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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.

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.

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 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.

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.

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.

Objectives Trichinella spiralis is a zoonotic parasite with a complex parasitic life cycle and exposed to animals or humans by infectious meat. To control transmissions of T. spiralis through the food chain to humans, sensitive and selective multihost sera-diagnosis is urgent needed for monitoring T. spiralis exposure. Methods A competition enzyme-linked immunosorbent assay (cELISA) for T. spiralis infection diagnosis in multihost sera was developed based on recombinant cystatin-like protein (rCLP-cELISA) as well as monoclonal antibodies. The sensitivity and accuracy of the rCLP-cELISA were quantified using swine (n = 1316), mice (n = 189) and human (n = 157) serum samples. T. spiralis-antibody targeting test ability of the rCLP-cELISA in swine (n = 22) and human (n = 36), instead of other parasites or viruses antibodies, was evaluated. Results The rCLP-cELISA showed high agreement with commercial ELISA kits in field swine sera assessed by Cohen’s kappa value (κ = 0.7963). And it showed 100% specificity in human trichinellosis detection with sensitivity of 96.49%, no cross-reaction with other parasite or virus infections, and high positive detection rate of 87.5% in low-dose infected swine. Besides, the rCLP-cELISA exhibited potential in the detection of T. spiralis, T. nelsoni and Trichinella T8 infections. Conclusions The rCLP-cELISA can be used for T. spiralis-associated antibody test in multihost sera., Author summary Infections with T. spiralis that lives in host muscles for long periods of time are commonly difficult to diagnosis without causing a strong immune response or symptoms. The habit of eating raw/undercooked pork meat accidentally introduces humans into the exposure of T. spiralis that circulates between herds and rodents. There is an urgent need for serological antibody test of T. spiralis to monitor the infection of humans as well as hosts in the food chain, which is restricted by the mixture type of current used antigens and species-specific secondary antibodies for different hosts. We developed a novel recombinant cystatin-like protein-based competition enzyme-linked immunosorbent assay (rCLP-cELISA) employing monoclonal antibody. The proposed method showed considerable sensitivity and specificity in filed swine sera and human serum samples with good versatility in mice. Taking advantage of its controllable quality stability, the rCLP-cELISA had potential applications for screening of T. spiralis infections for multihost sera in one test. With the development of monoclonal antibody modification strategies and the discovery of antigenic proteins from parasitic pathogens, the proposed competition ELISA also provides useful reference for the improvement of serological assay for monitoring the exposure of zoonotic parasites.

The development of efficient vaccines against COVID-19 is an emergent need for global public health. The spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a major target for the COVID-19 vaccine. To quickly respond to the outbreak of the SARS-CoV-2 pandemic, a nucleic acid-based vaccine is a novel option, beyond the traditional inactivated virus vaccine or recombinant protein vaccine. Here, we report a DNA vaccine containing the spike gene for delivery via electroporation. The spike genes of SARS-CoV and SARS-CoV-2 were codon optimized for mammalian cell expression and then cloned into mammalian cell expression vectors, called pSARS-S and pSARS2-S, respectively. Spike protein expression was confirmed by immunoblotting after transient expression in HEK293T cells. After immunization, sera were collected for antigen-specific antibody and neutralizing antibody titer analyses. We found that both pSARS-S and pSARS2-S immunization induced similar levels of antibodies against S2 of SARS-CoV-2. In contrast, only pSARS2-S immunization induced antibodies against the receptor-binding domain of SARS-CoV-2. We further found that pSARS2-S immunization, but not pSARS-S immunization, could induce very high titers of neutralizing antibodies against SARS-CoV-2. We further analyzed SARS-CoV-2 S protein-specific T cell responses and found that the immune responses were biased toward Th1. Importantly, pSARS2-S immunization in hamsters could induce protective immunity against SARS-CoV-2 challenge in vivo. These data suggest that DNA vaccination could be a promising approach for protecting against COVID-19., Author summary SARS-CoV-2 continues to threaten global health, and development of SARS-CoV-2 vaccine is an urgent priority to halt COVID-19 epidemic. Here, we developed DNA vaccines containing the spike gene from SARS-CoV-2 and delivered via electroporation. We observed that the DNA vaccine induced a long-lasting humoral response against SARS-CoV-2, and the elicited serum antibodies efficiently blocked the binding of SARS-CoV-2 RBD to its entry receptor (ACE2). Importantly, the DNA vaccine induced strong Th1-biased immune responses against SARS-CoV-2, which could provide protective effect with low side effects. Finally, we demonstrated that SARS-CoV-2 DNA vaccine conferred protective efficacy against SARS-CoV-2 infection in Syrian hamsters. Our findings suggested that DNA vaccination could be a useful approach for controlling COVID-19 pandemic in near future.

Background Serological diagnosis of Zika virus (ZIKV) infection is challenging because of the antibody cross-reactivity among flaviviruses. At the same time, the role of Nucleic Acid Testing (NAT) is limited by the low proportion of symptomatic infections and the low average viral load. Here, we compared the diagnostic performance of commercially available IgM, IgAM, and IgG ELISAs in sequential samples during the ZIKV and chikungunya (CHIKV) epidemics and co-circulation of dengue virus (DENV) in Brazil and Venezuela. Methodology/Principal findings Acute (day of illness 1–5) and follow-up (day of illness ≥ 6) blood samples were collected from nine hundred and seven symptomatic patients enrolled in a prospective multicenter study between June 2012 and August 2016. Acute samples were tested by RT-PCR for ZIKV, DENV, and CHIKV. Acute and follow-up samples were tested for IgM, IgAM, and IgG antibodies to ZIKV using commercially available ELISAs. Among follow-up samples with a RT-PCR confirmed ZIKV infection, anti-ZIKV IgAM sensitivity was 93.5% (43/46), while IgM and IgG exhibited sensitivities of 30.3% (10/33) and 72% (18/25), respectively. An additional 24% (26/109) of ZIKV infections were detected via IgAM seroconversion in ZIKV/DENV/CHIKV RT-PCR negative patients. The specificity of anti-ZIKV IgM was estimated at 93% and that of IgAM at 85%. Conclusions/Significance Our findings exemplify the challenges of the assessment of test performance for ZIKV serological tests in the real-world setting, during co-circulation of DENV, ZIKV, and CHIKV. However, we can also demonstrate that the IgAM immunoassay exhibits superior sensitivity to detect ZIKV RT-PCR confirmed infections compared to IgG and IgM immunoassays. The IgAM assay also proves to be promising for detection of anti-ZIKV seroconversions in sequential samples, both in ZIKV PCR-positive as well as PCR-negative patients, making this a candidate assay for serological monitoring of pregnant women in future ZIKV outbreaks., Author summary Zika virus (ZIKV) is transmitted through the bite of infected Aedes mosquitos but can also be transmitted sexually or vertically from mother-to-child. The same mosquitoes transmit dengue virus (DENV) and chikungunya virus (CHIKV), which cause similar clinical syndromes. The ZIKV epidemics in the Pacific and the Americas that occurred between 2015 and 2017 were linked to congenital abnormalities, most prominently microcephaly, in newborns. Because most infections are asymptomatic, diagnosis via indirect serological assays is an important strategy. On the other hand, many serological assays are affected by cross-reactivity resulting from prior infections by closely related viruses, such as DENV. This study evaluated three commercially available and widely used immunoassays that detect IgG, IgM or IgA and M (IgAM) antibodies to ZIKV. Our results suggest that the IgAM test performs best by detecting around 90% of RT-PCR confirmed infections. We also detected additional infections that were not detected by RT-PCR. The strength of this study is that it was carried out in two different countries of the American region where several arboviruses are endemic and that sequential blood samples from individual patients were available to evaluate the performance of the tests over time.

Giardia duodenalis, also known as G. intestinalis or G. lamblia, is the major cause of giardiasis leading to diarrheal disease with 280 million people infections annually worldwide. Extracellular vesicles (EVs) have emerged as a ubiquitous mechanism participating in cells communications. The aim of this study is to explore the roles of G. duodenalis EVs (GEVs) in host-pathogen interactions using primary mouse peritoneal macrophages as a model. Multiple methods of electron microscopy, nanoparticle tracking analysis, proteomic assays, flow cytometry, immunofluorescence, qPCR, western blot, ELISA, inhibition assays, were used to characterize GEVs, and explore its effects on the host cell innate immunity as well as the underlying mechanism using primary mouse peritoneal macrophages. Results showed that GEVs displayed typical cup-shaped structure with 150 nm in diameter. GEVs could be captured by macrophages and triggered immune response by increasing the production of inflammatory cytokines Il1β, Il6, Il10, Il12, Il17, Ifng, Tnf, Il18, Ccl20 and Cxcl2. Furthermore, activation of TLR2 and NLRP3 inflammasome signaling pathways involved in this process. In addition, CA-074 methyl ester (an inhibitor of cathepsin B) or zVAD-fmk (an inhibitor of pan-caspase) pretreatment entirely diminished these effects triggered by GEVs exposure. Taken together, these findings demonstrated that GEVs could be internalized into mouse peritoneal macrophages and regulate host cell innate immunity via TLR2 and NLRP3 inflammasome signaling pathways., Author summary G. duodenalis, one of the most common cause of diarrheal diseases, is widely existed in the contaminated water and threatening the public health especially in developing countries. Along with the increasing resistance to anti-G. duodenalis drugs occurs, new targets against giardiasis are of urgently needed. The innate immune system is the first defense line of organism to resist multiple pathogens invasion through recognizing pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors (PRRs), termed Toll-like receptors (TLRs) on the surface of cell membrane and nucleotide oligomerization domain (Nod)-like receptors (NLRs) inside immune cells. Recently, extracellular vesicles have emerged as a ubiquitous mechanism participating in cells communications. In this study, EVs secreted by extracellular protozoan G. duodenalis were obtained and displayed typical cup-shaped structure with 150 nm in diameter. Moreover, GEVs could enter into primary mouse peritoneal macrophages and regulate host cell innate immunity by up-regulation of various inflammatory cytokines expression. Furthermore, TLR2 and NLRP3 inflammasome signaling pathways involved in this process. This study demonstrated that GEVs could be internalized into primary mouse peritoneal macrophages, regulate host cell innate immunity via TLR2 and NLRP3 inflammasome signaling pathways, and may provide new targets against giardiasis.

