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Background: The human malaria parasite Plasmodium vivax infects red blood cells through a key pathway that requires interaction between Duffy binding protein II (DBPII) and its receptor on reticulocytes, the Duffy antigen/receptor for chemokines (DARC). A high proportion of P. vivax-exposed individuals fail to develop antibodies that inhibit DBPII-DARC interaction, and genetic factors that modulate this humoral immune response are poorly characterized. Here, we investigate if DBPII responsiveness could be HLA class II-linked. Methodology/Principal Findings: A community-based open cohort study was carried out in an agricultural settlement of the Brazilian Amazon, in which 336 unrelated volunteers were genotyped for HLA class II (DRB1, DQA1 and DQB1 loci), and their DBPII immune responses were monitored over time (baseline, 6 and 12 months) by conventional serology (DBPII IgG ELISA-detected) and functional assays (inhibition of DBPII–erythrocyte binding). The results demonstrated an increased susceptibility of the DRB1*13:01 carriers to develop and sustain an anti-DBPII IgG response, while individuals with the haplotype DRB1*14:02-DQA1*05:03-DQB1*03:01 were persistent non-responders. HLA class II gene polymorphisms also influenced the functional properties of DBPII antibodies (BIAbs, binding inhibitory antibodies), with three alleles (DRB1*07:01, DQA1*02:01 and DQB1*02:02) comprising a single haplotype linked with the presence and persistence of the BIAbs response. Modelling the structural effects of the HLA-DRB1 variants revealed a number of differences in the peptide-binding groove, which is likely to lead to altered antigen binding and presentation profiles, and hence may explain the differences in subject responses. Conclusions/Significance: The current study confirms the heritability of the DBPII antibody response, with genetic variation in HLA class II genes influencing both the development and persistence of IgG antibody responses. Cellular studies to increase knowledge of the binding affinities of DBPII peptides for class II molecules linked with good or poor antibody responses might lead to the development of strategies for controlling the type of helper T cells activated in response to DBPII. [ABSTRACT FROM AUTHOR]

Background: Schistosomiasis is a disease of major public health importance in sub-Saharan Africa. Immunoregulation begins early in schistosome infection and is characterized by hyporesponsiveness to parasite and bystander antigens, suggesting that a schistosome infection at the time of immunization could negatively impact the induction of protective vaccine responses. This study examined whether having a Schistosoma mansoni infection at the time of immunization with hepatitis B and tetanus toxoid (TT) vaccines impacts an individual’s ability to achieve and maintain protective antibody levels against hepatitis B surface antigen or TT. Methods: Adults were recruited from Kisumu Polytechnic College in Western Kenya. At enrollment, participants were screened for schistosomiasis and soil transmitted helminths (STHs) and assigned to groups based on helminth status. The vaccines were then administered and helminth infections treated a week after the first hepatitis B boost. Over an 8 month period, 3 blood specimens were obtained for the evaluation of humoral and cytokine responses to the vaccine antigens and for immunophenotyping. Results: 146 individuals were available for final analysis and 26% were S. mansoni positive (Sm+). Schistosomiasis did not impede the generation of initial minimum protective antibody levels to either hepatitis B or TT vaccines. However, median hepatitis B surface antibody levels were significantly lower in the Sm+ group after the first boost and remained lower, but not significantly lower, following praziquantel (PZQ) treatment and final boost. In addition, 8 months following TT boost and 7 months following PZQ treatment, Sm+ individuals were more likely to have anti-TT antibody levels fall below levels considered optimal for long term protection. IL-5 levels in response to in vitro TT stimulation of whole blood were significantly higher in the Sm+ group at the 8 month time period as well. Conclusions: Individuals with schistosomiasis at the start the immunizations were capable of responding appropriately to the vaccines as measured by antibody responses. However, they may be at risk of a more rapid decline in antibody levels over time, suggesting that treating schistosome infections with praziquantel before immunizations could be beneficial. The timing of the treatment as well as its full impact on the maintenance of antibodies against vaccine antigens remains to be elucidated. [ABSTRACT FROM AUTHOR]

Background: The pig parasite Ascaris suum plays and important role in veterinary medicine and represents a suitable model for A. lumbricoides, which infects over 800 million people. In pigs, continued exposure to Ascaris induces immunity at the level of the gut, protecting the host against migrating larvae. The objective of this study was to identify and characterize parasite antigens targeted by this local immune response that may be crucial for parasite invasion and establishment and to evaluate their protective and diagnostic potential. Methodology/Principal Findings: Pigs were immunized by trickle infection for 30 weeks, challenged with 2,000 eggs at week 32 and euthanized two weeks after challenge. At necropsy, there was a 100% reduction in worms recovered from the intestine and a 97.2% reduction in liver white spots in comparison with challenged non-immune control animals. Antibodies purified from the intestinal mucus or from the supernatant of cultured antibody secreting cells from mesenteric lymph nodes of immune pigs were used to probe L3 extracts to identify antibody targets. This resulted in the recognition of a 12kDa antigen (As12) that is actively shed from infective Ascaris L3. As12 was characterized as a phosphorylcholine-containing glycolipid-like antigen that is highly resistant to different enzymatic and chemical treatments. Vaccinating pigs with an As12 fraction did not induce protective immunity to challenge infection. However, serological analysis using sera or plasma from experimentally infected pigs or naturally infected humans demonstrated that the As12 ELISA was able to detect long-term exposure to Ascaris with a high diagnostic sensitivity (98.4% and 92%, respectively) and specificity (95.5% and 90.0%) in pigs and humans, respectively. Conclusions/Significance: These findings show the presence of a highly stage specific, glycolipid-like component (As12) that is actively secreted by infectious Ascaris larvae and which acts as a major antibody target in infected humans and pigs. [ABSTRACT FROM AUTHOR]

Introduction: Evidence for minimally symptomatic Ebola virus (EBOV) infection is limited. During the 2013–16 outbreak in West Africa, it was not considered epidemiologically relevant to published models or projections of intervention effects. In order to improve our understanding of the transmission dynamics of EBOV in humans, we investigated the occurrence of minimally symptomatic EBOV infection in quarantined contacts of reported Ebola virus disease cases in a recognized ‘hotspot.’ Methodology/Principal Findings: We conducted a cross-sectional serosurvey in Sukudu, Kono District, Sierra Leone, from October 2015 to January 2016. A blood sample was collected from 187 study participants, 132 negative controls (individuals with a low likelihood of previous exposure to Ebola virus), and 30 positive controls (Ebola virus disease survivors). IgG responses to Ebola glycoprotein and nucleoprotein were measured using Alpha Diagnostic International ELISA kits with plasma diluted at 1:200. Optical density was read at 450 nm (subtracting OD at 630nm to normalize well background) on a ChroMate 4300 microplate reader. A cutoff of 4.7 U/mL for the anti-GP ELISA yielded 96.7% sensitivity and 97.7% specificity in distinguishing positive and negative controls. We identified 14 seropositive individuals not known to have had Ebola virus disease. Two of the 14 seropositive individuals reported only fever during quarantine while the remaining 12 denied any signs or symptoms during quarantine. Conclusions/Significance: By using ELISA to measure Zaire Ebola virus antibody concentrations, we identified a significant number of individuals with previously undetected EBOV infection in a ‘hotspot’ village in Sierra Leone, approximately one year after the village outbreak. The findings provide further evidence that Ebola, like many other viral infections, presents with a spectrum of clinical manifestations, including minimally symptomatic infection. These data also suggest that a significant portion of Ebola transmission events may have gone undetected during the outbreak. Further studies are needed to understand the potential risk of transmission and clinical sequelae in individuals with previously undetected EBOV infection. [ABSTRACT FROM AUTHOR]

