of its putative outer-membrane protein TprK. In TprK, amino acid diversity is confined to seven variable (V) regions, and generation of sequence diversity within the V regions occurs via a non-reciprocal segmental gene conversion mechanism where donor cassettes recombine into the tprK expression site. Although previous studies have shown the significant role of immune selection in driving accumulation of TprK variants, the contribution of baseline gene conversion activity to variant diversity is less clear. Here, combining longitudinal tprK deep sequencing of near clonal Chicago C from immunocompetent and immunosuppressed rabbits along with the newly developed in vitro cultivation system for T. pallidum, we directly characterized TprK alleles in the presence and absence of immune selection. Our data confirm significantly greater sequence diversity over time within the V6 region during syphilis infection in immunocompetent rabbits compared to immunosuppressed rabbits, consistent with previous studies on the role of TprK in evasion of the host immune response. Compared to strains grown in immunocompetent rabbits, strains passaged in vitro displayed low level changes in allele frequencies of TprK variable region sequences similar to that of strains passaged in immunosuppressed rabbits. Notably, we found significantly increased rates of V6 allele generation relative to other variable regions in in vitro cultivated T, pallidum strains, illustrating that the diversity within these hypervariable regions occurs in the complete absence of immune selection. Together, our results demonstrate antigenic variation in T. pallidum can be studied in vitro and occurs even in the complete absence of immune pressure, allowing the T. pallidum population to continuously evade the immune system of the infected host., Author summary Syphilis continues to be a disease of global and public health concern, even though the infection can be easily diagnosed and effectively treated with penicillin. Although infected individuals often develop a strong immunity to the pathogen, repeated infection with the syphilis agent, Treponema pallidum subspecies pallidum (T. pallidum), is possible. Several studies point at antigenic variation of the T. pallidum TprK protein as the mechanism responsible for evasion of the immunity that develops during infection, pathogen persistence, and re-infection. Past studies have highlighted the importance of immune clearance of dominant variants that, in turn, allows less represented variants to emerge. The contribution of an immunity-independent baseline generation of variability in the tprK gene is less clear. Here, we used deep sequencing to profile tprK variants using a laboratory-isolated T. pallidum strain nearly isogenic for tprK that was propagated over time in vitro, where no immune pressure is exerted on the pathogen, as well as in samples obtained from immunosuppressed and immunocompetent rabbits infected with the same strain. We confirmed that tprK accumulates significantly more diversity under immune pressure, and demonstrated a low but discernible basal rate of gene conversion in complete absence of immune pressure.

Background Adverse reactions to antivenom considerably complicate the clinical management of snakebite envenomed patients because it necessitates a temporary suspension of life-saving antivenom, increases costs and can compromise patient outcomes. This study sought to explore the association between cattle-herding occupation and ethnic group and the occurrence of early adverse reactions to antivenom. Methods This cross-sectional study was conducted between the 25th April and 11th July 2011 at the Kaltungo General Hospital in north east Nigeria. The exposure variable of cattle-herding occupation showed a strong correlation with the ethnic group variable, thus these were combined into a new variable with three categories (Fulani and herder, either Fulani or herder, and neither Fulani nor herder). The outcome variable was the occurrence of early adverse reactions, defined as any new symptoms occurring within 6 hours of antivenom administration. Odds Ratios were estimated using multivariable logistic regression models controlling for potential confounders. Results Among 231 envenomed snakebite victims, the overall incidence of early adverse reactions was 11.9% (95% confidence intervals: 8.0–16.9%). Patients who were Fulani and herders had a higher incidence of early adverse reactions compared to patients who were neither Fulani nor herders (20% vs 5.7%). After adjusting for age and gender, victims who were Fulani and herders were 5.9 times more likely to have an early adverse reaction, compared to victims who were neither Fulani nor herders (95% CI: 1.88–18.59; p = 0.002). Interpretation To the best of our knowledge, this is the first study to provide evidence of higher odds of early adverse reactions among patients from a particular occupation and/or ethnic group. We recommend that snake envenomed patients of Fulani origin be especially closely monitored for adverse reactions, that hospitals receiving these patients be appropriately resourced to manage both envenoming and adverse reactions and that premedication with adrenaline should be considered. Our findings provide an argument for speculation on the influence of immunological or lifestyle-related differences on the occurrence of early adverse reactions to antivenom., Author summary Antivenom is the first-choice treatment of systemic snake envenoming that annually affects between 1.8–2.7 million victims globally. Access to antivenom is especially poor for those in greatest need because they typically reside in impoverished, rural tropical communities dependent upon health facilities with limited resources. In addition, clinical treatment of snakebite victims is further complicated by early adverse reactions (EARs) to antivenom-treatment. The causes of antivenom-associated EARs are poorly understood and under-researched. Despite antivenom producers instituting costly remedial manufacturing steps (removal of pyrogens and other impurities) to make their products safer, EARs still affect a high proportion of antivenom-treated patients. Instigated by anecdotal observations to the corresponding author from clinicians in rural Nigerian hospitals that snakebite victims of cattle-herding occupation, and especially those of Fulani ethnicity, suffer more frequent EARs than other groups, this cross-sectional study identified that risks of developing EARs to antivenom treatment include the ethnicity and pastoral lifestyle of snakebite patients. To our knowledge, this is the first study to identify that EARs to antivenom-treatment include factors associated with the victim, as well as factors related to the antivenom itself. We emphasise the need for more research on the causes of adverse reactions to antivenom so that strategies to reduce incidence can be implemented.