Background As the three major arthropod-borne viruses, dengue virus (DENV), chikungunya virus (CHIKV), and zika virus (ZIKV) are posing a growing threat to global public health and socioeconomic development. Our study aimed to systematically review the global seroprevalences of these arboviruses from existing publications. Methods Articles published between Jan 01, 2000 and Dec 31, 2019 in the databases of Embase, Pubmed and Web of Science were searched and collected. Countries or areas with known local presence of Aedes vector mosquitoes were included. Random effects model was utilized to estimate the pooled seroprevalences and the proportion of inapparent infection. Results Out of 1375, a total of 133 articles involving 176,001 subjects were included for our analysis. The pooled seroprevalences of DENV, CHIKV and ZIKV were 38%, 25% and 18%, respectively; and their corresponding proportions of inapparent infections were 80%, 40% and 50%. The South-East Asia Region had the highest seroprevalences of DENV and CHIKV, while the Region of the Americas had the highest seroprevalence of ZIKV. The seroprevalences of DENV and CHIKV were similar when comparing developed and developing countries, urban and rural areas, or among different populations. In addition, we observed a decreased global seroprevalences in the new decade (2010–2019) comparing to the decade before (2000–2009) for CHIKV. For ZIKV, the positive rates tested with the nucleic acid detection method were lower than those tested with the antibody detection method. Lastly, numerous cases of dual seropositivity for CHIKV and DENV were reported. Conclusions Our results revealed a varied prevalence of arbovirus infections in different geographical regions and countries, and the inapparent infection accounted an unneglected portion of infections that requires more attention. This study will shed lights on our understanding of the true burden of arbovirus infections and promote appropriate vaccination in the future., Author summary DENV, CHIKV, and ZIKV infections are the three major neglected tropical diseases continually posing threats to the public health and the socioeconomic development in recent decades. Given a high fraction of inapparent infections of these viruses, it is necessary to obtain informative knowledge of the seroepidemiology of DENV, CHIKV, and ZIKV worldwide, so as to estimate the true burden of these diseases and evaluate effects on vaccine efficacy and uptake. Given the fact that the reported infection rates may vary greatly due to the measurement, criteria of inclusion, and etc, a systematic review with meta-analysis is essential to collect and summarize the seroepidemiologic evidence of these arboviruses. In this study, the geographical distributions of DENV, CHIKV, and ZIKV and the proportions of their inapparent infections were analyzed, which revealed an overall high disease burden to the world, especially in specific regions. The epidemiological features of these arboviruses were also characterized via comparisons made in different countries/areas, populations, and time periods. In addition, evidence on the co-infection of DENV and CHIKV was assessed.

Mayaro virus (MAYV) is an alphavirus endemic to South and Central America associated with sporadic outbreaks in humans. MAYV infection causes severe joint and muscle pain that can persist for weeks to months. Currently, there are no approved vaccines or therapeutics to prevent MAYV infection or treat the debilitating musculoskeletal inflammatory disease. In the current study, a prophylactic MAYV vaccine expressing the complete viral structural polyprotein was developed based on a non-replicating human adenovirus V (AdV) platform. Vaccination with AdV-MAYV elicited potent neutralizing antibodies that protected WT mice against MAYV challenge by preventing viremia, reducing viral dissemination to tissues and mitigating viral disease. The vaccine also prevented viral-mediated demise in IFN⍺R1-/- mice. Passive transfer of immune serum from vaccinated animals similarly prevented infection and disease in WT mice as well as virus-induced demise of IFN⍺R1-/- mice, indicating that antiviral antibodies are protective. Immunization with AdV-MAYV also generated cross-neutralizing antibodies against two related arthritogenic alphaviruses–chikungunya and Una viruses. These cross-neutralizing antibodies were protective against lethal infection in IFN⍺R1-/- mice following challenge with these heterotypic alphaviruses. These results indicate AdV-MAYV elicits protective immune responses with substantial cross-reactivity and protective efficacy against other arthritogenic alphaviruses. Our findings also highlight the potential for development of a multi-virus targeting vaccine against alphaviruses with endemic and epidemic potential in the Americas., Author summary Mayaro virus is an understudied alphavirus that is currently circulating in tropical environments in South and Central America without an approved vaccine. Recent outbreaks have suggested a broadening range and higher likelihood of urban outbreaks, increasing the public health risk. Mayaro virus is closely related to other arthritogenic alphaviruses with overlapping circulation such as chikungunya and Una viruses, both of which also lack clinically approved vaccines. Identification of a safe, easily manufactured, and effective strategy to vaccinate at risk populations is important to control outbreak potential. Here we report on a vaccination approach using a non-replicating adenovirus viral vector that encodes Mayaro virus structural proteins that assemble into non-infectious virus-like particles upon expression following vaccination. These particles stimulate strong immune responses against Mayaro virus. Upon testing against other alphaviruses, it was determined that the vaccine elicits cross-reactive neutralizing antibodies against chikungunya and Una viruses, significantly diminishes disease severity, and protects immunocompromised, highly susceptible mice from death following viral challenge. Our study provides new approaches to protect against these co-circulating viruses using a single vaccine. This approach is highly amenable to other virus targets for vaccine development and its ability to provide protection against chikungunya virus has global ramifications.

Crimean-Congo hemorrhagic fever virus (CCHFV) is one of the most widespread zoonotic arthropod-borne viruses in many parts of Africa, Europe and Asia. It belongs to the family of Nairoviridae in the genus of Orthonairovirus. The main reservoir and vector are ticks of the genus Hyalomma. Livestock animals (such as cattle, small ruminants and camels) develop a viremias lasting up to two weeks with absence of clinical symptoms, followed by seroconversion. This study was carried out to assess risk factors that affect seroprevalence rates in different species. In total, 928 livestock animal samples (cattle = 201; sheep = 247; goats = 233; camels = 247) from 11 out of 13 regions in Mauritania were assayed for CCHFV-specific immunoglobulin G (IgG) antibodies using enzyme-linked immunosorbent assays (ELISA) (including a novel indirect camel-IgG-specific CCHFV ELISA). Inconclusive results were resolved by an immunofluorescence assay (IFA). A generalized linear mixed-effects model (GLMM) was used to draw conclusions about the impact of certain factors (age, species, sex and region) which might have influenced the CCHFV antibody status of surveyed animals. In goats and sheep, about 15% of the animals were seropositive, whereas in cattle (69%) and camels (81%), the prevalence rate was significantly higher. On average, cattle and camels were up to twice to four times older than small ruminants. Interestingly, the seroprevalence in all species was directly linked to the age of the animals, i.e. older animals had significantly higher seroprevalence rates than younger animals. The highest CCHFV seroprevalence in Mauritania was found in camels and cattle, followed by small ruminants. The large proportion of positive animals in cattle and camels might be explained by the high ages of the animals. Future CCHFV prevalence studies should at least consider the age of surveyed animals in order to avoid misinterpretations., Author summary Crimean-Congo hemorrhagic fever virus (CCHFV) is a silent threat that repeatedly causes severe hemorrhagic disease in humans who have been in close contact with livestock of endemic countries. The detection of CCHFV IgG antibodies in livestock can be a first indication whether the virus circulates in a given region and is thus a valuable diagnostic tool for determining the endemic status. Interestingly, earlier data from Mauritania showed a noticeable difference in IgG prevalence between sheep (18%) and cattle (67%). In contrast to sheep and cattle, current monitoring data on CCHFV IgG antibody presence in camels and goats in Mauritania is very limited. This study was conducted to provide a comprehensive up-to-date overview of CCHFV seroprevalences in the four most important Mauritanian livestock species (cattle, sheep, goats and camels). It attempts to highlight the role of potential risk factors responsible for deviating prevalences. In addition, we developed a camel-specific IgG ELISA, which can be used in future CCHFV seroprevalence studies. Furthermore, findings of this study contribute to a better understanding of the current epidemiological CCHFV situation in sub-Saharan Africa and which role different livestock species play regarding the viral circulation in endemic regions.

