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In sub-Saharan Africa, invasive nontyphoidal Salmonella (iNTS) infections with serovars S. Enteritidis, S. Typhimurium and I 4,[],12:i:- are widespread in children < 5 years old. Development of an efficacious vaccine would provide an important public health tool to prevent iNTS disease in this population. Glycoconjugates of S. Enteritidis core and O-polysaccharide (COPS) coupled to the homologous serovar phase 1 flagellin protein (FliC) were previously shown to be immunogenic and protected adult mice against death following challenge with a virulent Malian S. Enteritidis blood isolate. This study extends these observations to immunization of mice in early life and also assesses protection with partial and full regimens. Anti-COPS and anti-FliC serum IgG titers were assessed in infant and adult mice after immunization with 1, 2 or 3 doses of S. Enteritidis COPS:FliC alone or co-formulated with aluminum hydroxide or monophosphoryl lipid A (MPL) adjuvants. S. Enteritidis COPS:FliC was immunogenic in both age groups, although the immune responses were quantitatively lower in infants. Kinetics of antibody production were similar for the native and adjuvanted formulations after three doses; conjugates formulated with MPL elicited significantly increased anti-COPS IgG titers in adult but not infant mice. Nevertheless, robust protection against S. Enteritidis challenge was seen for all three formulations when three doses were given either during infancy or as adults. We further found that significant protection could be achieved with two COPS:FliC doses, despite elicitation of modest serum anti-COPS IgG antibody titers. These findings guide potential immunization strategies that may be translated to develop a human pediatric iNTS vaccine for sub-Saharan Africa. [ABSTRACT FROM AUTHOR]

Rift Valley fever virus (RVFV) is an important mosquito-borne veterinary and human pathogen that has caused large outbreaks of severe disease throughout Africa and the Arabian Peninsula. Currently, no licensed vaccine or therapeutics exists to treat this potentially deadly disease. The explosive nature of RVFV outbreaks and the severe consequences of its accidental or intentional introduction into RVFV-free areas provide the impetus for the development of novel vaccine candidates for use in both livestock and humans. Rationally designed vaccine candidates using reverse genetics have been used to develop deletion mutants of two known RVFV virulence factors, the NSs and NSm genes. These recombinant viruses were demonstrated to be protective and immunogenic in rats, mice, and sheep, without producing clinical illness in these animals. Here, we expand upon those findings and evaluate the single deletion mutant (ΔNSs rRVFV) and double deletion mutant (ΔNSs-ΔNSm rRVFV) vaccine candidates in the common marmoset (Callithrix jacchus), a non-human primate (NHP) model resembling severe human RVF disease. We demonstrate that both the ΔNSs and ΔNSs-ΔNSm rRVFV vaccine candidates were found to be safe and immunogenic in the current study. The vaccinated animals received a single dose of vaccine that led to the development of a robust antibody response. No vaccine-induced adverse reactions, signs of clinical illness or infectious virus were detected in the vaccinated marmosets. All vaccinated animals that were subsequently challenged with RVFV were protected against viremia and liver disease. In summary, our results provide the basis for further development of the ΔNSs and ΔNSs-ΔNSm rRVFV as safe and effective human RVFV vaccines for this significant public health threat. [ABSTRACT FROM AUTHOR]

A reliable and effective human challenge model is needed to help down-select the most promising ETEC vaccines currently under development. Such a model would need to reliably induce diarrhea in a high proportion of volunteers using the lowest possible inoculum to maximize safety and sensitivity. Previously we validated a challenge model that utilized a dose of 2x107 CFU of ETEC strain H10407 (LT+, ST+, CFA/I+ and O78+) to induce attack rates for moderate to severe diarrhea (MSD) of ~60–70%. Here we detail efforts to further refine the model in an attempt to determine if a lower challenge dose of H10407 can be used. Thirty subjects were randomized 1:1 to receive an oral administration of H10407 at doses of 106 or 105 CFU in bicarbonate buffer. After challenge, subjects were monitored for signs and symptoms of enteric illness and stool samples were collected to detect shedding of the challenge strain. Systemic and mucosal immune responses were measured using serum, antibody in lymphocyte supernatant and fecal samples. The attack rate was 13.3% (2/15) and 26.7% (4/15) for MSD in the 105 and 106 groups, respectively. Four MSD cases met criteria for early antibiotic treatment. All subjects but one shed the challenge strain in fecal samples. The frequency and magnitude of anti-LT toxin, CFA/I and LPS O78 immune responses were antigen, dose, severity of diarrhea and shedding levels dependent. Notably, although of lower magnitude, there were considerable immune responses in the subjects with no diarrhea. This may indicate that immune responses to asymptomatic infections of ETEC in children in the endemic countries may contribute to protection. Based on this and our prior studies, we conclude that a dose of 2x107 H10407 remains the lowest practical dose for use in future volunteer studies evaluating candidate vaccines and other preventive or therapeutic ETEC interventions. Trial registration: ClinicalTrials.gov . [ABSTRACT FROM AUTHOR]