Background: Previous works showed that immunization with saliva from Lutzomyia intermedia, a vector of Leishmania braziliensis, does not protect against experimental infection. However, L. braziliensis is also transmitted by Lutzomyia whitmani, a sand fly species closely related to Lu. intermedia. Herein we describe the immune response following immunization with Lu. whitmani saliva and the outcome of this response after L. braziliensis infection. Methods and findings: BALB/c mice immunized with Lu. whitmani saliva developed robust humoral and cellular immune responses, the latter characterized by an intense cellular infiltrate and production of IFN-γ and IL-10, by both CD4+ and CD8+ cells. Mice immunized as above and challenged with L. braziliensis plus Lu. whitmani saliva displayed significantly smaller lesions and parasite load at the challenge site. This protection was associated with a higher (p<0.05) IFN-γ production in response to SLA stimulation. Long-term persisting immunity was also detected in mice immunized with Lu. whitmani saliva. Furthermore, individuals residing in an endemic area for cutaneous leishmaniasis (CL) presented antibody responses to Lu. whitmani saliva. However CL patients, with active lesions, displayed a lower humoral response to Lu. whitmani saliva compared to individuals with subclinical Leishmania infection. Conclusion: Pre-exposure to Lu. whitmani saliva induces protection against L. braziliensis in a murine model. We also show that Lu. whitmani salivary proteins are immunogenic in naturally exposed individuals. Our results reinforce the importance of investigating the immunomodulatory effect of saliva from different species of closely related sand flies. [ABSTRACT FROM AUTHOR]

Dengue virus (DENV) is the causative agent of dengue fever and dengue hemorrhagic fever. The virus is endemic in over 120 countries, causing over 350 million infections per year. Dengue vaccine development is challenging because of the need to induce simultaneous protection against four antigenically distinct DENV serotypes and evidence that, under some conditions, vaccination can enhance disease due to specific immunity to the virus. While several live-attenuated tetravalent dengue virus vaccines display partial efficacy, it has been challenging to induce balanced protective immunity to all 4 serotypes. Instead of using whole-virus formulations, we are exploring the potentials for a particulate subunit vaccine, based on DENV E-protein displayed on nanoparticles that have been precisely molded using Particle Replication in Non-wetting Template (PRINT) technology. Here we describe immunization studies with a DENV2-nanoparticle vaccine candidate. The ectodomain of DENV2-E protein was expressed as a secreted recombinant protein (sRecE), purified and adsorbed to poly (lactic-co-glycolic acid) (PLGA) nanoparticles of different sizes and shape. We show that PRINT nanoparticle adsorbed sRecE without any adjuvant induces higher IgG titers and a more potent DENV2-specific neutralizing antibody response compared to the soluble sRecE protein alone. Antigen trafficking indicate that PRINT nanoparticle display of sRecE prolongs the bio-availability of the antigen in the draining lymph nodes by creating an antigen depot. Our results demonstrate that PRINT nanoparticles are a promising platform for delivering subunit vaccines against flaviviruses such as dengue and Zika. [ABSTRACT FROM AUTHOR]

Background: Major gaps in our understanding of Plasmodium vivax biology and the acquisition of immunity to this parasite hinder vaccine development. P. vivax merozoites exclusively invade reticulocytes, making parasite proteins that mediate reticulocyte binding and/or invasion potential key vaccine or drug targets. While protein interactions that mediate invasion are still poorly understood, the P. vivax Reticulocyte-Binding Protein family (PvRBP) is thought to be involved in P. vivax restricted host-cell selectivity. Methodology/Principal findings: We assessed the binding specificity of five members of the PvRBP family (PvRBP1a, PvRBP1b, PvRBP2a, PvRBP2b, PvRBP2-P2 and a non-binding fragment of PvRBP2c) to normocytes or reticulocytes. PvRBP2b was identified as the only reticulocyte-specific binder (P<0.001), whereas the others preferentially bound to normocytes (PvRBP1a/b P≤0.034), or showed comparable binding to both (PvRBP2a/2-P2, P = 0.38). Furthermore, we measured levels of total and IgG subclasses 1, 2, 3 and 4 to the six PvRBPs in a cohort of young Papua New Guinean children, and assessed their relationship with prospective risk of P. vivax malaria. Children had substantial, highly correlated (rho = 0.49–0.82, P<0.001) antibody levels to all six PvRBPs, with dominant IgG1 and IgG3 subclasses. Both total IgG (Incidence Rate Ratio [IRR] 0.63–0.73, P = 0.008–0.041) and IgG1 (IRR 0.56–0.69, P = 0.001–0.035) to PvRBP2b and PvRBP1a were strongly associated with reduced risk of vivax-malaria, independently of age and exposure. Conclusion/Significance: These results demonstrate a diversity of erythrocyte-binding phenotypes of PvRBPs, indicating binding to both reticulocyte-specific and normocyte-specific ligands. Our findings provide further insights into the naturally acquired immunity to P. vivax and highlight the importance of PvRBP proteins as targets of naturally acquired humoral immunity. In-depth studies of the role of PvRBPs in P. vivax invasion and functional validation of the role of anti-PvRBP antibodies in clinical immunity against P. vivax are now required to confirm the potential of the reticulocyte-binding PvRBP2b and PvRBP1a as vaccine candidate antigens. [ABSTRACT FROM AUTHOR]

Leprosy is caused by Mycobacterium leprae infection and remains a major public health problem in many areas of the world. Challenges to its timely diagnosis result in delay in treatment, which is usually associated with severe disability. Although phenolic glycolipid (PGL)-I has been reported as auxiliary diagnostic tool, currently there is no serological assay routinely used in leprosy diagnosis. The aim of this study was to evaluate the effectiveness of two related reagents, LID-1 and LID-NDO, for the detection of M. leprae infection. Sera from 98 leprosy patients, 365 household contacts (HHC) and 98 endemic controls from Rio Grande do Norte, Brazil, were evaluated. A subgroup of the HHC living in a hyperendemic area was followed for 7–10 years. Antigen-specific antibody responses were highest in multibacillary (MB) at the lepromatous pole (LL/BL) and lowest in paucibacillary (PB) at the tuberculoid pole (TT/BT). A positive correlation for both anti-LID-1 and anti-LID-NDO antibodies was found with bacterial burden (LID-1, r = 0.84, p<0.001; LID-NDO, r = 0.82, p<0.001), with higher sensitivity than bacilloscopy. According to Receiver Operating Curve, LID-1 and LID-NDO performed similarly. The sensitivity for MB cases was 89% for LID-1 and 95% for LID-NDO; the specificity was 96% for LID-1 and 88% for LID-NDO. Of the 332 HHC that were followed, 12 (3.6%) were diagnosed with leprosy in a median time of 31 (3–79) months after recruitment. A linear generalized model using LID-1 or LID-NDO as a predictor estimated that 8.3% and 10.4% of the HHC would become a leprosy case, respectively. Together, our findings support a role for the LID-1 and LID-NDO antigens in diagnosing MB leprosy and identifying people at greater risk of developing clinical disease. These assays have the potential to improve the diagnostic capacity at local health centers and aid development of strategies for the eventual control and elimination of leprosy from endemic areas. [ABSTRACT FROM AUTHOR]

Background: Animal control measures in Latin America have decreased the incidence of urban human rabies transmitted by dogs and cats; currently most cases of human rabies are transmitted by bats. In 2004–2005, rabies outbreaks in populations living in rural Brazil prompted widespread vaccination of exposed and at-risk populations. More than 3,500 inhabitants of Augusto Correa (Pará State) received either post-exposure (PEP) or pre-exposure (PrEP) prophylaxis. This study evaluated the persistence of rabies virus-neutralizing antibodies (RVNA) annually for 4 years post-vaccination. The aim was to evaluate the impact of rabies PrEP and PEP in a population at risk living in a rural setting to help improve management of vampire bat exposure and provide additional data on the need for booster vaccination against rabies. Methodology/Principal Findings: This prospective study was conducted in 2007 through 2009 in a population previously vaccinated in 2005; study participants were followed-up annually. An RVNA titer >0.5 International Units (IU)/mL was chosen as the threshold of seroconversion. Participants with titers ≤0.5 IU/mL or Equivalent Units (EU)/mL at enrollment or at subsequent annual visits received booster doses of purified Vero cell rabies vaccine (PVRV). Adherence of the participants from this Amazonian community to the study protocol was excellent, with 428 of the 509 (84%) who attended the first interview in 2007 returning for the final visit in 2009. The long-term RVNA persistence was good, with 85–88.0% of the non-boosted participants evaluated at each yearly follow-up visit remaining seroconverted. Similar RVNA persistence profiles were observed in participants originally given PEP or PrEP in 2005, and the GMT of the study population remained >1 IU/mL 4 years after vaccination. At the end of the study, 51 subjects (11.9% of the interviewed population) had received at least one dose of booster since their vaccination in 2005. Conclusions/Significance: This study and the events preceding it underscore the need for the health authorities in rabies enzootic countries to decide on the best strategies and timing for the introduction of routine rabies PrEP vaccination in affected areas. [ABSTRACT FROM AUTHOR]