Background Pteropine orthoreovirus (PRV) is an emerging bat-borne zoonotic virus that causes severe respiratory illness in humans. Although PRVs have been identified in fruit bats and humans in Australia and Asia, little is known about the prevalence of PRV infection in Africa. Therefore, this study performed an PRV surveillance in fruit bats in Zambia. Methods Egyptian fruit bats (Rousettus aegyptiacus, n = 47) and straw-colored fruit bats (Eidolon helvum, n = 33) captured in Zambia in 2017–2018 were screened for PRV infection using RT-PCR and serum neutralization tests. The complete genome sequence of an isolated PRV strain was determined by next generation sequencing and subjected to BLAST and phylogenetic analyses. Replication capacity and pathogenicity of the strain were investigated using Vero E6 cell cultures and BALB/c mice, respectively. Results An PRV strain, tentatively named Nachunsulwe-57, was isolated from one Egyptian fruit bat. Serological assays demonstrated that 98% of sera (69/70) collected from Egyptian fruit bats (n = 37) and straw-colored fruit bats (n = 33) had neutralizing antibodies against PRV. Genetic analyses revealed that all 10 genome segments of Nachunsulwe-57 were closely related to a bat-derived Kasama strain found in Uganda. Nachunsulwe-57 showed less efficiency in viral growth and lower pathogenicity in mice than another PRV strain, Miyazaki-Bali/2007, isolated from a patient. Conclusions A high proportion of Egyptian fruit bats and straw-colored fruit bats were found to be seropositive to PRV in Zambia. Importantly, a new PRV strain (Nachunsulwe-57) was isolated from an Egyptian fruit bat in Zambia, which had relatively weak pathogenicity in mice. Taken together, our findings provide new epidemiological insights about PRV infection in bats and indicate the first isolation of an PRV strain that may have low pathogenicity to humans., Author summary Pteropine orthoreovirus (PRV) is a causative agent of acute respiratory illness in humans in tropical and sub-tropical regions in Southeast Asia. PRVs have been originally isolated from fruit bats, and it is assumed that PRVs spread to humans by both bat-to-human and human-to-human transmission. Recently, an PRV was also detected from a fruit bat in the Afrotropical region and might potentially cause an emerging infection of the bat-borne zoonotic virus in Africa. However, little is known about the prevalence of PRV infection in Africa. In this study, we demonstrated the high prevalence of PRV infection in bat populations in Zambia and isolated a new strain of PRV from Egyptian fruit bats. In addition, we found that the bat-derived PRV strain had lower pathogenicity in mice than a human-derived PRV strain isolated from a patient in Southeast Asia. Our findings provide new epidemiological information about PRV in fruit bats in the Afrotropical region and indicate the first isolation of an PRV strain that may cause attenuated infection in humans.

Ebola virus (EBOV) is one of the most virulent pathogens that causes hemorrhagic fever and displays high mortality rates and low prognosis rates in both humans and nonhuman primates. The post-exposure antibody therapies to prevent EBOV infection are considered effective as of yet. However, owing to the poor thermal stability of mammalian antibodies, their application in the tropics has remained limited. Therefore, a thermostable therapeutic antibody against EBOV was developed modelled on the poultry(chicken) immunoglobulin Y (IgY). The IgY antibodies retaining their neutralising activity at 25°C for one year, displayed excellent thermal stability, opposed to conventional polyclonal antibodies (pAbs) or monoclonal antibodies (mAbs). Laying hens were immunised with a variety of EBOV vaccine candidates and it was confirmed that VSVΔG/EBOVGP encoding the EBOV glycoprotein could induce high titer neutralising antibodies against EBOV. The therapeutic efficacy of immune IgY antibodies in vivo was evaluated in the newborn Balb/c mice who have been challenged with the VSVΔG/EBOVGP model. Mice that have been challenged with a lethal dose of the pseudovirus were treated 2 or 24 h post-infection with different doses of anti-EBOV IgY. The group receiving a high dose of 106 NAU/kg (neutralising antibody units/kilogram) showed complete protection with no symptoms of a disease, while the low-dose group was only partially protected. Conversely, all mice receiving naive IgY died within 10 days. In conclusion, the anti-EBOV IgY exhibits excellent thermostability and protective efficacy. Anti-EBOV IgY shows a lot of promise in entering the realm of efficient Ebola virus treatment regimens., Author summary Despite the amount of efficient Ebola virus therapeutic antibodies reported in recent years, their application in tropical endemic areas has remained limited due to the low thermal stability of mammalian antibodies. A highly thermostable therapeutic polyclonal antibody against EBOV was developed based on chicken immunoglobulin Y (IgY). The EBOV specific IgY antibodies displayed excellent thermal stability, retaining their neutralising activity at 25°C for one year. The newborn mice receiving the passive transfer of IgY achieved complete protection against a lethal dose of virus challenge proving that the anti-EBOV IgY provides a promising recourse to solve some of the current clinical application hindrances of Ebola antibody-based treatments in Africa due to thermal stability.

Background Scrub typhus is a dominant cause of febrile illness in many parts of Asia. Immunity is limited by the great strain diversity of Orientia tsutsugamushi. It is unclear whether previous infection protects from severe infection or enhances the risk. Methods/principal findings We studied IgG antibody levels against O. tsutsugamushi at presentation in 636 scrub typhus patients using enzyme-linked immunosorbent assays (ELISA). The association between ELISA optical density (OD) and risk of severe infection was modelled using Poisson regression. OD was categorised as low (, Author summary In some viral infections, repeat infection is associated with a higher risk of severe disease than the first infection. Clear clinical and epidemiological evidence for this effect only exists for dengue virus infection, but it may also play a role in non-viral infections. Scrub typhus is caused by intracellular bacteria of the Orientia genus, and is among the most common causes of febrile illness in many parts of Asia. Despite being common and potentially fatal, it remains a neglected and under-diagnosed infection. Due to the great strain diversity of Orientia species (especially O. tsutsugamushi) people can experience multiple infections during life. In this study of 636 scrub typhus cases from South India, we found elevated initial IgG antibody levels, possibly from an earlier infection, to be strongly associated with severe disease. The effect was especially pronounced in younger patients and at moderately elevated antibody levels. The findings may be explained by severe infection eliciting an accelerated IgG response or, as in dengue fever, by previous scrub typhus infection enhancing the risk of severe subsequent episodes.

Salmonella and Shigella bacteria are food- and waterborne pathogens that are responsible for enteric infections in humans and are still the major cause of morbidity and mortality in the emerging countries. The existence of multiple Salmonella and Shigella serotypes as well as the emergence of strains resistant to antibiotics requires the development of broadly protective therapies. Recently, the needle tip proteins of the type III secretion system of these bacteria were successfully utilized (SipD for Salmonella and IpaD for Shigella) as vaccine immunogens to provide good prophylactic cross-protection in murine models of infections. From these experiments, we have isolated a cross-protective monoclonal antibody directed against a conserved region of both proteins. Its conformational epitope determined by Deep Mutational Scanning is conserved among needle tip proteins of all pathogenic Shigella species and Salmonella serovars, and are well recognized by this antibody. Our study provides the first in vivo experimental evidence of the importance of this common region in the mechanism of virulence of Salmonella and Shigella and opens the way to the development of cross-protective therapeutic agents., Author summary Salmonella and Shigella are responsible for gastrointestinal diseases and continue to remain a serious health hazard in South and South-East Asia and African countries, even more with the new emergence of multi drug resistances. Developed vaccines are either not commercialized (for Shigella) or cover only a limited number of serotypes (for Salmonella). There is thus a crucial need to develop cross-protective therapies. By targeting proteins SipD and IpaD belonging respectively to the injectisome of Salmonella and Shigella and necessary to their virulence, we have shown that a monoclonal antibody (mAb) directed against a conserved common region of their apical part provides good cross-protection prophylactic efficacy. We have determined the region targeted by this mAb which could explain why it is conserved among Salmonella and Shigella bacteria.

Crimean-Congo Hemorrhagic Fever Virus (CCHFV) is a geographically widespread tick-borne arbovirus that has been recognized by the WHO as an emerging pathogen needing urgent attention to ensure preparedness for potential outbreaks. Therefore, availability of accurate diagnostic tools for identification of acute cases is necessary. A panel comprising 121 sequential serum samples collected during acute, convalescent and subsided phase of PCR-proven CCHFV infection from 16 Kosovar patients was used to assess sensitivity. Serum samples from 60 healthy Kosovar blood donors were used to assess specificity. All samples were tested with two IgM/IgG immunofluorescence assays (IFA) from BNITM, the CCHFV Mosaic 2 IgG and IgM indirect immunofluorescence tests (IIFT) from EUROIMMUN, two BlackBox ELISAs for the detection of CCHFV-specific IgM and IgG antibodies (BNITM), two Anti-CCHFV ELISAs IgM and IgG from EUROIMMUN using recombinant structural proteins of CCHFV antigens, and two ELISAs from Vector-Best (IgM: μ-capture ELISA, IgG: indirect ELISA using immobilized CCHFV antigen). Diagnostic performances were compared between methods using sensitivity, specificity, concordance and degree of agreement with particular focus on the phase of the infection. In early and convalescent phases of infection, the sensitivities for detecting specific IgG antibodies differed for the ELISA test. The BlackBox IgG ELISA yielded the highest, followed by the EUROIMMUN IgG ELISA and finally the VectorBest IgG ELISA with the lowest sensitivities. In the subsided phase, the VectorBest IgM ELISA detected a high rate of samples that were positive for anti-CCHFV IgM antibodies. Both test systems based on immunofluorescence showed an identical sensitivity for detection of anti-CCHFV IgM antibodies in acute and convalescent phases of infection. Available serological test systems detect anti-CCHFV IgM and IgG antibodies accurately, but their diagnostic performances vary with respect to the phase of the infection., Author summary The Crimean-Congo Hemorrhagic Fever Virus (CCHFV) is the geographically most widespread tick-borne arbovirus and it is endemic in several countries. Because of its high case fatality rates, its potential for nosocomial outbreaks and the difficulties in treatment and prevention, CCHFV has been included in the WHO’s R&D Blueprint for Action to Prevent Epidemics. For fast implementation of infection control measures in CCHFV-affected countries, reliable diagnosis utilizing qualified molecular and serological tests is essential. Here we compared diagnostic performances of ten serological tests based on detection of IgM and IgG antibodies against CCHFV using samples from CCHFV patients and samples from healthy blood donors collected in Kosovo. The analyses focused on the phase of infection, because IgM and IgG tests have differing intended purposes. We concluded that the tests are suitable for accurate detection of anti-CCHFV IgM and IgG antibodies, but varied in their respective diagnostic performances with respect to the phase of the infection. IgM tests are mainly intended to support the diagnosis of acute infections and performed well in the early and convalescent phases of infection. IgG tests become relevant at later stages of disease progression and for disease surveillance and reached highest sensitivities in the subsided phase of infection.