Background: The mediators of protection against cholera, a severe dehydrating illness of humans caused by Vibrio cholerae, are unknown. We have previously shown that plasma IgA as well as memory B IgG cells targeting lipopolysaccharide (LPS) of Vibrio cholerae O1 correlate with protection against V. cholerae O1 infection among household contacts of cholera patients. Protection against cholera is serogroup specific, and serogroup specificity is defined by the O-specific polysaccharide (OSP) component of LPS. Therefore, we prospectively followed household contacts of cholera patients to determine whether OSP-specific immune responses present at the time of enrollment are associated with protection against V. cholerae infection. Methodology: In this study, we enrolled two hundred forty two household contacts of one hundred fifty index patients who were infected with Vibrio cholerae. We determined OSP-specific memory B cells and plasma IgA, IgG and IgM antibody responses on study entry (day 2). Principle findings: The presence of OSP-specific plasma IgA, IgM, and IgG antibody responses on study entry were associated with a decrease in the risk of infection in household contacts (IgA, p = 0.015; IgM, p = 0.01, and IgG, p = 0.024). In addition, the presence of OSP-specific IgG memory B cell responses in peripheral blood on study entry was also associated with a decreased risk of infection (44% reduction; 95% CI: 31.1 to 99.8) in contacts. No protection was associated with cholera toxin B subunit (CtxB)-specific memory B cell responses. Conclusion: These results suggest that immune responses that target OSP, both in plasma and memory responses, may be important in mediating protection against infection with V. cholerae O1. [ABSTRACT FROM AUTHOR]

Background: Evidence from recent studies assessing the impact of school water, sanitation and hygiene (WASH) interventions on child health has been mixed. Self-reports of disease are subject to bias, and few WASH impact evaluations employ objective health measures to assess reductions in disease and exposure to pathogens. We utilized antibody responses from dried blood spots (DBS) to measure the impact of a school WASH intervention on infectious disease among pupils in Mali. Methodology/Principal findings: We randomly selected 21 beneficiary primary schools and their 21 matched comparison schools participating in a matched-control trial of a comprehensive school-based WASH intervention in Mali. DBS were collected from 20 randomly selected pupils in each school (n = 807). We analyzed eluted IgG from the DBS using a Luminex multiplex bead assay to 28 antigens from 17 different pathogens. Factor analysis identified three distinct latent variables representing vector-transmitted disease (driven primarily by dengue), food/water-transmitted enteric disease (driven primarily by Escherichia coli and Vibrio cholerae), and person-to-person transmitted enteric disease (driven primarily by norovirus). Data were analyzed using a linear latent variable model. Antibody evidence of food/water-transmitted enteric disease (change in latent variable mean (β) = -0.24; 95% CI: -0.53, -0.13) and person-to-person transmitted enteric disease (β = -0.17; 95% CI: -0.42, -0.04) was lower among pupils attending beneficiary schools. There was no difference in antibody evidence of vector-transmitted disease (β = 0.11; 95% CI: -0.05, 0.33). Conclusions/Significance: Evidence of enteric disease was lower among pupils attending schools benefitting from school WASH improvements than students attending comparison schools. These findings support results from the parent study, which also found reduced incidence of self-reported diarrhea among pupils of beneficiary schools. DBS collection was feasible in this resource-poor field setting and provided objective evidence of disease at a low cost per antigen analyzed, making it an effective measurement tool for the WASH field. Trial registration: The trial was registered at ClinicalTrials.gov () [ABSTRACT FROM AUTHOR]