Background Snakebite is a neglected tropical disease that causes high global rates of mortality and morbidity. Although snakebite can cause a variety of pathologies in victims, haemotoxic effects are particularly common and are typically characterised by haemorrhage and/or venom-induced consumption coagulopathy. Antivenoms are the mainstay therapeutic for treating the toxic effects of snakebite, but despite saving thousands of lives annually, these therapies are associated with limited cross-snake species efficacy due to venom variation, which ultimately restricts their therapeutic utility to particular geographical regions. Methodology/Principal findings In this study we explored the feasibility of generating globally effective pathology-specific antivenoms to counteract the haemotoxic signs of snakebite envenoming. Two different immunogen mixtures, consisting of seven and twelve haemotoxic venoms sourced from geographically diverse and/or medically important snakes, were used to raise ovine polyclonal antibodies, prior to characterisation of their immunological binding characteristics and in vitro neutralisation profiles against each of the venoms. Despite variability of the immunogen mixtures, both experimental antivenoms exhibited broadly comparable in vitro venom binding and neutralisation profiles against the individual venom immunogens in immunological and functional assays. However, in vivo assessments using a murine preclinical model of antivenom efficacy revealed substantial differences in venom neutralisation. The experimental antivenom generated from the seven venom immunogen mixture outperformed the comparator, by providing protective effects against venom lethality caused by seven of the eight geographically diverse venoms tested, including three distinct venoms that were not used as immunogens to generate this antivenom. These findings suggest that a core set of venom immunogens may be sufficient to stimulate antibodies capable of broadly neutralising a geographically diverse array of haemotoxic snake venoms, and that adding additional venom immunogens may impact negatively on the dose efficacy of the resulting antivenom. Conclusions/Significance Although selection of appropriate immunogens that encapsulate venom toxin diversity without diluting antivenom potency remains challenging and further optimisation is required, the findings from this pilot study suggest that the generation of pathology-specific antivenoms with global utility is likely to feasible, thereby highlighting their promise as future modular treatments for the world’s tropical snakebite victims., Author summary Snakebite is a major public health concern that causes extensive death and disability in the tropical world. Variation in venom composition causes major challenges for snakebite therapy by typically restricting antivenom efficacy to only the few snake species that each product was designed against, meaning that many geographically restricted therapeutics are manufactured for different parts of the world. In this study, we explored the feasibility of generating a global pathology-specific antivenom by using two different sets of haemotoxic venoms as immunogens, one including seven venoms and the other twelve venoms, to generate ovine polyclonal antibodies. Our in vitro immunological cross-reactivity and venom functional-neutralisation findings demonstrated broadly comparable venom recognition and inhibition against the various venom immunogens, irrespective of whether they were included in the immunising mixture. However, assessments of murine in vivo protection against venom-induced lethality revealed that the antivenom generated with fewer immunogens outperformed the comparator, and provided broader preclinical efficacy, even against some venoms not used to raise the antibodies. These findings suggest that careful selection of venoms used as immunogens is crucial for maintaining antivenom potency, and that a small subset of venoms may be capable of generating an antivenom with potential global efficacy against a specific pathology commonly observed after snakebite.