The global spread of Zika virus (ZIKV), which caused a pandemic associated with Congenital Zika Syndrome and neuropathology in newborns and adults, prompted the pursuit of a safe and effective vaccine. Here, three kinds of recombinant rabies virus (RABV) encoding the prM-E protein of ZIKV were constructed: ZI-D (prM-E), ZI-E (transmembrane domain (TM) of prM-E replaced with RABV G) and ZI-F (signal peptide and TM domain of prM-E replaced with the region of RABV G). When the TM of prM-E was replaced with the region of RABV G (termed ZI-E), it promoted ZIKV E protein localization on the cell membrane and assembly on recombinant viruses. In addition, the change in the signal peptide with RABV G (termed ZI-F) was not conducive to foreign protein expression. The immunogenicity of recombinant viruses mixed with a complex adjuvant of ISA 201 VG and poly(I:C) was tested in BALB/c mice. After immunization with ZI-E, the anti-ZIKV IgG antibody lasted for at least 10 weeks. The titers of neutralizing antibodies (NAbs) against ZIKV and RABV at week 6 were all greater than the protective titers. Moreover, ZI-E stimulated the proliferation of splenic lymphocytes and promoted the secretion of cytokines. It also promoted the production of central memory T cells (TCMs) among CD4+/CD8+ T cells and stimulated B cell activation and maturation. These results indicate that ZI-E could induce ZIKV-specific humoral and cellular immune responses, which have the potential to be developed into a promising vaccine for protection against both ZIKV and RABV infections., Author summary There is no approved vaccine for Zika virus (ZIKV) disease. Many research efforts are ongoing, e.g., inactivated, DNA, or viral vector vaccines. However, due to the complexity of the pathogenesis and immunology of ZIKV, new ideas about vaccine development still need to be considered. Rabies virus (RABV) vectored vaccines have been developed for many viruses, such as against Lassa virus, canine distemper virus, Middle East respiratory syndrome coronavirus, and filovirus, based on the advantages of the vector. In this study, three recombinant RABVs expressing ZIKV prM-E, named ZI-D, ZI-E and ZI-F, are described. Since ZI-D and ZI-E could express foreign proteins successfully, the author evaluated the immunogenicity of ZI-D and ZI-E mixed with a complex adjuvant of ISA 201 VG and poly(I:C) in BALB/c mice. The study demonstrates that ZI-E induced mice to produce NAbs against both RABV and ZIKV and elicited specific cellular immune responses. The authors believe that the ZI-E vaccine based on the RABV vector has the potential to prevent ZIKV and RABV infections. It has the potential to be used in ZIKV-RABV binary vaccines in areas where both ZIKV and RABV are threats.

Background Reports have shown correlations between the immune response to vector saliva and Leishmaniasis outcome. We followed dogs in an endemic area for two years characterizing resistance or susceptibility to canine visceral leishmaniasis (CVL) according to Leishmania infantum diagnosis and clinical development criteria. Then, we aimed to identify a biosignature based on parasite load, serum biological mediators’ interactions, and vector exposure intensity associated with CVL resistance and susceptibility. Methodology/Principal findings A prospective two-year study was conducted in an area endemic for CVL. Dogs were evaluated at 6-month intervals to determine infection, clinical manifestations, immune profile, and sandfly exposure. CVL resistance or susceptibility was determined upon the conclusion of the study. After two years, 78% of the dogs were infected with L. infantum (53% susceptible and 47% resistant to CVL). Susceptible dogs presented higher splenic parasite load as well as persistence of the parasite during the follow-up, compared to resistant ones. Susceptible dogs also displayed a higher number of correlations among the investigated biological mediators, before and after infection diagnosis. At baseline, anti-saliva antibodies, indicative of exposure to the vector, were detected in 62% of the dogs, reaching 100% in one year. Higher sandfly exposure increased the risk of susceptibility to CVL by 1.6 times (CI: 1.11–2.41). We identified a discriminatory biosignature between the resistant and susceptible dogs assessing splenic parasite load, interaction of biological mediators, PGE2 serum levels and intensity of exposure to sandfly. All these parameters were elevated in susceptible dogs compared to resistant animals. Conclusions/Significance The biosignature identified in our study reinforces the idea that CVL is a complex multifactorial disease that is affected by a set of factors which are correlated and, for a better understanding of CVL, should not be evaluated in an isolated way., Author summary Visceral Leishmaniasis (VL) is a disease that can affect humans and dogs, caused by a parasite called Leishmania transmitted through the bite of sandfly insects. During the bite, together with the parasite, the insects also inoculate their saliva into the host. The host immune response produces molecules to the sandfly saliva, such as antibodies and cytokines that can impact VL resistance or susceptibility. The presence of these molecules also indicates if the insects bit the hosts. We followed dogs of a VL endemic area for two years to study Canine Visceral Leishmaniasis (CVL) and immune response to sandfly saliva. Dogs were evaluated at 6-month intervals to determine Leishmania infection, clinical manifestations, parasite load, immune response, and sandfly exposure. CVL resistance or susceptibility was determined upon the conclusion of the study. Dogs living in the endemic area were intensely bitten, as at the beginning of the study, 62% of the dogs present anti-saliva antibodies, reaching 100% after one year. Our findings revealed a biosignature of CVL susceptibility characterized by elevated parasite load, interaction of cytokines, and higher exposure to the sandfly. This data reinforced that CVL is a complex disease affected by several factors related to each other.

Dengue hemorrhagic fever (DHF) is caused by infection with dengue virus (DENV). Four different serotypes (DENV1-4) co-circulate in dengue endemic areas. The viral RNA genome-based reverse-transcription PCR (RT-PCR) is the most widely used method to identify DENV serotypes in patient specimens. However, the non-structural protein 1 (NS1) antigen as a biomarker for DENV serotyping is an emerging alternative method. We modified the serotyping-NS1-enzyme linked immunosorbent assay (stNS1-ELISA) from the originally established assay which had limited sensitivity overall and poor specificity for the DENV2 serotype. Here, four biotinylated serotype-specific antibodies were applied, including an entirely new design for detection of DENV2. Prediction of the infecting serotype of retrospective acute-phase plasma from dengue patients revealed 100% concordance with the standard RT-PCR method for all four serotypes and 78% overall sensitivity (156/200). The sensitivity of DENV1 NS1 detection was greatly improved (from 62% to 90%) by the addition of a DENV1/DENV3 sub-complex antibody pair. Inclusive of five antibody pairs, the stNS1-ELISA (plus) method showed an overall increased sensitivity to 85.5% (171/200). With the same clinical specimens, a commercial NS1 rapid diagnostic test (NS1-RDT) showed 72% sensitivity (147/200), significantly lower than the stNS1-ELISA (plus) performance. In conclusion, the stNS1-ELISA (plus) is an improved method for prediction of DENV serotype and for overall sensitivity. It could be an alternative assay not only for early dengue diagnosis, but also for serotype identification especially in remote resource-limited dengue endemic areas., Author summary Four serotypes of DENV co-circulate in dengue endemic areas. Secondary infection with a different DENV serotype is beleived to involve with severe dengue disease. Standard laboratory diagnosis to identify DENV serotypes in dengue patient specimens is performed by sophisticated genome-based RT-PCR method with serotype-specific oligoprimers. We have previously established an alternative protein-based NS1 assay for DENV serotyping namely, a serotyping-NS1-ELISA (stNS1-ELISA), with the use of serotype-specific monoclonal antibodies (Mabs) to NS1 protein. Due to its unsatisfactory performance, the stNS1-ELISA was modified in this study. The biotinylated serotype-specific detection Mabs were introduced to enhance the overall sensitivity. A new DENV2-specific antibody was applied to improve DENV serotype identification. Prediction of infecting serotype from NS1-positive samples by our modified assay was 100% concordant with the standard RT-PCR method for all four serotypes. The overall sensitivity was greatly improved by an additional DENV1/DENV3 sub-complex antibody. This modified assay is efficient not only for early dengue diagnosis, but also for serotype identification in epidemiological studies and disease surveillance.