Background: Lymphocytic choriomeningitis virus (LCMV) causes a variety of diseases, including asymptomatic infections, meningitis, and congenital infections in the fetus of infected mother. The development of a safe and effective vaccine against LCMV is imperative. This study aims to develop a new candidate vaccine against LCMV using a recombinant replication-incompetent rabies virus (RV) vector. Methodology/Principal findings: In this study, we have generated a recombinant deficient RV expressing the LCMV glycoprotein precursor (GPC) (RVΔP-LCMV/GPC) which is lacking the RV-P gene. RVΔP-LCMV/GPC is able to propagate only in cells expressing the RV-P protein. In contrast, the LCMV-GPC can be expressed in general cells, which do not express RV-P protein. The ability of RVΔP-LCMV/GPC to protect mice from LCMV infection and induce cellular immunity was assessed. Mice inoculated intraperitoneally with RVΔP-LCMV/GPC showed higher survival rates (88.2%) than those inoculated with the parental recombinant RV-P gene-deficient RV (RVΔP) (7.7%) following a LCMV challenge. Neutralizing antibody (NAb) against LCMV was not induced, even in the sera of surviving mice. CD8+ T-cell depletion significantly reduced the survival rates of RVΔP-LCMV/GPC-inoculated mice after the LCMV challenge. These results suggest that CD8+ T cells play a major role in the observed protection against LCMV. In contrast, NAbs against RV were strongly induced in sera of mice inoculated with either RVΔP-LCMV/GPC or RVΔP. In safety tests, suckling mice inoculated intracerebrally with RVΔP-LCMV/GPC showed no symptoms. Conclusions/Significance: These results show RVΔP-LCMV/GPC might be a promising candidate vaccine with dual efficacy, protecting against both RV and LCMV. [ABSTRACT FROM AUTHOR]

Brucellosis is an important zoonotic disease causing huge economic losses worldwide. Currently no effective immunotherapy for Brucellosis or any biomarker to monitor the efficacy of therapy is available. Treatment is ineffective and animals remain carrier lifelong. S19 and RB51 are live attenuated vaccine strains of Brucella abortus. However, S19 induces only antibody, ineffective for intracellular pathogen. RB51 induces cell mediated immunity (CMI) but it is Rifampicin resistant. Both organisms are secreted in milk and can infect humans and cause abortions in animals. Phage lysed bacteria (lysates) retain maximum immunogenicity as opposed to killing by heat or chemicals. We report here the successful immunotherapy of bovine Brucellosis by phage lysates of RB51 (RL) and S19 (SL). The SL induced strong antibody response and RL stimulated CMI. In vitro restimulation of leukocytes from RL immunized cattle induced interferon gamma production. A single subcutaneous dose of 2 ml of cocktail lysate (both RL and SL), eliminated live virulent Brucella from Brucellosis affected cattle with plasma level of Brucella specific 223 bp amplicon undetectable by RT-PCR and blood negative for live Brucella by culture in 3 monthspost-immunization. This is the first report on minimally invasive monitoring of the efficacy of antibacterial therapy employing plasma RNA specific for live bacteria as a biomarker as well as on the use of RB51 phage lysate for successful immunotherapy of Brucellosis in cattle. [ABSTRACT FROM AUTHOR]

West Nile virus (WNV), a zoonotic pathogen naturally transmitted by mosquitoes whose natural hosts are birds, has spread worldwide during the last few decades. Resident birds play an important role in flavivirus epidemiology, since they can serve as reservoirs and facilitate overwintering of the virus. Herein, we report the first experimental infection of magpie (Pica pica) with two strains of West Nile virus, lineages 1 (NY-99) and 2 (SRB Novi-Sad/12), which are currently circulating in Europe. Magpies were highly susceptible to WNV infection, with similar low survival rates (30% and 42.8%) for both lineages. All infected magpies developed viremia detectable at 3 days post-infection with titers above those necessary for successful transmission of WNV to a mosquito. Neutralizing antibodies were detected at all time points analyzed (from 7 to 17 days post-infection). WNV genome was detected in the brains and hearts of all magpies that succumbed to the infection, and, in some of the surviving birds. WNV-RNA was amplified from swabs (oral and cloacal) at 3, 6 and 7 days post-infection and feather pulps, from 3 to 17 days post-infection, of infected animals. Even more, infectious virus was recovered from swabs up to 7 days post-infection and from feather pulps up to 10 days post infection. Sham-infected control animals were negative for viremia, viral RNA, and antibodies. These results suggest that the magpie, which is one of the most abundant corvid species in Europe, could represent a source of WNV transmission for birds and humans. Our observations shed light on the pathogenesis, transmission, and ecology of WNV and can benefit the implementation of surveillance and control programs. [ABSTRACT FROM AUTHOR]