With global warming and lush forest change, vector-borne infections are expected to increase in the number and diversity of agents. Since the first report of severe fever with thrombocytopenia syndrome (SFTS) in 2013, the number of reported cases has increased annually in South Korea. However, although tick-borne encephalitis virus (TBEV) was detected from ticks and wild rodents, there is no human TBE case report in South Korea. This study aimed to determine the seroprevalence of TBEV and SFTS virus (SFTSV) among forest and field workers in South Korea. From January 2017 to August 2018, a total 583 sera were obtained from the forest and field workers in South Korea. IgG enzyme-linked immunosorbent assay (ELISA) and neutralization assay were conducted for TBEV, and indirect immunofluorescence assay (IFA) and neutralization assay were performed for SFTSV. Seroprevalence of TBEV was 0.9% (5/583) by IgG ELISA, and 0.3% (2/583) by neutralization assay. Neutralizing antibody against TBEV was detected in a forest worker in Jeju (1:113) and Hongcheon (1:10). Only 1 (0.2%) forest worker in Yeongju was seropositive for SFTSV by IFA (1:2,048) and neutralizing antibody was detected also. In conclusion, this study shows that it is necessary to raise the awareness of physicians about TBEV infection and to make efforts to survey and diagnose vector-borne diseases in South Korea., Author summary With global warming and lush forest change, vector-borne infections are expected to increase in the number and diversity of agents. Since the first report of severe fever with thrombocytopenia syndrome (SFTS) in 2013, the number of reported cases has increased annually in South Korea. However, although tick-borne encephalitis virus (TBEV) was detected from ticks and wild rodents, there is no human TBE case report in South Korea. This study aimed to determine the seroprevalence of TBEV and SFTS virus (SFTSV) among forest and field workers in South Korea. Among 583 forest/field workers, the seroprevalence of neutralizing antibodies against TBEV and SFTSV were 0.3% (2/583) and 0.2% (1/583), respectively. This study shows that it is necessary to raise the awareness of physicians about TBEV infection and to make efforts to survey and diagnose vector-borne diseases in South Korea.

As lymphatic filariasis (LF) programs move closer to established targets for validation elimination of LF as a public health problem, diagnostic tools capable of supporting the needs of the programs are critical for success. Known limitations of existing diagnostic tools make it challenging to have confidence that program endpoints have been achieved. In 2019, the World Health Organization (WHO) established a Diagnostic Technical Advisory Group (DTAG) for Neglected Tropical Diseases tasked with prioritizing diagnostic needs including defining use-cases and target product profiles (TPPs) for needed tools. Subsequently, disease-specific DTAG subgroups, including one focused on LF, were established to develop TPPs and use-case analyses to be used by product developers. Here, we describe the development of two priority TPPs for LF diagnostics needed for making decisions for stopping mass drug administration (MDA) of a triple drug regimen and surveillance. Utilizing the WHO core TPP development process as the framework, the LF subgroup convened to discuss and determine attributes required for each use case. TPPs considered the following parameters: product use, design, performance, product configuration and cost, and access and equity. Version 1.0 TPPs for two use cases were published by WHO on 12 March 2021 within the WHO Global Observatory on Health Research and Development. A common TPP characteristic that emerged in both use cases was the need to identify new biomarkers that would allow for greater precision in program delivery. As LF diagnostic tests are rarely used for individual clinical diagnosis, it became apparent that reliance on population-based surveys for decision making requires consideration of test performance in the context of such surveys. In low prevalence settings, the number of false positive test results may lead to unnecessary continuation or resumption of MDA, thus wasting valuable resources and time. Therefore, highly specific diagnostic tools are paramount when used to measure low thresholds. The TPP process brought to the forefront the importance of linking use case, program platform and diagnostic performance characteristics when defining required criteria for diagnostic tools., Author summary High quality diagnostic tools are an essential component of lymphatic filariasis (LF) programs. Currently, diagnostic tools are used by national programs to establish baseline endemicity, monitor progress of program interventions, determine when interventions can be stopped, and surveillance. For years, LF programs have relied on diagnostic tools that have been central to informing key program decisions but were not necessarily designed to undertake the roles for which they are used. Known limitations of existing diagnostic tools make it challenging to have confidence that program endpoints have been achieved. Target product profiles (TPP) provide product requirements to guide developers and manufacturers in their efforts to design tools fit for purpose. However, development of diagnostic tools used in public health programs requires consideration of aspects beyond those considered when developing diagnostic tools used for clinical diagnosis. The TPP process for two LF use cases brought to the forefront the importance of linking use case, program platform and diagnostic performance characteristics when defining required criteria for diagnostic tools.