Background Pulmonary sequelae (PS) in patients with chronic paracoccidioidomycosis (PCM) typically include pulmonary fibrosis and emphysema. Knowledge of the molecular pathways involved in PS of PCM is required for treatment and biomarker identification. Methodology/Principal findings This non-concurrent cohort study included 29 patients with pulmonary PCM that were followed before and after treatment. From this group, 17 patients evolved to mild/ moderate PS and 12 evolved severe PS. Sera from patients were evaluated before treatment and at clinical cure, serological cure, and apparent cure. A nanoACQUITY UPLC-Xevo QT MS system and PLGS software were used to identify serum differentially expressed proteins, data are available via ProteomeXchange with identifier PXD026906. Serum differentially expressed proteins were then categorized using Cytoscape software and the Reactome pathway database. Seventy-two differentially expressed serum proteins were identified in patients with severe PS compared with patients with mild/moderate PS. Most proteins altered in severe PS were involved in wound healing, inflammatory response, and oxygen transport pathways. Before treatment and at clinical cure, signaling proteins participating in wound healing, complement cascade, cholesterol transport and retinoid metabolism pathways were downregulated in patients with severe PS, whereas signaling proteins in gluconeogenesis and gas exchange pathways were upregulated. At serological cure, the pattern of protein expression reversed. At apparent cure pathways related with tissue repair (fibrosis) became downregulated, and pathway related oxygen transport became upregulated. Additionally, we identified 15 proteins as candidate biomarkers for severe PS. Conclusions/Significance Development of severe PS is related to increased expression of proteins involved in glycolytic pathway and oxygen exchange), indicative of the greater cellular activity and replication associated with early dysregulation of wound healing and aberrant tissue repair. Our findings provide new targets to study mechanisms of PS in PCM, as well as potential biomarkers., Author summary Pulmonary fibrosis is the main sequel of paracoccidioidomycosis (PCM), a fungal disease that affects mainly men, rural workers. The development of pulmonary fibrosis is complex and involves several mechanisms that culminate in aberrant collagen production and deposition in the lungs making it became stiff and blocking the air passages. These changes lead to difficulty in breathing and in PCM patients dyspnea in response to high or low levels of exertion is common. Therefore, these patients show incapacity to work and the decreased quality of life. With the possibility of identifying some marker, for example, it could help the indication of respiratory physiotherapy, professional rehabilitation, or therapeutic intervention. This is the first study to examine the pulmonary sequelae (PS) in patients with paracoccidioidomycosis using an approach combining proteomics with bioinformatics. Here, we identify the specific proteome pattern found in PCM patients with severe sequelae that distinguishes these patients from that with mild/moderate sequelae. Our results showed that time points immediately before treatment and at clinical cure are key moments at which PS can progress to severe PS due a dysregulation in wound healing with consequent delayed in the healing processes resulting in an aberrant scar. As such, we suggest that the prognoses for severe PS should be considered as soon as possible and as early as diagnosis of PCM. Furthermore, we used proteomics to identify possible serum biomarkers with which to predict the likely development of severe PS, to be validated in future studies.

Background Thirty-four CCHF cases (17 fatal; 17 survived) were confirmed from Gujarat state, India during the year 2019. We aimed to find out the viral load, antibody kinetics, cytokine profile and phylogenetic analysis between fatal and non- fatal cases. Methods Thirty four cases were included in this study. Blood and urine samples were collected from all the cases on the day of admission to hospital. Non-fatal cases were followed weekly for understanding the profile of viral kinetics, anti-CCHFV IgM and IgG antibodies. We also quantified the cytokines in both fatal and non-fatal cases. For epidemiological correlation, livestock were screened for anti-CCHF IgG antibodies and the tick pool specimens were tested by real time RT-PCR. Virus isolation was attempted on tick pools and human specimens and phylogenetic analysis performed on human and ticks complete genome sequences. Results CCHF cases were detected throughout year in 2019 with the peak in August. Out of 34 cases, eight secondary CCHF cases were reported. Cases were predominantly detected in males and in 19–45 years age group (55.88%). The persistence of viremia was observed till 76th POD (post onset date) in one case whereas anti-CCHFV IgM and IgG was detected amongst these cases from the 2nd and 20th POD respectively. Positivity observed amongst livestock and tick pools were was 21.57% and 7.4% respectively. The cytokine analysis revealed a significant increase in the level of serum IL-6, IL-10 and IFN-γ during the acute phase of the infection, but interestingly IL-10 lowered to normal upon clearance of the virus in the clinically recovered case. Fatal cases had high viral RNA copy numbers. Bleeding from one or two mucosal sites was significantly associated with fatality (OR-16.47;p-0.0034 at 95% CI). We could do CCHF virus isolation from two cases. Phylogenetic analysis revealed circulation of re-assortment of Asian-West African genotypes in humans and ticks. Conclusions The persistence of CCHF viral RNA was detected till 76th POD in one of the survivors. The circulation of a re-assortment Asian-West African genotype in a CCHF case is also reported first time from India., Author summary Crimean Congo hemorrhagic fever is a zoonotic tick-borne viral hemorrhagic disease. This disease is reported from Europe, Mediterranean, north-western China, central Asia, Africa, and the Middle East. Several outbreaks of CCHF were reported from Gujarat and Rajasthan states, India from 2011 to 2019. In this study, we discuss the clinical, molecular, serological, and the cytokine data of 34 CCHF cases (17 fatal and 17 survived) which were detected from Gujarat state in the year 2019. A sequential weekly follow up of the CCHF survivors was performed to understand the viral kinetics and the antibody profile. Interestingly, the presence of persistence CCHF viral RNA was observed till 76th POD in one of the survivors. To our knowledge, we are reporting this long term persistence of viremia for the first time. We also observed that the anti-CCHFV IgM detection in the serum samples starts as soon as 2nd POD but anti-CCHFV IgG antibody could be detected in the majority of the cases only after the 28th POD. The cytokine analysis revealed a significant increase in the level of serum IL-6, IL-10 and IFN-γ during the acute phase of the infection, but interestingly IL-10 lowered to normal upon clearance of the virus in the clinically recovered case. We did the phylogenetic analysis and concluded the circulation of the Asian-West African re-assortment genotype in humans, which has not been reported from India prior to this study.

Background Brucellosis, as a serious zoonotic infectious disease, has been recognized as a re-emerging disease in the developing countries worldwide. In china, the incidence of brucellosis is increasing each year, seriously threatening the health of humans as well as animal populations. Despite a quite number of diagnostic methods currently being used for brucellosis, innovative technologies are still needed for its rapid and accurate diagnosis, especially in area where traditional diagnostic is unavailable. Methodology/Principal findings In this study, a total of 22 B cell linear epitopes were predicted from five Brucella outer membrane proteins (OMPs) using an immunoinformatic approach. These epitopes were then chemically synthesized, and with the method of indirect ELISA (iELISA), each of them displayed a certain degree of capability in identifying human brucellosis positive sera. Subsequently, a fusion protein consisting of the 22 predicted epitopes was prokaryotically expressed and used as diagnostic antigen in a newly established brucellosis testing method, nano-ZnO modified paper-based ELISA (nano-p-ELISA). According to the verifying test using a collection of sera collected from brucellosis and non-brucellosis patients, the sensitivity and specificity of multiepitope based nano-p-ELISA were 92.38% and 98.35% respectively. The positive predictive value was 98.26% and the negative predictive value was 91.67%. The multiepitope based fusion protein also displayed significantly higher specificity than Brucella lipopolysaccharide (LPS) antigen. Conclusions B cell epitopes are important candidates for serologically testing brucellosis. Multiepitope fusion protein based nano-p-ELISA displayed significantly sensitivity and specificity compared to Brucella LPS antigen. The strategy applied in this study will be helpful to develop rapid and accurate diagnostic method for brucellosis in human as well as animal populations., Author summary Brucellosis is one of the most important zoonosis in the world and has caused tremendous economic losses in agriculture and animal husbandry in many countries. Developing rapid, sensitive and specific diagnostic methods is very important for early detection and treatment of brucellosis patients. In this study, a novel diagnostic technique, nano-ZnO modified paper ELISA, was established. The antigen used in this technique was a fusion protein containing multiple B cell epitopes, which were predicted from Brucella major outer membrane proteins such as Bp26, Omp31, Omp16, Omp2b and Omp25. Comparing to traditional LPS antigen, this multiepitope based antigen displayed considerably higher sensitivity and higher specificity in laboratory. With the strategy described in this paper, more efficient epitopes and protein antigen can be identified in the future. Currently, LPS antigen is only prepared from live Brucella, while protein antigen can be produced in large quantities in prokaryotic expression system. In addition to nano-p-ELISA, this protein antigen can also be used for development other methods such as fluorescent polarization assay (FPA) and immunochromatographic assay (ICA) to meet the varied demand for brucellosis testing.

Chronic Chagas disease cardiomyopathy (CCC), an especially aggressive inflammatory dilated cardiomyopathy caused by lifelong infection with the protozoan Trypanosoma cruzi, is a major cause of cardiomyopathy in Latin America. Although chronic myocarditis may play a major pathogenetic role, little is known about the molecular mechanisms responsible for its severity. The aim of this study is to study the genes and microRNAs expression in tissues and their connections in regards to the pathobiological processes. To do so, we integrated for the first time global microRNA and mRNA expression profiling from myocardial tissue of CCC patients employing pathways and network analyses. We observed an enrichment in biological processes and pathways associated with the immune response and metabolism. IFNγ, TNF and NFkB were the top upstream regulators. The intersections between differentially expressed microRNAs and differentially expressed target mRNAs showed an enrichment in biological processes such as Inflammation, inflammation, Th1/IFN-γ-inducible genes, fibrosis, hypertrophy, and mitochondrial/oxidative stress/antioxidant response. MicroRNAs also played a role in the regulation of gene expression involved in the key cardiomyopathy-related processes fibrosis, hypertrophy, myocarditis and arrhythmia. Significantly, a discrete number of differentially expressed microRNAs targeted a high number of differentially expressed mRNAs (>20) in multiple processes. Our results suggest that miRNAs orchestrate expression of multiple genes in the major pathophysiological processes in CCC heart tissue. This may have a bearing on pathogenesis, biomarkers and therapy., Author summary Chronic Chagas disease cardiomyopathy (CCC), an aggressive dilated cardiomyopathy caused by Trypanosoma cruzi, is a major cause of cardiomyopathy in Latin America. Little is known about the molecular mechanisms responsible for its severity. Authors study the possible role of microRNAs in the regulation of gene expression in relevant pathways and pathobiological processes. Differentially expressed genes (DEGs) and differentially expressed miRNAs (DEMs) -small RNAs that can regulate gene expression—associated to severe cardiomyopathy development. The inflammatory mediator Interferon-γ was the most likely inducer of gene expression in CCC, and most genes belonged to the immune response, fibrosis, hypertrophy and mitochondrial metabolism. A discrete number of differentially expressed mRNAs targeted a high number of differentially expressed mRNAs in multiple processes. Moreover, several pathways had multiple targets regulated by microRNAs, suggesting synergic effect. Results suggest that microRNAs orchestrate expression of multiple genes in the major pathophysiological processes in CCC heart tissue.