Background: Cholera is an acute voluminous dehydrating diarrheal disease caused by toxigenic strains of Vibrio cholerae O1 and occasionally O139. A growing body of evidence indicates that immune responses targeting the O-specific polysaccharide (OSP) of V. cholerae are involved in mediating protection against cholera. We therefore assessed whether antibody responses against OSP occur after vaccination with live attenuated oral cholera vaccine CVD 103-HgR, and whether such responses correlate with protection against cholera. Methodology: We assessed adult North American volunteers (n = 46) who were vaccinated with 5 × 108 colony-forming units (CFU) of oral cholera vaccine CVD 103-HgR and then orally challenged with approximately 1 × 105 CFU of wild-type V. cholerae O1 El Tor Inaba strain N16961, either 10 or 90 days post-vaccination. Principal findings: Vaccination was associated with induction of significant serum IgM and IgA anti-OSP and vibriocidal antibody responses within 10 days of vaccination. There was significant correlation between anti-OSP and vibriocidal antibody responses. IgM and IgA anti-OSP responses on day 10 following vaccination were associated with lower post-challenge stool volume (r = −0.44, P = 0.002; r = −0.36, P = 0.01; respectively), and none of 27 vaccinees who developed a ≥1.5 fold increase in any antibody isotype targeting OSP on day 10 following vaccination compared to baseline developed moderate or severe cholera following experimental challenge, while 5 of 19 who did not develop such anti-OSP responses did (P = 0.01). Conclusion: Oral vaccination with live attenuated cholera vaccine CVD 103-HgR induces antibodies that target V. cholerae OSP, and these anti-OSP responses correlate with protection against diarrhea following experimental challenge with V. cholerae O1. Trial registration: ClinicalTrials.gov [ABSTRACT FROM AUTHOR]

Background: The classical method for detection of Lassa virus-specific antibodies is the immunofluorescence assay (IFA) using virus-infected cells as antigen. However, IFA requires laboratories of biosafety level 4 for assay production and an experienced investigator to interpret the fluorescence signals. Therefore, we aimed to establish and evaluate enzyme-linked immunosorbent assays (ELISA) using recombinant Lassa virus nucleoprotein (NP) as antigen. Methodology/Principal findings: The IgM ELISA is based on capturing IgM antibodies using anti-IgM, and the IgG ELISA is based on capturing IgG antibody–antigen complexes using rheumatoid factor or Fc gamma receptor CD32a. Analytical and clinical evaluation was performed with 880 sera from Lassa fever endemic (Nigeria) and non-endemic (Ghana and Germany) areas. Using the IFA as reference method, we observed 91.5–94.3% analytical accuracy of the ELISAs in detecting Lassa virus-specific antibodies. Evaluation of the ELISAs for diagnosis of Lassa fever on admission to hospital in an endemic area revealed a clinical sensitivity for the stand-alone IgM ELISA of 31% (95% CI 25–37) and for combined IgM/IgG detection of 26% (95% CI 21–32) compared to RT-PCR. The specificity of IgM and IgG ELISA was estimated at 96% (95% CI 93–98) and 100% (95% CI 99–100), respectively, in non-Lassa fever patients from non-endemic areas. In patients who seroconverted during follow-up, Lassa virus-specific IgM and IgG developed simultaneously rather than sequentially. Consistent with this finding, isolated IgM reactivity, i.e. IgM in the absence of IgG, had no diagnostic value. Conclusions/Significance: The ELISAs are not equivalent to RT-PCR for early diagnosis of Lassa fever; however, they are of value in diagnosing patients at later stage. The IgG ELISA may be useful for epidemiological studies and clinical trials due its high specificity, and the higher throughput rate and easier operation compared to IFA. [ABSTRACT FROM AUTHOR]

Background: Diagnosing scrub typhus clinically is difficult, hence laboratory tests play a very important role in diagnosis. As performing sophisticated laboratory tests in resource-limited settings is not feasible, accurate point-of-care testing (POCT) for scrub typhus diagnosis would be invaluable for patient diagnosis and management. Here we summarise the existing evidence on the accuracy of scrub typhus POCTs to inform clinical practitioners in resource-limited settings of their diagnostic value. Methodology/principal findings: Studies on POCTs which can be feasibly deployed in primary health care or outpatient settings were included. Thirty-one studies were identified through PubMed and manual searches of reference lists. The quality of the studies was assessed with the Quality Assessment of Diagnostic Accuracy Studies 2 (QUADAS-2). About half (n = 14/31) of the included studies were of moderate quality. Meta-analysis showed the pooled sensitivity and specificity of commercially available immunochromatographic tests (ICTs) were 66.0% (95% CI 0.37–0.86) and 92.0% (95% CI 0.83–0.97), respectively. There was a significant and high degree of heterogeneity between the studies (I2 value = 97.48%, 95% CI 96.71–98.24 for sensitivity and I2 value = 98.17%, 95% CI 97.67–98.67 for specificity). Significant heterogeneity was observed for total number of samples between studies (p = 0.01), study design (whether using case-control design or not, p = 0.01), blinding during index test interpretation (p = 0.02), and QUADAS-2 score (p = 0.01). Conclusions/significance: There was significant heterogeneity between the scrub typhus POCT diagnostic accuracy studies examined. Overall, the commercially available scrub typhus ICTs demonstrated better performance when ‘ruling in’ the diagnosis. There is a need for standardised methods and reporting of diagnostic accuracy to decrease between-study heterogeneity and increase comparability among study results, as well as development of an affordable and accurate antigen-based POCT to tackle the inherent weaknesses associated with serological testing. [ABSTRACT FROM AUTHOR]