Background: The World Health Organization (WHO) now requires a second surveillance survey for trachoma after an impact assessment has found follicular trachoma (TF) <5% to determine if re-emergence has occurred. Using new WHO guidelines, we undertook surveillance surveys, and determined the prevalence of infection and antibody positivity, in two districts in Nepal. Methods: 20 clusters were randomly selected within each district, 15 were randomly selected for antibody testing. In each cluster, we randomly selected 50 children ages 1–9 years and 100 adults ≥15 years. TF and trachomatous trichiasis (TT) were evaluated. Conjunctival swabs to test for chlamydial infection using GenXpert platform were obtained, and dried blood spots were collected to test for antibodies to Chlamydia Trachomatis pgp3 using the Luminex platform. Findings: 3 cases of TF were found in the two districts, and one case of infection. Pgp3 antibody positivity was 2·4% (95% confidence interval: 1·4%, 3·7%), and did not increase with age (P = 0.24). No clustering of antibody positivity within communities was found. TT prevalence was <1/1,000 population. Interpretation: The surveillance surveys, as proposed by WHO, showed no evidence for re-emergence of trachoma in two districts of Nepal. The low level and no significant increase by age in seroprevalence of antibodies to C trachomatis pgp3 antigen deserve further investigation as a marker of interruption of transmission. [ABSTRACT FROM AUTHOR]

Background: Bartonella bacilliformis is the causative agent of Carrion’s disease, a neglected illness with mortality rates of 40–85% in the absence of treatment. The lack of a diagnostic technique to overcome misdiagnosis and treat asymptomatic carriers is of note. This study aimed to identify new B. bacilliformis antigenic candidates that could lead to a new diagnostic tool able to be implemented in endemic rural areas. Methodology/Principal Findings: Blood (n = 198) and serum (n = 177) samples were collected in northern Peru. Clinical data were recorded. Specific 16S rRNA amplification by RT-PCR, IFA and ELISA for IgM/IgG with whole cells as antigens was done. Western blot analysis and N-terminal amino acid sequencing detected seroreactive proteins. ELISAs for IgM/IgG for the antigenic candidates were performed. Of the population 33.3% reported at least one symptom compatible with Carrion’s disease; 25.4% (IFA), 27.1% (ELISA-IgG), 33.9% (ELISA-IgM) and 38.9% (RT-PCR) of samples were positive. Four proteins were considered potential antigenic candidates, including two new antigenic candidates, succinyl-CoA synthetase subunit α (SCS-α) and succinyl-CoA synthetase subunit β (SCS-β). On Western blot both Pap31 and SCS-α interacted with IgM, while GroEL and SCS-β interacted with IgG. The presence of specific antibodies against the antigenic candidates varied from 34.5% (IgG against SCS-α) to 97.2% (IgM against Pap31). Conclusions/Significance: RT-PCR and the high levels of positivity for specific ELISAs demonstrate high levels of B. bacilliformis exposure and asymptomatic carriers among inhabitants. The new antigens identified might be used as a new rapid diagnostic tool to diagnose acute Carrion’s disease and identify asymptomatic carriers. [ABSTRACT FROM AUTHOR]

Machupo virus (MACV), a New World arenavirus, is the etiological agent of Bolivian hemorrhagic fever (BHF). Junin virus (JUNV), a close relative, causes Argentine hemorrhagic fever (AHF). Previously, we reported that a recombinant, chimeric MACV (rMACV/Cd#1-GPC) expressing glycoprotein from the Candid#1 (Cd#1) vaccine strain of JUNV is completely attenuated in a murine model and protects animals from lethal challenge with MACV. A rMACV with a single F438I substitution in the transmembrane domain (TMD) of GPC, which is equivalent to the F427I attenuating mutation in Cd#1 GPC, was attenuated in a murine model but genetically unstable. In addition, the TMD mutation alone was not sufficient to fully attenuate JUNV, indicating that other domains of the GPC may also contribute to the attenuation. To investigate the requirement of different domains of Cd#1 GPC for successful attenuation of MACV, we rescued several rMACVs expressing the ectodomain of GPC from Cd#1 either alone (MCg1), along with the TMD F438I substitution (MCg2), or with the TMD of Cd#1 (MCg3). All rMACVs exhibited similar growth curves in cultured cells. In mice, the MCg1 displayed significant reduction in lethality as compared with rMACV. The MCg1 was detected in brains and spleens of MCg1-infected mice and the infection was associated with tissue inflammation. On the other hand, all animals survived MCg2 and MCg3 infection without detectable levels of virus in various organs while producing neutralizing antibody against Cd#1. Overall our data suggest the indispensable role of each GPC domain in the full attenuation and immunogenicity of rMACV/Cd#1 GPC. [ABSTRACT FROM AUTHOR]

Background: Chikungunya virus (CHIKV) is a re-emerging mosquito-borne virus which causes epidemics of fever, severe joint pain and rash. Between 2005 and 2010, the East/Central/South African (ECSA) genotype was responsible for global explosive outbreaks across India, the Indian Ocean and Southeast Asia. From late 2013, Asian genotype CHIKV has caused outbreaks in the Americas. The characteristics of cross-antibody efficacy and epitopes are poorly understood. Methodology/Principal Findings: We characterized human immune sera collected during two independent outbreaks in Malaysia of the Asian genotype in 2006 and the ECSA genotype in 2008–2010. Neutralizing capacity was analyzed against representative clinical isolates as well as viruses rescued from infectious clones of ECSA and Asian CHIKV. Using whole virus antigen and recombinant E1 and E2 envelope glycoproteins, we further investigated antibody binding sites, epitopes, and antibody titers. Both ECSA and Asian sera demonstrated stronger neutralizing capacity against the ECSA genotype, which corresponded to strong epitope-antibody interaction. ECSA serum targeted conformational epitope sites in the E1-E2 glycoprotein, and E1-E211K, E2-I2T, E2-H5N, E2-G118S and E2-S194G are key amino acids that enhance cross-neutralizing efficacy. As for Asian serum, the antibodies targeting E2 glycoprotein correlated with neutralizing efficacy, and I2T, H5N, G118S and S194G altered and improved the neutralization profile. Rabbit polyclonal antibody against the N-terminal linear neutralizing epitope from the ECSA sequence has reduced binding capacity and neutralization efficacy against Asian CHIKV. These findings imply that the choice of vaccine strain may impact cross-protection against different genotypes. Conclusion/Significance: Immune serum from humans infected with CHIKV of either ECSA or Asian genotypes showed differences in binding and neutralization characteristics. These findings have implications for the continued outbreaks of co-circulating CHIKV genotypes and effective design of vaccines and diagnostic serological assays. [ABSTRACT FROM AUTHOR]

ELISA-based methods of detecting Fasciola cathepsins in feces are powerful techniques for diagnosing infections by F. hepatica and F. gigantica. In the last decade, the in-house MM3-COPRO ELISA and its commercial version BIO K 201 (BIO X Diagnostics, Belgium) have been recognized as useful tools for detecting early infections by such trematodes and for monitoring the efficacy of anthelmintic treatments in human and animal species, as they provide some advantages over classic fecal egg counts. However, the sensitivity of MM3-COPRO ELISA can sometimes be compromised by the high variability in the concentration of cathepsins in fecal samples throughout the biological cycle of Fasciola (mainly in cattle) and by differences in the between-batch performance of peroxidase-labeled anti-mouse IgG polyclonal antibodies. To prevent such problems, we investigated whether the incorporation of a commercial streptavidin-polymerized horseradish peroxidase conjugate, in order to reveal bound biotinylated monoclonal antibody MM3, can improve the sensitivity of the MM3-COPRO ELISA. We observed that inclusion of this reagent shifted the previous detection limit of the assay from 0.6 ng/mL to 150 pg/mL and that the modified test is able to identify infection in cows harboring only one fluke. Moreover, we demonstrated that maximal OD values can be achieved with short incubations (30 min each step) at RT with shaking, rather than standard incubations, which significantly accelerates the diagnostic procedure. Finally, we did not find a significant correlation between coproantigen concentration and parasite burden in cattle, which may be due to the low parasite burden (1–10 adult flukes) of the animals used in the present study. As the usefulness of the classic MM3-COPRO test for detecting animal and human infections has already been demonstrated, it is expected that the improvements reported in this study will add new insights into the diagnosis and control of fasciolosis. [ABSTRACT FROM AUTHOR]