Clonorchiasis caused by Clonorchis sinensis is endemic in East Asia; approximately 15 million people have been infected thus far. To diagnose the infection, serodiagnostic tests with excellent functionality should be performed. First, 607 expressed sequence tags encoding polypeptides with a secretory signal were expressed into recombinant proteins using an in vitro translation system. By protein array-based screening using C. sinensis-infected sera, 18 antigen candidate proteins were selected and assayed for cross-reactivity against Opisthorchis viverrini-infected sera. Of the six antigenic proteins selected, four were synthesized on large scale in vitro and evaluated for antigenicity against the flukes-infected human sera using ELISA. CsAg17 antigen showed the highest sensitivity (77.1%) and specificity (71.2%). The sensitivity and specificity of the bacterially produced CsAg17-28GST fusion antigen was similar to those of CsAg17 antigen. CsAg17 antigen can be used to develop point-of-care serodiagnostic tests for clonorchiasis., Author summary Human clonorchiasis is a parasitic disease caused by the Chinese liver fluke, Clonorchis sinensis. Humans are infected through eating raw freshwater fishes carrying C. sinensis metacercariae, the encysted larvae. They excyst in the duodenum, move into the liver via bile duct and grow to adult worms. Excretory-secretory products of the worm damage the liver causing various inflammatory pathological changes and may lead to bile duct cancer. Although there exists an anthelmintic choice praziquantel to kill the fluke, emphasis is placed on early diagnosis and prevention before the infection becomes disease. Microscopic stool examination is the standard diagnostic method but is cumbersome and time consuming. Blood serum antibodies from clonorchiasis patients could provide a simple and fast diagnosis. However, antibody detecting diagnostics developed so far have a low specificity and sensitivity. In the present study we selected 607 antigenic candidate proteins from the genomic database and synthesized them through an integrated high-throughput proteogenomic tools. We identified several antigenic proteins and evaluated their diagnostic potential for clonorchiasis. One of them, CsAg17, showed a high sensitivity and specificity. This antigen deserves development of point-of-care serodiagnostics for C. sinensis infections.

The development of vaccines against flaviviruses, including Zika virus (ZIKV) and dengue virus (DENV), continues to be a major challenge, hindered by the lack of efficient and reliable methods for screening neutralizing activity of sera or antibodies. To address this need, we previously developed a plasmid-based, replication-incompetent DENV reporter virus particle (RVP) production system as an efficient and safe alternative to the Plaque Reduction Neutralization Test (PRNT). As part of the response to the 2015–2016 ZIKV outbreak, we developed pseudo-infectious ZIKV RVPs by modifying our DENV RVP system. The use of ZIKV RVPs as critical reagents in human clinical trials requires their further validation using stability and reproducibility metrics for large-scale applications. In the current study, we validated ZIKV RVPs using infectivity, neutralization, and enhancement assays with monoclonal antibodies (MAbs) and human ZIKV-positive patient serum. ZIKV RVPs are antigenically equivalent to live virus based on binding ELISA and neutralization results and are nonreplicating based on the results of live virus replication assays. We demonstrate reproducible neutralization titer data (NT50 values) across different RVP production lots, volumes, time frames, and laboratories. We also show RVP stability across experimentally relevant time intervals and temperatures. Our results demonstrate that ZIKV RVPs provide a safe, high-throughput, and reproducible reagent for large-scale, long-term studies of neutralizing antibodies and sera, which can facilitate large-scale screening and epidemiological studies to help expedite ZIKV vaccine development., Author summary ZIKV is a mosquito-borne virus that can cause severe birth defects and other disorders. Large outbreaks of ZIKV occurred in 2015 and 2016 and there are still no drugs or vaccines available to protect against ZIKV infection. Vaccine development has been hindered by the lack of safe and efficient methods to screen ZIKV neutralizing properties of patient sera or antibodies, especially in the context of large clinical trials. To address this unmet need, we developed and validated the use of ZIKV reporter virus particles (RVPs), a safe, high-throughput, and quantitative alternative to using live virus for neutralization studies. We show that ZIKV RVPs are stable, show lot-to-lot consistency, and provide reproducible neutralization data that is suitable for the large-scale studies needed for development of a ZIKV vaccine, epidemiologic surveillance, and high-throughput screening.

The 2013–2016 Ebola virus (EBOV) outbreak in West Africa and the ongoing cases in the Democratic Republic of the Congo have spurred development of a number of medical countermeasures, including rapid Ebola diagnostic tests. The likelihood of transmission increases as the disease progresses due to increasing viral load and potential for contact with others. Early diagnosis of EBOV is essential for halting spread of the disease. Polymerase chain reaction assays are the gold standard for diagnosing Ebola virus disease (EVD), however, they rely on infrastructure and trained personnel that are not available in most resource-limited settings. Rapid diagnostic tests that are capable of detecting virus with reliable sensitivity need to be made available for use in austere environments where laboratory testing is not feasible. The goal of this study was to produce candidate lateral flow immunoassay (LFI) prototypes specific to the EBOV glycoprotein and viral matrix protein, both targets known to be present during EVD. The LFI platform utilizes antibody-based technology to capture and detect targets and is well suited to the needs of EVD diagnosis as it can be performed at the point-of-care, requires no cold chain, provides results in less than twenty minutes and is low cost. Monoclonal antibodies were isolated, characterized and evaluated in the LFI platform. Top performing LFI prototypes were selected, further optimized and confirmed for sensitivity with cultured live EBOV and clinical samples from infected non-human primates. Comparison with a commercially available EBOV rapid diagnostic test that received emergency use approval demonstrates that the glycoprotein-specific LFI developed as a part of this study has improved sensitivity. The outcome of this work presents a diagnostic prototype with the potential to enable earlier diagnosis of EVD in clinical settings and provide healthcare workers with a vital tool for reducing the spread of disease during an outbreak., Author summary Ebola virus (EBOV) causes a severe hemorrhagic fever and has an extremely high fatality rate that ranges from 60%-90%. There is no approved treatment or vaccine for this infectious disease and halting spread of the virus relies on identifying and isolating infected patients quickly. The current gold standard, polymerase chain reaction assay, requires patient samples be transported to regional reference laboratories where it often takes days to get results. A handful of Ebola rapid diagnostic tests have been developed, but lack the sensitivity required to detect the virus in earlier stages of the disease. There is great need for more sensitive rapid diagnostic tests that can identify the EBOV infected patients when they first become symptomatic. This study focused on production of high affinity mAbs to two target EBOV proteins for development of a more sensitivity rapid diagnostic test. Efforts have resulted in production of prototype detecting the EBOV glycoprotein that shows a notable improvement in sensitivity and offers the potential for earlier diagnosis of infection.

Over the last decades, rickettsioses are emerging worldwide. These diseases are caused by intracellular bacteria. Although rickettsioses can be treated with antibiotics, a vaccine against rickettsiae is highly desired for several reasons. Rickettsioses are highly prevalent, especially in poor countries, and there are indications of the development of antibiotic resistance. In addition, some rickettsiae can persist and cause recurrent disease. The development of a vaccine requires the understanding of the immune mechanisms that are involved in protection as well as in immunopathology. Knowledge about these immune responses is accumulating, and efforts have been undertaken to identify antigenic components of rickettsiae that may be useful as a vaccine. This review provides an overview on current knowledge of adaptive immunity against rickettsiae, which is essential for defense, rickettsial antigens that have been identified so far, and on vaccination strategies that have been used in animal models of rickettsial infections.

Background Clonorchis sinensis, a fluke dwelling in the intrahepatic bile ducts causes clonorchiasis, which affect about 15 million people wide-distributed in eastern Asia. During C. sinensis infection, worm-host interaction results in activation of patterns recognition receptors (PRRs) such as Toll-like receptors (TLRs) and further triggers immune responses, which determines the outcome of the infection. However, the mechanisms by which pathogen-associated molecules patterns from C. sinensis interact with TLRs were poorly understood. In the present study, we assumed that the molecules from C. sinensis may regulate host immune responses via TLR2 signaling pathway. Methodology/Principal findings In the present study, we have identified a ~34 kDa CsHscB from C. sinensis which physically bound with TLR2 as demonstrated by molecular docking and pull-down assay. We also found that recombinant CsHscB (rCsHscB) potently activates macrophage to express various proteins including TLR2, CD80, MHCII, and cytokines like IL-6, TNF-α, and IL-10, but rCsHscB failed to induce IL-10 in macrophages from Tlr2-/- mice. Moreover, ERK1/2 activation was required for rCsHscB-induced IL-10 production in macrophages. In vivo study revealed that rCsHscB triggered a high production of IL-10 in the wild-type (WT) but not in Tlr2-/- mice. Consistently, the phosphorylation of ERK1/2 was also attenuated in Tlr2-/- mice compared to the WT mice, after the treatment with rCsHscB. Conclusions/Significance Our data thus demonstrate that rCsHscB from C. sinensis interacts with TLR2 to be endowed with immune regulatory activities, and may have some therapeutic implications in future beyond parasitology., Author summary Clonorchis sinensis is a kind of liver fluke whose infection can cause inflammation and, fibrosis around the bile ducts. As type I biological agents (carcinogens) Type, it can also cause bile ductal cancer. However, the mechanisms by which the parasite interacts with host cells and further induce serious sequences are poorly understood. Here, we found a~34 kDa rCsHscB from C. sinensis that can bind with TLR2, a well-known pattern recognition receptor. Importantly, we found that rCsHscB could lead to the activation of macrophage and induce a strong production of cytokines such as IL-10 and IL-6 through the TLR2-mediated signaling pathway. Besides, the animal study demonstrated that rCsHscB induce a high production of IL-10 in the wild-type (WT) but not in Tlr2-/- mice. These data showed that rCsHscB from C. sinensis interacted with TLR2 have immune-regulatory abilities, which can be used for other inflammatory disorders in the future beyond parasitology.