Background Individuals infected with SARS-CoV-2 develop neutralising antibodies. We investigated the proportion of individuals with SARS-CoV-2 neutralising antibodies after infection and how this proportion varies with selected covariates. Methodology/Principal findings This systematic review and meta-analysis examined the proportion of individuals with SARS-CoV-2 neutralising antibodies after infection and how these proportions vary with selected covariates. Three models using the maximum likelihood method assessed these proportions by study group, covariates and individually extracted data (protocol CRD42020208913). A total of 983 reports were identified and 27 were included. The pooled (95%CI) proportion of individuals with neutralising antibodies was 85.3% (83.5–86.9) using the titre cut off >1:20 and 83.9% (82.2–85.6), 70.2% (68.1–72.5) and 54.2% (52.0–56.5) with titres >1:40, >1:80 and >1:160, respectively. These proportions were higher among patients with severe COVID-19 (e.g., titres >1:80, 84.8% [80.0–89.2], >1:160, 74.4% [67.5–79.7]) than those with mild presentation (56.7% [49.9–62.9] and 44.1% [37.3–50.6], respectively) and lowest among asymptomatic infections (28.6% [17.9–39.2] and 10.0% [3.7–20.1], respectively). IgG and neutralising antibody levels correlated poorly. Conclusions/Significance 85% of individuals with proven SARS-CoV-2 infection had detectable neutralising antibodies. This proportion varied with disease severity, study setting, time since infection and the method used to measure antibodies., Author summary Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) elicits adaptive immunological responses, including immunoglobulins A, M, and G and neutralising antibodies. Neutralising antibodies are considered markers of functional immunity and protection. However, not all individuals with proven infections have detectable neutralising antibodies. In this systematic review, we investigated the proportion of individuals with former SARS-CoV-2 infections who develop neutralising antibodies, whether their titres vary with disease severity, and their correlation with Immunoglobulin G. We found that approximately 85% of individuals with SARS-CoV-2 infection have detectable neutralising antibodies. This proportion was higher among patients with severe Coronavirus Disease 19 and lower in asymptomatic infections. The variation across studies reflected the wide range of methods used to measure both immunoglobulins and neutralising antibodies, and highlight the need for an international reference standard to measure SARS-CoV-2 antibodies.

Concerns have arisen that pre-existing immunity to dengue virus (DENV) could enhance Zika virus (ZIKV) disease, due to the homology between ZIKV and DENV and the observation of antibody-dependent enhancement (ADE) among DENV serotypes. To date, no study has examined the impact of pre-existing DENV immunity on ZIKV pathogenesis during pregnancy in a translational non-human primate model. Here we show that macaques with a prior DENV-2 exposure had a higher burden of ZIKV vRNA in maternal-fetal interface tissues as compared to DENV-naive macaques. However, pre-existing DENV immunity had no detectable impact on ZIKV replication kinetics in maternal plasma, and all pregnancies progressed to term without adverse outcomes or gross fetal abnormalities detectable at delivery. Understanding the risks of ADE to pregnant women worldwide is critical as vaccines against DENV and ZIKV are developed and licensed and as DENV and ZIKV continue to circulate., Author summary Zika virus (ZIKV) gained global attention during an explosive outbreak in the Americas in 2015–16 when it was causally associated with the constellation of birth defects now termed congenital Zika syndrome (CZS). However, a substantial proportion of gestational ZIKV infections result in babies without apparent birth defects. Could there be other factors that influence ZIKV pathogenicity? For example, it is well-established that pre-existing immunity to one dengue virus (DENV) serotype can enhance the severity of a secondary DENV infection. ZIKV is antigenically closely related to DENV, but whether DENV-specific antibodies enhance the severity of ZIKV infection is unclear. To answer this question, we used our non-human primate model of ZIKV to assess the impact of pre-existing immunity to DENV on ZIKV pathogenesis during pregnancy. We did not observe any difference in ZIKV replication in plasma between macaques that were immune to DENV and those that were not. However, there was more ZIKV vRNA detected in the placenta of macaques immune to DENV, suggesting DENV immunity could enhance ZIKV infection of the placenta. As vaccines to both DENV and ZIKV are developed, it remains critical to understand the risks of DENV immunity for pregnant women exposed to ZIKV.