Dengue virus (DENV) is the most common mosquito-borne flavivirus; it can either cause mild dengue fever or the more severe dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). One of the characteristic features of DHF/DSS is vascular leakage; although DENV nonstructural protein 1 (NS1) has been proved to induce vascular leakage after binding to Toll-like receptor 4, the down-stream mechanism has not yet been fully understood. In the sera of DENV-infected patients, the concentrations of DENV NS1 and inflammatory cytokine macrophage migration inhibitory factor (MIF) are positively correlated with disease severity, but whether DENV NS1 induces vascular leakage through MIF secretion remains unknown. We demonstrated that recombinant NS1 induced vascular leakage and MIF secretion both in human endothelial cell line HMEC-1 and in mice. Furthermore, these phenomena were inhibited in the presence of anti-NS1 antibodies both in vitro and in vivo. DENV NS1 also induced LC3-I to LC3-II conversion and p62 degradation in endothelial cell line, which indicated the formation of autophagy. To clarify whether MIF or autophagy mediated DENV NS1-induced vascular leakage, various inhibitors were applied. The results showed that DENV NS1-induced vascular leakage and VE-cadherin disarray were blocked in the presence of MIF inhibitors, anti-MIF-antibodies or autophagy inhibitors. An Atg5 knockdown clone further confirmed that autophagy formation of endothelial cells was required in NS1-induced vascular leakage. Furthermore, DENV NS1-induced LC3 puncta were also decreased in the presence of MIF inhibitors, indicating that MIF mediated DENV NS1-induced autophagy. Taken together, the results suggest a potential mechanism of DENV-induced vascular leakage and provide possible therapeutic targets against DHF/DSS. [ABSTRACT FROM AUTHOR]

Chikungunya virus (CHIKV) has emerged as a global health concern due to its recent spread in both old and new world. So far, no CHIKV specific drug or vaccine is licensed for human use. In this study, we report production of Chikungunya virus like particles (CHIK-VLPs) using novel yeast expression system (Pichia pastoris) and its evaluation as vaccine candidate. The gene encoding structural polyprotein of CHIKV from a recent epidemic strain was cloned into yeast expression system. The multicopy integrants were processed for expression of CHIK-VLPs. The VLPs were purified and confirmed through electron microscopic analysis for their morphological identity with CHIKV. The in vitro and in vivo evaluation of CHIK-VLPs as vaccine candidate was determined in Balb/c mice. Induction of both humoral and cellular immune response was observed with different doses of CHIK-VLPs. The humoral immune response was studied through different techniques like enzyme linked immunosorbent assay, IgG Isotyping and plaque reduction neutralization test. CHIK-VLPs were found to elicit high titer of antibodies that are able to recognize native CHIKV. Higher level of IgG2a and IgG1 subtypes was identified suggestive of balanced Th1/Th2 response. Both in vitro and in vivo neutralization activity of CHIK-VLPs antibodies was observed even with low concentration, which shows its high specificity and neutralizing activity against two different CHIKV strains. Neonatal mice receiving anti-CHIK-VLPs antibodies were protected from CHIKV challenge. Induction of cellular immune response was confirmed through higher level of TNF-α, IL-10 and substantial level of IL-2, IL-4 and IFN-γ indicating a balanced response. This is the first report, where CHIK-VLPs has been expressed by Pichia pastoris and evaluated for neutralizing activity against CHIKV. These promising results indicate the utility of CHIK-VLPs as a promising vaccine candidate against emerging CHIKV. [ABSTRACT FROM AUTHOR]

Background: Mycolactone, the macrolide exotoxin produced by Mycobacterium ulcerans, causes extensive tissue destruction by inducing apoptosis of host cells. In this study, we aimed at the production of antibodies that could neutralize the cytotoxic activities of mycolactone. Methodology/Principal Findings: Using the B cell hybridoma technology, we generated a series of monoclonal antibodies with specificity for mycolactone from spleen cells of mice immunized with the protein conjugate of a truncated synthetic mycolactone derivative. L929 fibroblasts were used as a model system to investigate whether these antibodies can inhibit the biological effects of mycolactone. By measuring the metabolic activity of the fibroblasts, we found that anti-mycolactone mAbs can completely neutralize the cytotoxic activity of mycolactone. Conclusions/Significance: The toxin neutralizing capacity of anti-mycolactone mAbs supports the concept of evaluating the macrolide toxin as vaccine target. [ABSTRACT FROM AUTHOR]

Dengue virus (DENV) causes a spectrum of diseases ranging from self-limiting dengue fever to severe conditions such as haemorrhagic fever and dengue shock syndrome. Antibody-dependent enhancement (ADE) is thought to explain the occurrence of severe dengue whereby pre-existing binding but non-neutralising antibodies enhance DENV infection. The ADE phenomenon is supported by epidemiological findings that infants that born to dengue immune mothers are at greater risk to develop severe dengue upon primary infection. The role of maternally acquired dengue-specific antibodies in disease enhancement was recently recapitulated in a mouse model where mice born to DENV1-immune mothers experienced enhanced disease severity upon DENV2 infection. Here, this study investigates the relative contribution of maternal dengue-specific antibodies acquired during gestation and breastfeeding in dengue disease. Using a surrogate breastfeeding mother experimental approach, we showed that majority of the maternal dengue-specific antibodies were acquired during breastfeeding and conferred an extended enhancement window. On the other hand, in the context of homologous infection, breastfeeding conferred protection. Furthermore, measurement of dengue-specific antibody titres over time in mice born to dengue immune mothers revealed a biphasic pattern of antibody decay as reported in humans. Our work provides evidence of the potential contribution of breast milk-acquired dengue-specific IgG antibodies in enhancement and protection against dengue. Should such contribution be established in humans as well, it may have important implications for the development of guidelines to dengue-immune breastfeeding mothers. [ABSTRACT FROM AUTHOR]

Chikungunya is a viral disease transmitted by Aedes aegypti and Ae. albopictus mosquitoes. In late 2013, chikungunya virus (CHIKV) was introduced into the Caribbean island of St. Martin. Since then, approximately 2 million chikungunya cases have been reported by the Pan American Health Organization, and most countries in the Americas report autochthonous transmission of CHIKV. In Nicaragua, the first imported case was described in July 2014 and the first autochthonous case in September 2014. Here, we conducted two studies to analyze the seroprevalence of anti-CHIKV antibodies after the first chikungunya epidemic in a community-based cohort study (ages 2–14 years) and in a cross-sectional survey of persons aged ≥15 years in the same area of Managua, Nicaragua. Routine annual serum samples collected from 3,362 cohort participants in March/April 2014 and 2015, and 848 age-stratified samples collected from persons ≥15 years old at the end of May-beginning of June 2015 were used to estimate the seroprevalence of anti-CHIKV antibodies after the first epidemic (October 2014 to February 2015 in the study population). Using an Inhibition ELISA assay that measures total anti-CHIKV antibodies, the seroprevalence was significantly higher in those aged ≥15 (13.1% (95%CI: 10.9, 15.5)) than in the pediatric population (6.1% (95%CI: 5.3, 6.9)). The proportion of inapparent infections was 58.3% (95%CI: 51.5, 65.1) in children and 64.9% (95%CI: 55.2, 73.7) in the ≥15 study population. We identified age, water availability, household size, and socioeconomic status as factors associated with the presence of anti-CHIKV antibodies. Overall, this is the first report of CHIKV seropositivity in continental Latin America and provides useful information for public health authorities in the region. [ABSTRACT FROM AUTHOR]