Infecting approximately one-third of the world’s human population, Toxoplasma gondii has been associated with cognitive function. Here, we sought to further characterize the association between Toxoplasma gondii and cognitive function in a community sample of adults aged approximately 40 to70 years. Using adjusted linear regression models, we found associations of Toxoplasma gondii seropositivity with worse reasoning (b = -.192, p < .05) and matrix pattern completion (b = -.681, p < .01), of higher anti-Toxoplasma gondii p22 antibody levels with worse reasoning (b = -.078, p < .01) and slower Trails (numeric) performance (b = 5.962, p < .05), of higher anti-Toxoplasma gondii sag1 levels with worse reasoning (b = -.081, p < .05) and worse matrix pattern completion (b = -.217, p < .05), and of higher mean of the anti-Toxoplasma gondii p22 and sag1 levels with worse reasoning (b = -.112, p < .05), slower Trails (numeric) performance (b = 9.195, p < .05), and worse matrix pattern completion (b = -.245, p < .05). Neither age nor educational attainment moderated associations between the measures of Toxoplasma gondii seropositivity or serointensity. Sex, however, moderated the association between the sag1 titer and digit-symbol substitution and the association between the mean of the p22 and sag1 levels and digit-symbol substitution, and income moderated the association between Toxoplasma gondii seropositivity and numeric memory and the association between the p22 level and symbol-digit substitution. Based on the available neuropsychological tasks in this study, Toxoplasma gondii seropositivity and serointensity were associated with some aspects of poorer executive function in adults., Author summary Toxoplasma gondii is a parasite that reproduces in cats but that can infect other animals and humans. Toxoplasma gondii has an affinity for the brain and infection results in a long-term immune response that may have negative effects. Animal models have shown that Toxoplasma gondii can affect behavior and cognition (thinking). In humans, the association is less clear, but there are associations between infection and variety of psychiatric and neurological conditions such as schizophrenia and epilepsy. Toxoplasma gondii may even increase the risk of dementia. If true, it is possible that the presence of this infection may affect thinking long before the onset of dementia. We sought to determine if the presence of antibodies in the blood to Toxoplasma gondii, which is the immune system’s response to past infections, might be associated with performance on measures of cognition. We analyzed a large dataset, the UK Biobank, which includes measures of cognition and blood test results for antibodies to Toxoplasma gondii. We found that both the presence of antibodies as well as the amount of these antibodies were related to some aspects of cognition, particularly problem solving and reasoning in otherwise healthy adults ages 40 to 69 years.

Background Despite identification 50 years ago, the true burden of Lassa Fever (LF) across Africa remains undefined for reasons including research focus on hospitalised patients, lack of validated field-feasible tools which reliably identify past infection, and the fact that all assays require blood samples making large-scale surveys difficult. Designated a priority pathogen of epidemic potential requiring urgent research by the World Health Organisation, a better understanding of LF sero-epidemiology is essential to developing and evaluating new interventions including vaccines. We describe the first field testing of a novel species-neutral Double Antigen Binding Assay (DABA) designed to detect antibodies to LF in plasma and oral fluid. Methodology/Principal findings Paired plasma and oral fluid were collected in Sierra Leone from survivors discharged from Kenema Government Hospital Lassa Fever Unit between 1980 and 2018, and from controls recruited in Freetown in 2019. Epidemiological sensitivity and specificity of the DABA measured against historical diagnosis in survivors and self-declared non-exposed controls was 81.7% (95% CI 70.7%– 89.9%) and 83.3% (72.7%- 91.1%) respectively in plasma, and 71.8% (60.0%– 81.9%) and 83.3% (72.7%– 91.1%) respectively in oral fluid. Antibodies were identified in people infected up to 15 years and, in one case, 40 years previously. Participants found oral fluid collection easy and painless with 80% happy to give an oral fluid sample regularly. Conclusions/Significance Given the difficulties of assay validation in a resource-limited setting, including unexpected exposures and diagnostics of varying accuracy, the new assay performed well in both plasma and oral fluid. Sensitivity and specificity are expected to be higher when case/control ascertainment is more definitive and further work is planned to investigate this. Even at the performance levels achieved, the species-neutral DABA has the potential to facilitate the large-scale seroprevalence surveys needed to underpin essential developments in LF control, as well as support zoonotic investigations., Author summary Lassa Fever (LF) is a life-threatening rodent-borne viral haemorrhagic disease of epidemic potential particularly affecting some West African communities. Although it is estimated to cause 100,000–300,000 infections and 5,000 deaths annually, the true burden of the disease in the community is not well understood since research has largely focussed on hospitalised cases (thought to account for ~15% of infections) because of the difficulty of identifying mild or asymptomatic cases in the community. Gaining a better understanding of LF epidemiology, particularly the exposure and immunity status of populations in countries where the virus is endemic in the rodent hosts, is critical to developing control measures including vaccines which can protect against disease and/or reduce risk of epidemic transmission. A key step is to be able to identify who has been previously infected by virus, and therefore likely immune, and who remains susceptible by examining the prevalence of antibodies to the virus in the community. However, to date all tests require blood to be drawn which can be problematic for community participation as well as risky for collectors. This study reports on a project to develop and validate an antibody test that can be used with an easy-to-give oral fluid sample.

Background Chikungunya (CHIK) and yellow fever (YF) are becoming major public health threats in East African countries including Ethiopia. In Ethiopia, there is no reliable information about the epidemiology of CHIK. This study aimed to assess a community-based sero-prevalence of CHIK and YF in the South Omo Valley, an endemic area for YF. Methods Between February and June 2018, blood samples were collected from study participants and screened for IgG antibody against CHIK virus (CHIKV) and YF virus (YFV) infections using ELISA. Data were computerized using Epi Data Software v.3.1 and analyzed using SPSS. Results A total of 360 participants (51.7% males, age range from 6 to 80, mean age ± SD = 31.95 ± 14.05 years) participated in this study. The overall sero-prevalence of IgG antibody was 43.6% (157/360) against CHIKV, while it was 49.5% (155/313) against YFV. Out of 155 samples which were positive for IgG antibody to YFV, 93 (60.0%) were positive for IgG antibody to CHIKV. Out of 158 samples which were negative for IgG antibody to YFV, 64(40.5%) were positive for IgG antibody to CHIKV. There was a significant positive correlation between IgG antibodies to CHIKV and YFV (sr = 0.82; P, Author summary Mosquito-borne viral diseases including yellow fever and chikungunya are becoming major public health problem in Africa. Community-based sero-epidemiological studies of mosquito-borne viral infections are important to known the occurrence of these diseases and to design appropriate prevention and control strategies. In this study, we assessed the sero-prevalence of IgG antibody against Chikungunya virus and Yellow fever virus in 360 study participants and associated risk factors among the community members of South Omo, Southern Ethiopia. Our study showed a sero-prevalence of IgG antibody; 43.6%, (157 out of 360) to Chikungunya virus, and 49.5% (155 out of 313) against Yellow fever virus in the study participants.Further studies on active case detection of chikungunya and raising awareness, advocating policies to mitigate the risk of arboviral infections have paramount importance in the present study area.

Novel diagnostic tools are a major challenge for brucellosis research, especially in developing countries. Herein, we established a handheld quantum dot (QD) immunochromatographic device for the fast detection of brucellosis antibodies in the field. Total bacterial protein extracted from Brucella 104M served as labelling and coating antigen. QD labelling and immunochromatography methods were used to optimise reaction conditions, labelling conditions, reaction temperature and storage temperature. QD test strips were employed to test brucellosis serum to determine their sensitivity, specificity and stability. Test strips were compared with Rose Bengal test, standard agglutination test and colloidal gold immunochromatographic assay. Labelled Brucella total protein displayed good specificity and no cross-reactivity. The concentration of labelled total bacterial protein was 3.9 mg/ml, the coating concentration was 2.0 mg/ ml, and the serum titre with the lowest detection sensitivity was 1:25. The optimal reaction temperature for the test strip was 25−30°C. The test strip was stable after storage at room temperature and the repeatability was high, with a coefficient of variation of 4.0%. After testing 199 serum samples, the sensitivity of the QD test strip was 98.53%, the specificity was 93.57%, and the coincidence rate with the standard agglutination test was 96.98%. The developed QD immunochromatographic method can be used for rapid detection and preliminary screening of brucellosis in the field., Author summary Brucellosis is a neglected infection that has a widespread geographic distribution. Based on an evaluation from the World Health Organization (WHO), brucellosis cases have been reported in more than 170 countries with about 500,000 new cases being reported each year. However, the actual number of brucellosis patients is much higher, and it is believed to be approximately 10–25 times the number of reported cases. This big discrepancy between the reported rate and the actual incidence rate is largely due to misdiagnosis and underdiagnosis, especially in endemic areas. In recent years, considerable effort has been mobilized toward the development of rapid, reliable field diagnostic assays and molecular diagnostic approaches. In this study, we established a handheld quantum dot (QD) immunochromatographic device for the fast detection of brucellosis antibodies in the field. This QD test strip does not require extensive laboratory infrastructure or technical expertise, and has high sensitivity (98.53%) and specificity (93.57%), as well as a high coincidence rate (96.98%) with SAT. This new tool is expected to provide fast and simple point-of-care testing at county-level clinics and CDC labs.