Mongooses, a nonnative species, are a known reservoir of rabies virus in the Caribbean region. A cross-sectional study of mongooses at 41 field sites on the US Virgin Islands of St. Croix, St. John, and St. Thomas captured 312 mongooses (32% capture rate). We determined the absence of rabies virus by antigen testing and rabies virus exposure by antibody testing in mongoose populations on all three islands. USVI is the first Caribbean state to determine freedom-from-rabies for its mongoose populations with a scientifically-led robust cross-sectional study. Ongoing surveillance activities will determine if other domestic and wildlife populations in USVI are rabies-free., Author summary Mongooses in the Caribbean region are known to carry rabies and infect and kill humans with the deadly virus. While many countries in the Caribbean region assume they are rabies-free, there is a lack of scientifically based surveillance. We prospectively designed a statistically valid study to determine that mongoose populations in the US Virgin Islands are rabies-free and pose no risk of transmission to human or animal populations. We are pursuing further surveillance efforts to establish rabies freedom in all wildlife and domestic animal population in the US Virgin Islands.

Background India has set a goal to eliminate measles and rubella/Congenital Rubella Syndrome (CRS) by 2023. Towards this goal, India conducted nationwide supplementary immunization activity (SIA) with measles-rubella containing vaccine (MRCV) targeting children aged between 9 months to, Author summary Rubella infection during the first trimester of pregnancy can affect fetus, resulting in spontaneous abortion, stillbirth or birth of a baby with a combination of birth defects known as congenital rubella syndrome (CRS). Vaccination with rubella containing vaccine (RCV) is recommended as one of the strategies for eliminating rubella/CRS. The Southeast Asia region has set a target to eliminate rubella/CRS by 2023. Towards this goal, India completed nationwide immunization campaigns using measles-rubella vaccine during 2017–19, targeting children aged 9 months to

With increasing geographic spread, frequency, and magnitude of outbreaks, dengue continues to pose a major public health threat worldwide. Dengvaxia, a dengue live-attenuated tetravalent vaccine, was licensed in 2015, but post hoc analyses of long-term data showed serostatus-dependent vaccine performance with an excess risk of hospitalized and severe dengue in seronegative vaccine recipients. The World Health Organization (WHO) recommended that only persons with evidence of past dengue infection should receive the vaccine. A test for pre-vaccination screening for dengue serostatus is needed. To develop the target product profile (TPP) for a dengue pre-vaccination screening test, face-to-face consultative meetings were organized with follow-up regional consultations. A technical working group was formed to develop consensus on a reference test against which candidate pre-vaccination screening tests could be compared. The group also reviewed current diagnostic landscape and the need to accelerate the evaluation, regulatory approval, and policy development of tests that can identify seropositive individuals and maximize public health impact of vaccination while avoiding the risk of hospitalization in dengue-naive individuals. Pre-vaccination screening strategies will benefit from rapid diagnostic tests (RDTs) that are affordable, sensitive, and specific and can be used at the point of care (POC). The TPP described the minimum and ideal characteristics of a dengue pre-vaccination screening RDT with an emphasis on high specificity. The group also made suggestions for accelerating access to these RDTs through streamlining regulatory approval and policy development. Risk and benefit based on what can be achieved with RDTs meeting minimal and optimal characteristics in the TPP across a range of seroprevalences were defined. The final choice of RDTs in each country will depend on the performance of the RDT, dengue seroprevalence in the target population, tolerance of risk, and cost-effectiveness., Author summary This paper describes the consensus on the minimum and ideal performance and operational characteristics of rapid tests that would be used for dengue pre-vaccination screening. This profile will incentivize industry to develop better pre-vaccination screening tests. The choice of which test to use depends on the seroprevalence of the population targeted for vaccination and the optimal balance between benefit and risks. The group also made suggestions for accelerating access to these pre-vaccination screening tests through streamlining regulatory approval and policy development.