Background: The bivalent whole-cell (BivWC) oral cholera vaccine (Shanchol) is effective in preventing cholera. However, evaluations of immune responses following vaccination with BivWC have been limited. To determine whether BivWC induces significant mucosal immune responses, we measured V. cholerae O1 antigen-specific antibody secreting cell (ASC) responses following vaccination. Methodology/Principal Findings: We enrolled 24 Haitian adults in this study, and administered doses of oral BivWC vaccine 14 days apart (day 0 and day 14). We drew blood at baseline, and 7 days following each vaccine dose (day 7 and 21). Peripheral blood mononuclear cells (PBMCs) were isolated, and ASCs were enumerated using an ELISPOT assay. Significant increases in Ogawa (6.9 cells per million PBMCs) and Inaba (9.5 cells per million PBMCs) OSP-specific IgA ASCs were detected 7 days following the first dose (P < 0.001), but not the second dose. The magnitude of V. cholerae-specific ASC responses did not appear to be associated with recent exposure to cholera. ASC responses measured against the whole lipolysaccharide (LPS) antigen and the OSP moiety of LPS were equivalent, suggesting that all or nearly all of the LPS response targets the OSP moiety. Conclusions/Significance: Immunization with the BivWC oral cholera vaccine induced ASC responses among a cohort of healthy adults in Haiti after a single dose. The second dose of vaccine resulted in minimal ASC responses over baseline, suggesting that the current dosing schedule may not be optimal for boosting mucosal immune responses to V. cholerae antigens for adults in a cholera-endemic area. [ABSTRACT FROM AUTHOR]

Background: Scrub typhus is a common cause of undiagnosed febrile illness in certain tropical regions, but can be easily treated with antibiotics. The causative agent, Orientia tsutsugamushi, is antigenically variable which complicates diagnosis and efforts towards vaccine development. Methodology/Principal Findings: This study aimed to dissect the antigenic and genetic relatedness of O. tsutsugamushi strains and investigate sero-diagnostic reactivities by titrating individual patient sera against their O. tsutsugamushi isolates (whole-cell antigen preparation), in homologous and heterologous serum-isolate pairs from the same endemic region in NE Thailand. The indirect immunofluorescence assay was used to titrate Orientia tsutsugamushi isolates and human sera, and a mathematical technique, antigenic cartography, was applied to these data to visualise the antigenic differences and cross-reactivity between strains and sera. No functional or antigen-specific analyses were performed. The antigenic variation found in clinical isolates was much less pronounced than the genetic differences found in the 56kDa type-specific antigen genes. The Karp-like sera were more broadly reactive than the Gilliam-like sera. Conclusions/Significance: Antigenic cartography worked well with scrub typhus indirect immunofluorescence titres. The data from humoral responses suggest that a Karp-like strain would provide broader antibody cross-reactivity than a Gilliam-like strain. Although previous exposure to O. tsutsugamushi could not be ruled out, scrub typhus patient serum antibody responses were characterised by strong homologous, but weak heterologous antibody titres, with little evidence for cross-reactivity by Gilliam-like sera, but a broader response from some Karp-like sera. This work highlights the importance of antigenic variation in O. tsutsugamushi diagnosis and determination of new serotypes. [ABSTRACT FROM AUTHOR]

Background: There is no point of care diagnostic test for infection with M. Leprae or for leprosy, although ELISA anti PGL-1 has been considered and sometimes used as a means to identify infection. Methods: A systematic review of all cohort studies, which classified healthy leprosy contacts, at entry, according to anti-PGL1 positivity, and had at least one year follow up. The outcome was clinical diagnosis of leprosy by an experienced physician. The meta-analysis used a fixed model to estimated OR for the association of PGL-1 positivity and clinical leprosy. A fixed model also estimated the sensibility of PGL-1 positivity and positive predictive value. Results: Contacts who were anti PGL-1 positive at baseline were 3 times as likely to develop leprosy; the proportion of cases of leprosy that were PGL-1 positive at baseline varied but was always under 50%. Conclusions: Although there is a clear and consistent association between positivity to anti PGL-1 and development of leprosy in healthy contacts, selection of contacts for prophylaxis based on anti PGL1 response would miss more than half future leprosy cases. Should chemoprophylaxis of controls be incorporated into leprosy control programmes, PGL1 appears not to be a useful test in the decision of which contacts should receive chemoprophylaxis. [ABSTRACT FROM AUTHOR]

Background: Elimination of Plasmodium vivax malaria would be greatly facilitated by the development of an effective vaccine. A comprehensive and systematic characterization of antibodies to P. vivax antigens in exposed populations is useful in guiding rational vaccine design. Methodology/Principal Findings: In this study, we investigated antibodies to a large library of P. vivax entire ectodomain merozoite proteins in 2 Asia-Pacific populations, analysing the relationship of antibody levels with markers of current and cumulative malaria exposure, and socioeconomic and clinical indicators. 29 antigenic targets of natural immunity were identified. Of these, 12 highly-immunogenic proteins were strongly associated with age and thus cumulative lifetime exposure in Solomon Islanders (P<0.001–0.027). A subset of 6 proteins, selected on the basis of immunogenicity and expression levels, were used to examine antibody levels in plasma samples from a population of young Papua New Guinean children with well-characterized individual differences in exposure. This analysis identified a strong association between reduced risk of clinical disease and antibody levels to P12, P41, and a novel hypothetical protein that has not previously been studied, PVX_081550 (IRR 0.46–0.74; P<0.001–0.041). Conclusion/Significance: These data emphasize the benefits of an unbiased screening approach in identifying novel vaccine candidate antigens. Functional studies are now required to establish whether PVX_081550 is a key component of the naturally-acquired protective immune response, a biomarker of immune status, or both. [ABSTRACT FROM AUTHOR]

Entamoeba histolytica causes intestinal disease in endemic settings throughout the world. Diagnosis of E. histolytica infection would be improved by the identification of biomarkers that are expressed by cysts of E. histolytica, but not by cysts of closely related commensal species of Entamoeba. Herein, we describe two novel monoclonal antibodies (1A4 and 1D3) produced against a spacer region of the E. histolytica Jacob2 lectin, an outer cyst wall protein. These reagents demonstrated no cross-reaction to E. dispar recombinant antigen and low picomolar molecular detection limits when paired in ELISA sandwich assays. In an immunofluorescence microscopy assay, the α-Jacob2 murine antibodies labeled cysts of three xenically cultured E. histolytica isolates but did not label cysts of three E. bangladeshi isolates. Monoclonal antibody 1A4 did not cross-react with xenic cultures of three E. dispar isolates, demonstrating specificity to E. histolytica, while monoclonal antibody 1D3 cross-reacted with two out of three E. dispar isolates. Both antibodies labeled cysts in formalin-fixed slides, a potential logistical advantage in some settings. The monoclonal antibody 1A4 was also used in an immunofluorescence microscopy assay with formalin-fixed stool specimens. Seven out of ten ELISA-positive stool specimens exhibited 1A4-labeled cyst-like objects, compared to one out of seven ELISA-negative specimens. These results demonstrate that antibodies generated against the flexible spacer of E. histolytica Jacob2 lectin recognize and bind to Jacob2 protein in whole cysts and are capable of differentiating Entamoeba species in fixed specimens. Thus, Jacob2 is a promising biomarker for use in diagnosing E. histolytica infection. [ABSTRACT FROM AUTHOR]

Background: The current Japanese encephalitis (JE) vaccine derived from G3 JE virus (JEV) can induce protective immunity against G1–G4 JEV genotypes. However, protective efficacy against the emerging G5 genotype has not been reported. Methods/Principal Findings: Using in vitro and in vivo tests, biological phenotype and cross-immunoreactions were compared between G3 JEV and G5 JEV (wild strains). The PRNT90 method was used to detect neutralizing antibodies against different genotypes of JEV in JE vaccine-immunized subjects and JE patients. In JE vaccine-immunized mice, the lethal challenge protection rates against G3 and G5 JEV wild strains were 100% and 50%, respectively. The seroconversion rates (SCRs) of virus antibodies against G3 and G5 JEV among vaccinated healthy subjects were 100% and 35%, respectively. All clinically identified JE patients showed high levels of G3 JEV neutralizing antibodies (≥1:10–1280) with positive serum geometric mean titers (GMTs) of 43.2, while for G5 JEV, neutralizing antibody conversion rates were only 64% with positive serum GMTs of 11.14. Moreover, the positive rate of JEV neutralizing antibodies against G5 JEV in pediatric patients was lower than in adults. Conclusions/Significance: Low levels of neutralizing/protective antibodies induced by the current JE vaccine, based on the G3 genotype, were observed against the emerging G5 JEV genotype. Our results demonstrate the need for more detailed studies to reevaluate whether or not the apparent emergence of G5 JEV can be attributed to failure of the current vaccine to induce appropriate immune protectivity against this genotype of JEV. [ABSTRACT FROM AUTHOR]