Environmental enteric dysfunction (EED) is an intestinal disorder common among children in low-resource settings and is associated with increased risk of growth stunting, cognitive deficits, and reduced oral vaccine immunogenicity. The Micronutrient and EED Assessment Tool (MEEDAT) is a multiplexed immunoassay that measures biomarkers previously associated with child growth faltering and/or oral vaccine immunogenicity: intestinal fatty acid–binding protein (I-FABP), soluble CD14 (sCD14), insulin-like growth factor 1 (IGF-1), and fibroblast growth factor 21 (FGF21). MEEDAT also measures systemic inflammation (α1-acid glycoprotein, C-reactive protein), ferritin, soluble transferrin receptor, retinol binding protein 4, thyroglobulin, and Plasmodium falciparum antigenemia (histidine-rich protein 2). The performance of MEEDAT was compared with commercially available enzyme-linked immunosorbent assays (ELISAs) using 300 specimens from Malian infant clinical trial participants. Regression methods were used to test if MEEDAT biomarkers were associated with seroconversion to meningococcal A conjugate vaccine (MenAV), yellow fever vaccine (YFV), and pentavalent rotavirus vaccine (PRV) after 28 days, or with growth faltering over 12 weeks. The Pearson correlations between the MEEDAT and ELISA results were 0.97, 0.86, 0.80, and 0.97 for serum I-FABP, sCD14, IGF-1, and FGF21, respectively. There were significant associations between I-FABP concentration and the probability of PRV IgG seroconversion and between IGF-1 concentration and the probability of YFV seroconversion. In multivariable models neither association remained significant, however there was a significant negative association between AGP concentration and YFV seroconversion. GLP-2 and sCD14 concentrations were significantly negatively associated with 12-week change in weight-for-age z-score and weight-for-height z-score in multivariable models. MEEDAT performed well in comparison to commercially-available ELISAs for the measurement of four analytes for EED and growth hormone resistance. Adoption of MEEDAT in low-resource settings could help accelerate the identification of interventions that prevent or treat child stunting and interventions that boost the immunogenicity of child vaccinations., Author summary Environmental enteric dysfunction (EED) is an intestinal disorder common among children in low-resource settings and has been associated with increased risk of growth stunting, cognitive deficits, and reduced oral vaccine immunogenicity. A key challenge to identifying children with EED at highest risk of morbid sequelae is the lack of validated predictive biomarkers. Ongoing clinical studies are testing and validating EED biomarkers in child populations at risk for stunting, yet testing multiple biomarkers commonly requires specialized equipment, complex methods, resources, and considerable effort. The Micronutrient and EED Assessment Tool (MEEDAT) is a multiplexed immunoassay that measures biomarkers associated with child growth faltering and oral vaccine immunogenicity, and biomarkers indicative of systemic inflammation and micronutrient deficiencies. The performance of MEEDAT was well-correlated with commercial monoplex assays in specimens from children living in a low-resource setting in the present study. MEEDAT biomarkers were associated with growth outcomes and seroconversion in response to several vaccines. MEEDAT has the potential to reduce the time and cost of evaluating impact of interventions targeting EED.

Russell’s vipers (RVs) envenoming is an important public health issue in South-East Asia. Disseminated intravascular coagulopathy, systemic bleeding, hemolysis, and acute renal injury are obvious problems that develop in most cases, and neuromuscular junction blocks are an additional problem caused by western RV snakebite. The complex presentations usually are an obstacle to early diagnosis and antivenom administration. Here, we tried to produce highly specific antibodies in goose yolks for use in a paper-based microfluidic diagnostic kit, immunochromatographic test of viper (ICT-Viper), to distinguish RVs from other vipers and even cobra snakebite in Asia. We used indirect ELISA to monitor specific goose IgY production and western blotting to illustrate the interaction of avian or mammal antibody with venom proteins. The ICT-Viper was tested not only in prepared samples but also in stored patient serum to demonstrate its preliminary efficacy. The results revealed that specific anti-Daboia russelii IgY could be raised in goose eggs effectively without inducing adverse effects. When it was collocated with horse anti-Daboia siamensis antibody, which broadly reacted with most of the venom proteins of both types of Russell’s viper, the false cross-reactivity was reduced, and the test showed good performance. The limit of detection was reduced to 10 ng/ml in vitro, and the test showed good detection ability in clinical snake envenoming case samples. The ICT-Viper performed well and could be combined with a cobra venom detection kit (ICT-Cobra) to create a multiple detection strip (ICT-VC), which broadens its applications while maintaining its detection ability for snake envenomation identification. Nonetheless, the use of the ICT-Viper in the South-East Asia region is pending additional laboratory and field investigations and regional collaboration. We believe that the development of this practical diagnostic tool marks the beginning of positive efforts to face the global snakebite issue., Author summary For a long time, many scientists have made efforts to develop more effective antivenoms or to expand the limits of existing antivenoms. Such endeavors are challenging but worth the effort in the long run. However, to use precious antivenom more effectively, a snake species discrimination tool might be helpful for treatment by ensuring accurate antivenom usage and dosage. In this study, we revealed that avian IgY might be an economical alternative for collocation with the equine F(ab’)2 fragment in the development of a diagnostic device with improved performance. Only a few avian eggs (about 10–15) would be necessary to produce 10,000 kits. Immunochromatographic test (ICT) is a simple, easy, fast, and low-cost point-of-care lateral flow assay device, and especially adapted to perform in resource-deficient areas. Such an antibody collocation strategy might be beneficial to overcome the urgent need for snakebite diagnosis as soon as possible. More cooperative investigations across Asia continent might be initiated in the future to face the challenge of snakebites together.

Accurate data on the Lassa virus (LASV) human case fatality rate (CFR) and the prevalence of LASV in humans, rodents and other mammals are needed for better planning of actions that will ultimately reduce the burden of LASV infection in sub-Saharan Africa. In this systematic review with meta-analysis, we searched PubMed, Scopus, Africa Journal Online, and African Index Medicus from 1969 to 2020 to obtain studies that reported enough data to calculate LASV infection CFR or prevalence. Study selection, data extraction, and risk of bias assessment were conducted independently. We extracted all measures of current, recent, and past infections with LASV. Prevalence and CFR estimates were pooled using a random-effect meta-analysis. Factors associated with CFR, prevalence, and sources of between-study heterogeneity were determined using subgroup and metaregression analyses. This review was registered with PROSPERO, CRD42020166465. We initially identified 1,399 records and finally retained 109 reports that contributed to 291 prevalence records from 25 countries. The overall CFR was 29.7% (22.3–37.5) in humans. Pooled prevalence of LASV infection was 8.7% (95% confidence interval: 6.8–10.8) in humans, 3.2% (1.9–4.6) in rodents, and 0.7% (0.0–2.3) in other mammals. Subgroup and metaregression analyses revealed a substantial statistical heterogeneity explained by higher prevalence in tissue organs, in case-control, in hospital outbreak, and surveys, in retrospective studies, in urban and hospital setting, in hospitalized patients, and in West African countries. This study suggests that LASV infections is an important cause of death in humans and that LASV are common in humans, rodents and other mammals in sub-Saharan Africa. These estimates highlight disparities between sub-regions, and population risk profiles. Western Africa, and specific key populations were identified as having higher LASV CFR and prevalence, hence, deserving more attention for cost-effective preventive interventions., Author summary Lassa virus (LASV) infection constitutes a major public health threat as it has a direct impact on the mortality of febrile patients, healthcare workers, pregnant women, visitors of endemic countries with a consequential negative impact on national and individual economies. It is necessary to have accurate epidemiological data on LASV infection, in order to prioritize the policies, funding for public health interventions, and health-care planning, especially in sub-Saharan Africa (SSA). Data concerning virus occurrence in rodents and other mammal species can also assist in guiding control of Lassa fever from an ecological perspective. This could include preventive measures such as the protection of food fields by anti-rodent barriers, the protection of rodent predatory fauna, the sanitation of home and particularly the food storage place and the blocking of rodent access to home and food stored. Our systematic review and meta-analysis of LASV infection in SSA has contemporarily and comprehensively summarized the prevalence of current, recent, and past infection both in humans, rodents and other mammals. Broadly, the study showed relatively high CFR estimates in humans. Additionally, Western Africa presented the highest burden of LASV infections compared to other regions. This study emphasized various populations of SSA with relatively high burden of LASV infection. We have identified specific populations at high risk of LASV infection, who may urgently benefit from routine screening, detection and management programmes. Beyond, preventive strategies should be promoted, by educating and raising people’s awareness about LASV infection, and strengthening practitioners’ capacities towards adequate diagnosis and proper management of this infection in SSA.

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.