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.

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.

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.

Introduction To date, eleven countries have been validated as having eliminated trachoma as a public health problem, including Ghana in 2018. Surveillance for recrudescence is needed both pre- and post-validation but evidence-based guidance on appropriate strategies is lacking. We explored two potential surveillance strategies in Ghana. Methodology/principal findings Amongst randomly-selected communities enrolled in pre-validation on-going surveillance between 2011 and 2015, eight were identified as having had trachomatous-inflammation follicular (TF) prevalence ≥5% in children aged 1–9 years between 2012 and 2014. These eight were re-visited in 2015 and 2016 and neighbouring communities were also added (“TF trigger” investigations). Resident children aged 1–9 years were then examined for trachoma and had a conjunctival swab to test for Chlamydia trachomatis (Ct) and a dried blood spot (DBS) taken to test for anti-Pgp3 antibodies. These investigations identified at least one community with evidence of probable recent Ct ocular transmission. However, the approach likely lacks sufficient spatio-temporal power to be reliable. A post-validation surveillance strategy was also evaluated, this reviewed the ocular Ct infection and anti-Pgp3 seroprevalence data from the TF trigger investigations and from the pre-validation surveillance surveys in 2015 and 2016. Three communities identified as having ocular Ct infection >0% and anti-Pgp3 seroprevalence ≥15.0% were identified, and along with three linked communities, were followed-up as part of the surveillance strategy. An additional three communities with a seroprevalence ≥25.0% but no Ct infection were also followed up (“antibody and infection trigger” investigations). DBS were taken from all residents aged ≥1 year and ocular swabs from all children aged 1–9 years. There was evidence of transmission in the group of communities visited in one district (Zabzugu-Tatale). There was no or little evidence of continued transmission in other districts, suggesting previous infection identified was transient or potentially not true ocular Ct infection. Conclusions/significance There is evidence of heterogeneity in Ct transmission dynamics in northern Ghana, even 10 years after wide-scale MDA has stopped. There is added value in monitoring Ct infection and anti-Ct antibodies, using these indicators to interrogate past or present surveillance strategies. This can result in a deeper understanding of transmission dynamics and inform new post-validation surveillance strategies. Opportunities should be explored for integrating PCR and serological-based markers into surveys conducted in trachoma elimination settings., Author summary The goal for trachoma programmes is elimination of trachoma as a public health problem. This means that ongoing low-level eye-to-eye transmission of the causative bacterium, Chlamydia trachomatis (Ct), is acceptable. Countries need to implement a suitable surveillance system to identify any return to higher transmission levels. The best methodology for doing this is not known. We first explored the approach used by Ghana in its standard programme, which involved monitoring a limited number of randomly selected communities for evidence of active (inflammatory) trachoma visible in children’s eyes on examination by trained observers. Although this strategy led to identification of at least one community that had probably had recent Ct transmission, the approach is unlikely to consistently identify places where return to higher levels of transmission is a risk. We also explored using information on infection (detected in eye swabs) and antibodies to Ct (detected in the blood) to identify communities at risk. We found evidence of both persistent eye-to-eye Ct transmission and areas where infection was transient and has now gone away. We conclude that the use of infection and antibody data for surveillance of trachoma appears promising.