Background: Neurotropic arboviral infections are an important cause of encephalitis. A zoonotic, vector-borne alphavirus, Madariaga virus (MADV; formerly known as South American eastern equine encephalitis virus), caused its first documented human outbreak in 2010 in Darien, Panama, where the genetically similar Venezuelan equine encephalitis virus (VEEV) is endemic. We report the results of a seroprevalence survey of animals and humans, illustrating contrasting features of MADV and VEEV ecology and epidemiology. Methods: Small mammals were trapped in 42 sites in Darien, Panama, using Sherman traps, Tomahawk traps, and mist nets for bats. Blood was tested for the presence of neutralizing antibodies to MADV and VEEV. In addition, bird sera collected in 2007 in Chagres, Panama, were tested for MADV and VEEV neutralizing antibodies. Viremia was ascertained by RT-PCR. Human exposure to these two viruses was determined by IgG ELISA, followed by plaque reduction neutralization tests. To identify relevant risk factors for MADV or VEEV exposure, logistic regression analysis was performed, and the most parsimonious model was selected based on the Akaike information criterion. Results: The animal survey yielded 32 bats (16 species), 556 rodents (12 species), and 20 opossums (4 species). The short-tailed cane mouse (Zygodontomys brevicauda) found abundantly in pasture and farms, had the highest MADV seroprevalence (8.3%). For VEEV, the shrub and forest-dwelling long-whiskered rice rat (Transandinomys bolivaris) had the highest seroprevalence (19.0%). Viremia was detected in one animal (Z. brevicauda). Of the 159 bird sera (50 species) tested, none were positive for either virus. In humans (n = 770), neutralizing antibodies to MADV and VEEV were present in 4.8% and 31.5%, respectively. MADV seropositivity was positively associated with cattle ranching, farming, and fishing. Having VEEV antibodies and shrubs near the house diminished risk. Age, forest work, farming and fishing were risk factors for VEEV, while having MADV antibodies, glazed windows, waste pick-up and piped water were protective. Conclusion: Our findings suggest that the short-tailed cane mouse and the long-whiskered rice rat serve as hosts for MADV and VEEV, respectively. The preferred habitat of these rodent species coincides with areas associated with human infection risk. Our findings also indicate that MADV emerged recently in humans, and that the transmission cycles of these two sympatric alphaviruses differ spatially and in host utilization. [ABSTRACT FROM AUTHOR]

Background: Nontyphoidal strains of Salmonella are a leading cause of death among HIV-infected Africans. Antibody-induced complement-mediated killing protects healthy Africans against Salmonella, but increased levels of anti-lipopolysaccharide (LPS) antibodies in some HIV-infected African adults block this killing. The objective was to understand how these high levels of anti-LPS antibodies interfere with the killing of Salmonella. Methodology/Principal Findings: Sera and affinity-purified antibodies from African HIV-infected adults that failed to kill invasive S. Typhimurium D23580 were compared to sera from HIV-uninfected and HIV-infected subjects with bactericidal activity. The failure of sera from certain HIV-infected subjects to kill Salmonella was found to be due to an inherent inhibitory effect of anti-LPS antibodies. This inhibition was concentration-dependent and strongly associated with IgA and IgG2 anti-LPS antibodies (p<0.0001 for both). IgG anti-LPS antibodies, from sera of HIV-infected individuals that inhibit killing at high concentration, induced killing when diluted. Conversely, IgG, from sera of HIV-uninfected adults that induce killing, inhibited killing when concentrated. IgM anti-LPS antibodies from all subjects also induced Salmonella killing. Finally, the inhibitory effect of high concentrations of anti-LPS antibodies is seen with IgM as well as IgG and IgA. No correlation was found between affinity or avidity, or complement deposition or consumption, and inhibition of killing. Conclusion/Significance: IgG and IgM classes of anti-S. Typhimurium LPS antibodies from HIV-infected and HIV-uninfected individuals are bactericidal, while at very high concentrations, anti-LPS antibodies of all classes inhibit in vitro killing of Salmonella. This could be due to a variety of mechanisms relating to the poor ability of IgA and IgG2 to activate complement, and deposition of complement at sites where it cannot insert in the bacterial membrane. Vaccine trials are required to understand the significance of lack of in vitro killing by anti-LPS antibodies from a minority of HIV-infected individuals with impaired immune homeostasis. [ABSTRACT FROM AUTHOR]

Background: Identification of species-specific trypanosome molecules is important for laboratory- and field-based research into epidemiology and disease diagnosis. Although Trypanosoma congolense is the most important trypanosome pathogen of cattle in Africa, no species-specific molecules found in infective bloodstream forms (BSF) of the parasites have been identified, thus limiting development of diagnostic tests. Methods: Immuno-mass spectrometric methods were used to identify a protein that is recognized by a T. congolense-specific monoclonal antibody (mAb) Tc6/42.6.4. The identified molecule was expressed as a recombinant protein in E. coli and was tested in several immunoassays for its ability to interact with the mAb. The three dimensional structure of the protein was modeled and compared to crystal- and NMR-structures of the homologous proteins from T. cruzi and T. brucei respectively, in order to examine structural differences leading to the different immunoreactivity of the T. congolense molecule. Enzyme-linked immunosorbent assays (ELISA) were used to measure antibodies produced by trypanosome-infected African cattle in order to assess the potential for use of T. congolense calflagin in a serodiagnostic assay. Results: The antigen recognized by the T. congolense-specific mAb Tc6/42.6.4 was identified as a flagellar calcium-binding protein, calflagin. The recombinant molecule showed immunoreactivity with the T. congolense-specific mAb confirming that it is the cognate antigen. Immunofluorescence experiments revealed that Ca2+ modulated the localization of the calflagin molecule in trypanosomes. Structural modelling and comparison with calflagin homologues from other trypanosomatids revealed four non-conserved regions on the surface of the T. congolense molecule that due to differences in surface chemistry and structural topography may form species-specific epitopes. ELISAs using the recombinant calflagin as antigen to detect antibodies in trypanosome-infected cattle showed that the majority of cattle had antibody responses. Area under the Receiver-Operating Characteristic (ROC) curves, associated with host IgG and IgM, were calculated to be 0.623 and 0.709 respectively, indicating a positive correlation between trypanosome infection and the presence of anti-calflagin antibodies. Conclusions: While calflagin is conserved among different species of African trypanosomes, our results show that T. congolense calflagin possesses unique epitopes that differentiate this protein from homologues in other trypanosome species. MAb Tc6/42.6.4 has clear utility as a laboratory tool for identifying T. congolense. T. congolense calflagin has potential as a serodiagnostic antigen and should be explored further for its utility in antigen-detection assays for diagnosis of cattle infections. [ABSTRACT FROM AUTHOR]

Background: Severe fever with thrombocytopenia syndrome (SFTS) is a tick-borne infectious disease with a high case fatality rate, and is caused by the SFTS virus (SFTSV). SFTS is endemic to China, South Korea, and Japan. The viral RNA level in sera of patients with SFTS is known to be strongly associated with outcomes. Virological SFTS diagnosis with high sensitivity and specificity are required in disease endemic areas. Methodology/Principal Findings: We generated novel monoclonal antibodies (MAbs) against the SFTSV nucleocapsid (N) protein and developed a sandwich antigen (Ag)-capture enzyme-linked immunosorbent assay (ELISA) for the detection of N protein of SFTSV using MAb and polyclonal antibody as capture and detection antibodies, respectively. The Ag-capture system was capable of detecting at least 350–1220 TCID50/100 μl/well from the culture supernatants of various SFTSV strains. The efficacy of the Ag-capture ELISA in SFTS diagnosis was evaluated using serum samples collected from patients suspected of having SFTS in Japan. All 24 serum samples (100%) containing high copy numbers of viral RNA (>105 copies/ml) showed a positive reaction in the Ag-capture ELISA, whereas 12 out of 15 serum samples (80%) containing low copy numbers of viral RNA (<105 copies/ml) showed a negative reaction in the Ag-capture ELISA. Among these Ag-capture ELISA-negative 12 samples, 9 (75%) were positive for IgG antibodies against SFTSV. Conclusions: The newly developed Ag-capture ELISA is useful for SFTS diagnosis in acute phase patients with high levels of viremia. [ABSTRACT FROM AUTHOR]

Background: Severe fever with thrombocytopenia syndrome (SFTS) is caused by SFTS virus (SFTSV), a tick-borne phlebovirus in family Bunyaviridae. Studies have found that humans, domestic and wildlife animals can be infected by SFTSV. However, the viral ecology, circulation, and transmission remain largely unknown. Methodology/Principal Findings: Sixty seven human SFTS cases were reported and confirmed by virus isolation or immunofluorescence assay between 2011 and 2014. In 2013–2014 we collected 9,984 ticks from either vegetation or small wild mammals in the endemic area in Jiangsu, China, and detected SFTSV-RNA by real-time RT-PCR in both questing and feeding Haemaphysalis longicornis and H. flava. Viral RNA was identified in larvae of H. longicornis prior to a first blood meal, which has never been confirmed previously in nature. SFTSV-RNA and antibodies were also detected by RT-PCR and ELISA, respectively, in wild mammals including Erinaceus europaeus and Sorex araneus. A live SFTSV was isolated from Erinaceus europaeus captured during the off tick-feeding season and with a high SFTSV antibody titer. Furthermore, SFTSV antibodies were detected in the migratory birds Anser cygnoides and Streptopelia chinensis using ELISA. Conclusions/Significance: The detection of SFTSV-RNA in non-engorged larvae indicated that vertical transmission of SFTSV in H. longicornis might occur in nature, which suggests that H. longicornis is a putative reservoir host of SFTSV. Small wild mammals such as Erinaceus europaeus and Sorex araneus could be infected by SFTSV and may serve as natural amplifying hosts. Our data unveiled that wild birds could be infected with SFTSV or carry SFTSV-infected ticks and thus might contribute to the long-distance spread of SFTSV via migratory flyways. These findings provide novel insights for understanding SFTSV ecology, reservoir hosts, and transmission in nature and will help develop new measures in preventing its rapid spread both regionally and globally. [ABSTRACT FROM AUTHOR]

Rift Valley fever virus (RVFV) is a mosquito-borne pathogen that affects domesticated ruminants and occasionally humans. Classical RVF vaccines are based on formalin-inactivated virus or the live-attenuated Smithburn strain. The inactivated vaccine is highly safe but requires multiple administrations and yearly re-vaccinations. Although the Smithburn vaccine provides solid protection after a single vaccination, this vaccine is not safe for pregnant animals. An alternative live-attenuated vaccine, named Clone 13, carries a large natural deletion in the NSs gene which encodes the major virulence factor of the virus. The Clone 13 vaccine was previously shown to be safe for young lambs and calves. Moreover, a study in pregnant ewes suggested that the vaccine could also be applied safely during gestation. To anticipate on a possible future incursion of RVFV in Europe, we have evaluated the safety of Clone 13 for young lambs and pregnant ewes. In line with the guidelines from the World Organisation for Animal health (Office International des Epizooties, OIE) and regulations of the European Pharmacopeia (EP), these studies were performed with an overdose. Our studies with lambs showed that Clone 13 dissemination within vaccinated animals is very limited. Moreover, the Clone 13 vaccine virus was not shed nor spread to in-contact sentinels and did not revert to virulence upon animal-to-animal passage. Importantly, a large experiment with pregnant ewes demonstrated that the Clone 13 virus is able to spread to the fetus, resulting in malformations and stillbirths. Altogether, our results suggest that Clone 13 can be applied safely in lambs, but that caution should be taken when Clone 13 is used in pregnant animals, particularly during the first trimester of gestation. [ABSTRACT FROM AUTHOR]

Epidemiological studies have reported that most of the severe dengue cases occur upon a secondary heterologous infection. Furthermore, babies born to dengue immune mothers are at greater risk of developing severe disease upon primary infection with a heterologous or homologous dengue virus (DENV) serotype when maternal antibodies reach sub-neutralizing concentrations. These observations have been explained by the antibody mediated disease enhancement (ADE) phenomenon whereby heterologous antibodies or sub-neutralizing homologous antibodies bind to but fail to neutralize DENV particles, allowing Fc-receptor mediated entry of the virus-antibody complexes into host cells. This eventually results in enhanced viral replication and heightened inflammatory responses. In an attempt to replicate this ADE phenomenon in a mouse model, we previously reported that upon DENV2 infection 5-week old type I and II interferon (IFN) receptors-deficient mice (AG129) born to DENV1-immune mothers displayed enhancement of disease severity characterized by increased virus titers and extensive vascular leakage which eventually led to the animals’ death. However, as dengue occurs in immune competent individuals, we sought to reproduce this mouse model in a less immunocompromised background. Here, we report an ADE model that is mediated by maternal antibodies in type I IFN receptor-deficient A129 mice. We show that 5-week old A129 mice born to DENV1-immune mothers succumbed to a DENV2 infection within 4 days that was sub-lethal in mice born to naïve mothers. Clinical manifestations included extensive hepatocyte vacuolation, moderate vascular leakage, lymphopenia, and thrombocytopenia. Anti-TNFα therapy totally protected the mice and correlated with healthy hepatocytes. In contrast, blocking IL-6 did not impact the virus titers or disease outcome. This A129 mouse model of ADE may help dissecting the mechanisms involved in dengue pathogenesis and evaluate the efficacy of vaccine and therapeutic candidates. [ABSTRACT FROM AUTHOR]

Background: Conventional diagnostic methods for human ascariasis are based on the detection of Ascaris lumbricoides eggs in stool samples. However, studies of ascariasis in pigs have shown that the prevalence and the number of eggs detected in the stool do not correlate well with exposure of the herd to the parasite. On the other hand, an ELISA test measuring antibodies to Ascaris suum haemoglobin (AsHb) has been shown to be useful for estimating transmission intensity on pig farms. In this study, we further characterized the AsHb antigen and screened samples from a population-based study conducted in an area that is endemic for Ascaris lumbricoides in Indonesia to assess changes in AsHb antibody rates and levels in humans following mass drug administration (MDA). Methodology/Principal findings: We developed and evaluated an ELISA to detect human IgG4 antibodies to AsHb. We tested 1066 plasma samples collected at different times from 599 subjects who lived in a village in rural Indonesia that was highly endemic for ascariasis. The community received 6 rounds of MDA for lymphatic filariasis with albendazole plus diethylcarbamazine between 2002 and 2007. While the AsHb antibody assay was not sensitive for detecting all individuals with Ascaris eggs in their stools, the percentage of seropositive individuals decreased rapidly following MDA. Reductions in antibody rates reflected decreased mean egg output per person both at the community level and in different age groups. Two years after the last round of MDA the community egg output and antibody prevalence rate were reduced by 81.6% and 78.9% respectively compared to baseline levels. Conclusion/Significance: IgG4 antibody levels to AsHb appear to reflect recent exposure to Ascaris. The antibody prevalence rate may be a useful indicator for Ascaris transmission intensity in communities that can be used to assess the impact of control measures on the force of transmission. [ABSTRACT FROM AUTHOR]

Background: A vaccine to prevent infection and disease caused by Plasmodium vivax is needed both to reduce the morbidity caused by this parasite and as a key component in efforts to eradicate malaria worldwide. Vivax malaria protein 1 (VMP001), a novel chimeric protein that incorporates the amino- and carboxy- terminal regions of the circumsporozoite protein (CSP) and a truncated repeat region that contains repeat sequences from both the VK210 (type 1) and the VK247 (type 2) parasites, was developed as a vaccine candidate for global use. Methods: We conducted a first-in-human Phase 1 dose escalation vaccine study with controlled human malaria infection (CHMI) of VMP001 formulated in the GSK Adjuvant System AS01B. A total of 30 volunteers divided into 3 groups (10 per group) were given 3 intramuscular injections of 15μg, 30μg, or 60μg respectively of VMP001, all formulated in 500μL of AS01B at each immunization. All vaccinated volunteers participated in a P. vivax CHMI 14 days following the third immunization. Six non-vaccinated subjects served as infectivity controls. Results: The vaccine was shown to be well tolerated and immunogenic. All volunteers generated robust humoral and cellular immune responses to the vaccine antigen. Vaccination did not induce sterile protection; however, a small but significant delay in time to parasitemia was seen in 59% of vaccinated subjects compared to the control group. An association was identified between levels of anti-type 1 repeat antibodies and prepatent period. Significance: This trial was the first to assess the efficacy of a P. vivax CSP vaccine candidate by CHMI. The association of type 1 repeat-specific antibody responses with delay in the prepatency period suggests that augmenting the immune responses to this domain may improve strain-specific vaccine efficacy. The availability of a P. vivax CHMI model will accelerate the process of P. vivax vaccine development, allowing better selection of candidate vaccines for advancement to field trials. [ABSTRACT FROM AUTHOR]