Vibrio cholerae is a bacterial pathogen which causes the severe acute diarrheal disease cholera. Given that a symptomatic incident of cholera can lead to long term protection, a thorough understanding of the immune response to this pathogen is needed to identify parameters critical to the generation and durability of immunity. To approach this, we utilized a live attenuated cholera vaccine to model the response to V. cholerae infection in 12 naïve subjects. We found that this live attenuated vaccine induced durable vibriocidal antibody titers that were maintained at least one year after vaccination. Similar to what we previously reported in infected patients from Bangladesh, we found that vaccination induced plasmablast responses were primarily specific to the two immunodominant antigens lipopolysaccharide (LPS) and cholera toxin (CT). Interestingly, the magnitude of the early plasmablast response at day 7 predicted the serological outcome of vaccination at day 30. However, this correlation was no longer present at later timepoints. The acute responses displayed preferential immunoglobulin isotype usage, with LPS specific cells being largely IgM or IgA producing, while cholera toxin responses were predominantly IgG. Finally, CCR9 was highly expressed on vaccine induced plasmablasts, especially on IgM and IgA producing cells, suggesting a role in migration to the gastrointestinal tract. Collectively, these findings demonstrate that the use of a live attenuated cholera vaccine is an effective tool to examine the primary and long-term immune response following V. cholerae exposure. Additionally, it provides insight into the phenotype and specificity of the cells which likely return to and mediate immunity at the intestinal mucosa. A thorough understanding of these properties both in peripheral blood and in the intestinal mucosae will inform future vaccine development against both cholera and other mucosal pathogens. Trial Registration:NCT03251495., Author summary Cholera is a severe diarrheal illness which affects millions of people annually. Those most affected reside in developing countries where outbreaks can cause a devastating healthcare crisis. Despite this, much is still unknown regarding the longevity of protective immune responses or how they are generated and maintained. This includes aspects of the mucosal response where immunity is induced, as well as features of the early immune response that could predict long-term immunity. To address these issues, we examined primary humoral immune responses following administration of a live attenuated cholera vaccine to 12 human participants. This study provides insight into the development of humoral immunity and information that may prove valuable for improving cholerae vaccines and treatments.

Background Cystic echinococcosis (CE) is a complex disease for which clear understanding of clinical manifestations is needed to avoid misdiagnosis, inappropriate treatment, and severe complications. We evaluated the accuracy of a whole-blood stimulation test based on Interleukin (IL)-4 detection in response to Antigen B (AgB) of Echinococcus granulosus sensu lato to discriminate cyst viability and detect cyst reactivation in patients with hepatic CE. Methodology/Principal findings Thirty patients with CE3b cysts and 37 patients with spontaneously-inactivated CE4-CE5 cysts were recruited (T0). After enrollment, 5 patients with CE3b cysts received albendazole, resulting in cyst solidification (CE4) in 4/5. Within a two-year follow-up, the whole-blood test was repeated at two time-points, in ≥14 (T1) and in ≥4 (T2) patients per group. IL-4 and a panel of other soluble factors were measured in the stimulated plasma. Baseline IL-4 levels were significantly higher in patients with CE3b compared to those with CE4 cysts (p = 0.006). Test accuracy for CE3b diagnosis had a sensitivity of 33–60% and a specificity of 76–95%, depending on the cut-off applied. Overall, IL-4 levels did not change significantly over time in either group; however, patients within the CE3b group showed a significant decrease of IL-1ra, IL-6, IL-8, G-CSF, IFN-γ, IP-10, MCP-1, MIP-1α, FGF at T1 compared to T0 (p≤0.042). Conclusions/Significance Whole-blood IL-4-response to AgB is significantly higher in patients with active compared to inactive CE but apparently not modulated over time after treatment. On the contrary, the levels of IL-1ra, IL-6, IL-8, G-CSF, IFN-γ, IP-10, MCP-1, MIP-1α, FGF significantly decreased in active CE during follow-up. Additional studies are needed to understand whether these findings might have a clinical significance for patients’ follow-up., Author summary Cystic echinococcosis (CE) is a complex disease for which a clear understanding of clinical manifestations is needed to avoid misdiagnosis, inappropriate treatment, and severe complications. The development of a laboratory test able to assess CE cyst viability and reactivation over time would facilitate the classification of patients and cysts, their rational assignment to a clinical management option and the implementation of a regular follow-up. Here, we evaluated the accuracy of a cytokine-based whole-blood test to detect the reactivation and the viability of hepatic CE cysts. The results showed that IL-4-response to AgB is not modulated over-time after treatment, while, IL-1ra, IL-6, IL-8, G-CSF, IFN-γ, IP-10, MCP-1, MIP-1α, FGF levels significantly decreased in active CE during follow-up. Additional studies are needed to understand whether these findings might have a clinical significance for patients’ follow-up.