Borrelia persica, a bacterium transmitted by the soft tick Ornithodoros tholozani, causes tick-borne relapsing fever in humans in the Middle East, Central Asia and the Indian peninsula. Immunocompetent C3H/HeOuJ mice were infected intradermally with B. persica at varying doses: 1 x 106, 1 x 104, 1 x 102 and 4 x 100 spirochetes/mouse. Subsequently, blood samples were collected and screened for the presence of B. persica DNA. Spirochetes were detected in all mice infected with 1 x 106, 1 x 104 and 1 x 102 borrelia by real-time PCR targeting the flaB gene of the bacterium. Spirochetemia developed with a one- to two-day delay when 1 x 104 and 1 x 102 borrelia were inoculated. Mice injected with only four organisms were negative in all tests. No clinical signs were observed when infected mice were compared to negative control animals. Organs (heart, spleen, urinary bladder, tarsal joint, skin and brain) were tested for B. persica-specific DNA and cultured for the detection of viable spirochetes. Compiled data show that the target organs of B. persica infections are the brain and the skin. A newly developed serological two-tiered test system (ELISA and western blot) for the detection of murine IgM, IgG and IgA antibody titers against B. persica showed a vigorous antibody response of the mice during infection. In conclusion, the infection model described here for B. persica is a platform for in vivo studies to decipher the so far unexplored survival strategies of this Borrelia species. [ABSTRACT FROM AUTHOR]

Background: Dengue has emerged as one of the most important infectious diseases in the last five decades. Evidence indicates the expansion of dengue virus endemic areas and consequently the exponential increase of dengue virus infections across the subtropics. The clinical manifestations of dengue virus infection include sudden fever, rash, headache, myalgia and in more serious cases, spontaneous bleeding. These manifestations occur in children as well as in adults. Defining the epidemiology of dengue in a given area is critical to understanding the disease and devising effective public health strategies. Methodology/Principal Findings: Here, we report the results from a prospective cohort study of 4380 adults in West Java, Indonesia, from 2000–2004 and 2006–2009. A total of 2167 febrile episodes were documented and dengue virus infections were confirmed by RT-PCR or serology in 268 cases (12.4%). The proportion ranged from 7.6 to 41.8% each year. The overall incidence rate of symptomatic dengue virus infections was 17.3 cases/1,000 person years and between September 2006 and April 2008 asymptomatic infections were 2.6 times more frequent than symptomatic infections. According to the 1997 WHO classification guidelines, there were 210 dengue fever cases, 53 dengue hemorrhagic fever cases (including one dengue shock syndrome case) and five unclassified cases. Evidence for sequential dengue virus infections was seen in six subjects. All four dengue virus serotypes circulated most years. Inapparent dengue virus infections were predominantly associated with DENV-4 infections. Conclusions/Significance: Dengue virus was responsible for a significant percentage of febrile illnesses in an adult population in West Java, Indonesia, and this percentage varied from year to year. The observed incidence rate during the study period was 43 times higher than the reported national or provincial rates during the same time period. A wide range of clinical severity was observed with most infections resulting in asymptomatic disease. The circulation of all four serotypes of dengue virus was observed in most years of the study. [ABSTRACT FROM AUTHOR]

Background: Buruli ulcer, caused by infection with Mycobacterium ulcerans, is a chronic ulcerative neglected tropical disease of the skin and subcutaneous tissue that is most prevalent in West African countries. M. ulcerans produces a cytotoxic macrolide exotoxin called mycolactone, which causes extensive necrosis of infected subcutaneous tissue and the development of characteristic ulcerative lesions with undermined edges. While cellular immune responses are expected to play a key role against early intracellular stages of M. ulcerans in macrophages, antibody mediated protection might be of major relevance against advanced stages, where bacilli are predominantly found as extracellular clusters. Methodology/Principal Findings: To assess whether vaccine induced antibodies against surface antigens of M. ulcerans can protect against Buruli ulcer we formulated two surface vaccine candidate antigens, MUL_2232 and MUL_3720, as recombinant proteins with the synthetic Toll-like receptor 4 agonist glucopyranosyl lipid adjuvant-stable emulsion. The candidate vaccines elicited strong antibody responses without a strong bias towards a TH1 type cellular response, as indicated by the IgG2a to IgG1 ratio. Despite the cross-reactivity of the induced antibodies with the native antigens, no significant protection was observed against progression of an experimental M. ulcerans infection in a mouse footpad challenge model. Conclusions: Even though vaccine-induced antibodies have the potential to opsonise the extracellular bacilli they do not have a protective effect since infiltrating phagocytes might be killed by mycolactone before reaching the bacteria, as indicated by lack of viable infiltrates in the necrotic infection foci. [ABSTRACT FROM AUTHOR]

Background: Infectious diseases pose a severe worldwide threat to human and livestock health. While early diagnosis could enable prompt preventive interventions, the majority of diseases are found in rural settings where basic laboratory facilities are scarce. Under such field conditions, point-of-care immunoassays provide an appropriate solution for rapid and reliable diagnosis. The limiting steps in the development of the assay are the identification of a suitable target antigen and the selection of appropriate high affinity capture and detection antibodies. To meet these challenges, we describe the development of a Nanobody (Nb)-based antigen detection assay generated from a Nb library directed against the soluble proteome of an infectious agent. In this study, Trypanosoma congolense was chosen as a model system. Methodology/Principal Findings: An alpaca was vaccinated with whole-parasite soluble proteome to generate a Nb library from which the most potent T. congolense specific Nb sandwich immunoassay (Nb474H-Nb474B) was selected. First, the Nb474-homologous sandwich ELISA (Nb474-ELISA) was shown to detect experimental infections with high Positive Predictive Value (98%), Sensitivity (87%) and Specificity (94%). Second, it was demonstrated under experimental conditions that the assay serves as test-of-cure after Berenil treatment. Finally, this assay allowed target antigen identification. The latter was independently purified through immuno-capturing from (i) T. congolense soluble proteome, (ii) T. congolense secretome preparation and (iii) sera of T. congolense infected mice. Subsequent mass spectrometry analysis identified the target as T. congolense glycosomal aldolase. Conclusions/Significance: The results show that glycosomal aldolase is a candidate biomarker for active T. congolense infections. In addition, and by proof-of-principle, the data demonstrate that the Nb strategy devised here offers a unique approach to both diagnostic development and target discovery that could be widely applied to other infectious diseases. [ABSTRACT FROM AUTHOR]

Background: Entamoeba histolytica is an intestinal protozoan parasite that causes amoebiasis, including amebic dysentery and liver abscesses. E. histolytica invades host tissues by adhering onto cells and phagocytosing them depending on the adaptation and expression of pathogenic factors, including Gal/GalNAc lectin. We have previously reported that E. histolytica possesses multiple CXXC sequence motifs, with the intermediate subunit of Gal/GalNAc lectin (i.e., Igl) as a key factor affecting the amoeba's pathogenicity. The present work showed the effect of immunization with recombinant Igl on amebic liver abscess formation and the corresponding immunological properties. Methodology/Principal Findings: A prokaryotic expression system was used to prepare the full-length Igl and the N-terminal, middle, and C-terminal fragments (C-Igl) of Igl. Vaccine efficacy was assessed by challenging hamsters with an intrahepatic injection of E. histolytica trophozoites. Hamsters intramuscularly immunized with full-length Igl and C-Igl were found to be 92% and 96% immune to liver abscess formation, respectively. Immune-response evaluation revealed that C-Igl can generate significant humoral immune responses, with high levels of antibodies in sera from immunized hamsters inhibiting 80% of trophozoites adherence to mammalian cells and inducing 80% more complement-mediated lysis of trophozoites compared with the control. C-Igl was further assessed for its cellular response by cytokine-gene qPCR analysis. The productions of IL-4 (8.4-fold) and IL-10 (2-fold) in the spleen cells of immunized hamsters were enhanced after in vitro stimulation. IL-4 expression was also supported by increased programmed cell death 1 ligand 1 gene. Conclusions/Significance: Immunobiochemical characterization strongly suggests the potential of recombinant Igl, especially the C-terminal fragment, as a vaccine candidate against amoebiasis. Moreover, protection through Th2-cell participation enabled effective humoral immunity against amebic liver abscesses. [ABSTRACT FROM AUTHOR]