Background Ebola virus (EBOV) is a zoonotic filovirus spread through exposure to infected bodily fluids of a human or animal. Though EBOV is capable of causing severe disease, referred to as Ebola Virus Disease (EVD), individuals who have never been diagnosed with confirmed, probable or suspected EVD can have detectable EBOV antigen-specific antibodies in their blood. This study aims to identify risk factors associated with detectable antibody levels in the absence of an EVD diagnosis. Methodology Data was collected from September 2015 to August 2017 from 1,366 consenting individuals across four study sites in the DRC (Boende, Kabondo-Dianda, Kikwit, and Yambuku). Seroreactivity was determined to EBOV GP IgG using Zaire Ebola Virus Glycoprotein (EBOV GP antigen) ELISA kits (Alpha Diagnostic International, Inc.) in Kinshasa, DRC; any result above 4.7 units/mL was considered seroreactive. Among the respondents, 113 (8.3%) were considered seroreactive. Several zoonotic exposures were associated with EBOV seroreactivity after controlling for age, sex, healthcare worker status, location, and history of contact with an EVD case, namely: ever having contact with bats, ever having contact with rodents, and ever eating non-human primate meat. Contact with monkeys or non-human primates was not associated with seroreactivity. Conclusions This analysis suggests that some zoonotic exposures that have been linked to EVD outbreaks can also be associated with EBOV GP seroreactivity in the absence of diagnosed EVD. Future investigations should seek to clarify the relationships between zoonotic exposures, seroreactivity, asymptomatic infection, and EVD., Author summary Ebola virus (EBOV) is spread through exposure to infected bodily fluids of a human or animal. While EBOV can lead to a severe disease, Ebola Virus Disease (EVD), it is possible for individuals to have anti-EBOV antibodies without ever getting sick with EVD. Seroreactivity (the detection of antigen-specific antibodies) suggests that that person has been exposed to EBOV or a similar virus in the past. Our study looked for seroreactive individuals who have never received an EVD diagnosis in four sites across the Democratic Republic of the Congo. Then we checked if animal exposures previously linked to EVD were more common among seroreactive individuals than non-seroreactive individuals. Among respondents from all four sites, 113 (8.3%) were seroreactive to EBOV. Additionally, contact with bats, rodents, and eating non-human primate meat were associated with seroreactivity, indicating these factors may be predictors of undocumented EBOV exposure events. These findings show that some EVD risk factors may be associated with EBOV seroreactivity without EVD diagnosis. Future research is needed to clarify the relationships between zoonotic exposures, seroreactivity, asymptomatic infection, and EVD.

Chagas disease, caused by the parasite Trypanosoma cruzi, is considered endemic in more than 20 countries but lacks both an approved vaccine and limited treatment for its chronic stage. Chronic infection is most harmful to human health because of long-term parasitic infection of the heart. Here we show that immunization with a virus-like particle vaccine displaying a high density of the immunogenic α-Gal trisaccharide (Qβ-αGal) induced several beneficial effects concerning acute and chronic T. cruzi infection in α1,3-galactosyltransferase knockout mice. Approximately 60% of these animals were protected from initial infection with high parasite loads. Vaccinated animals also produced high anti-αGal IgG antibody titers, improved IFN-γ and IL-12 cytokine production, and controlled parasitemia in the acute phase at 8 days post-infection (dpi) for the Y strain and 22 dpi for the Colombian strain. In the chronic stage of infection (36 and 190 dpi, respectively), all of the vaccinated group survived, showing significantly decreased heart inflammation and clearance of amastigote nests from the heart tissue., Author summary Chagas disease, caused by the protozoan parasite Trypanosoma cruzi, is a significant endemic infectious disease in Latin America and is spreading in the U.S. and Europe with the presence of its insect transmission vector. No approved vaccine against Chagas disease exists. We describe a vaccine candidate based on a carbohydrate found on the T. cruzi cell surface, linked in the vaccine to a virus-like particle that provides a strong and focused immune response. Mice were immunized and challenged with the Trypanosoma cruzi parasites from two strains (Y and Colombian). Vaccination conferred substantial protection of mice against infection, compared with the unvaccinated group. Vaccinated animals presented low parasitemia, increased production of pro-inflammatory cytokines IL-12 and IFN-γ, decreased cardiomyocyte damage, and rapid clearance of parasite nests from heart tissue. These effects were especially significant at time points modeling chronic disease, an important consideration for this pathogen. We, therefore, believe this is a valuable path to pursue in the development of vaccines against Chagas disease.