The article focuses on the need to change the serological threshold to prevent mass drug administration (MDA) and develop an assessment strategy using accurate diagnostic tools for onchocerciasis, which is suggested by the World Health Organization (WHO). Topics discussed include the use of enzyme-linked immunosorbent assays (ELISA) and lateral flow-based assay, the importance of the sensitivity of the tools, and sample size. The transmission dynamics of the parasite is emphasized.

Zika virus (ZIKV) is an emerging arbovirus belonging to the genus flavivirus that comprises other important public health viruses, such as dengue (DENV) and yellow fever (YFV). In general, ZIKV infection is a self-limiting disease, however cases of Guillain-Barré syndrome and congenital brain abnormalities in newborn infants have been reported. Diagnosing ZIKV infection remains a challenge, as viral RNA detection is only applicable until a few days after the onset of symptoms. After that, serological tests must be applied, and, as expected, high cross-reactivity between ZIKV and other flavivirus serology is observed. Plaque reduction neutralization test (PRNT) is indicated to confirm positive samples for being more specific, however it is laborious intensive and time consuming, representing a major bottleneck for patient diagnosis. To overcome this limitation, we developed a high-throughput image-based fluorescent neutralization test for ZIKV infection by serological detection. Using 226 human specimens, we showed that the new test presented higher throughput than traditional PRNT, maintaining the correlation between results. Furthermore, when tested with dengue virus samples, it showed 50.53% less cross reactivity than MAC-ELISA. This fluorescent neutralization test could be used for clinical diagnosis confirmation of ZIKV infection, as well as for vaccine clinical trials and seroprevalence studies. [ABSTRACT FROM AUTHOR]

Recognizing cholera cases early, especially in the initial phase of an outbreak and in areas where cholera has not previously circulated, is a high public health priority. Laboratory capacity in such settings is often limited. To address this, we have developed a rapid diagnostic test (RDT) termed Cholkit that is based on an immunochromatographic lateral flow assay for the diagnosis of cholera cases using stool. Cholkit contains a monoclonal antibody (ICL-33) to the O-specific polysaccharide (OSP) component of V. cholerae O1 lipopolysaccharide, and recognizes both Inaba and Ogawa serotypes. We tested the Cholkit dipstick using fresh stool specimens of 76 adults and children presenting with acute watery diarrhea at the icddr,b hospital in Dhaka, Bangladesh. We compared Cholkit’s performance with those of microbial culture, PCR (targeting the rfb and ctxA genes of V. cholerae) and the commercially available RDT, Crystal VC (Span Diagnostics; Surat, India). We found that all stool specimens with a positive culture for V. cholerae O1 (n = 19) were positive by Cholkit as well as Crystal VC. We then used Bayesian latent class modeling to estimate the sensitivity and specificity of each diagnostic assay. The sensitivity of Cholkit, microbiological culture, PCR and Crystal VC was 98% (95% CI: 88–100), 71% (95% CI: 59–81), 74% (95% CI: 59–86) and 98% (95% CI: 88–100), respectively. The specificity for V. cholerae O1 was 97% (95% CI: 89–100), 100%, 97% (95% CI: 93–99) and 98% (95% CI: 92–100), respectively. Of note, two Crystal VC dipsticks were positive for V. cholerae O139 but negative by culture and PCR in this area without known circulating epidemic V. cholerae O139. In conclusion, the Cholkit dipstick is simple to use, requires no dedicated laboratory capacity, and has a sensitivity and specificity for V. cholerae O1 of 98% and 97%, respectively. Cholkit warrants further evaluation in other settings. [ABSTRACT FROM AUTHOR]

West Nile virus (WNV) and St. Louis encephalitis (SLEV) virus are enzootically maintained in North America in cycles involving the same mosquito vectors and similar avian hosts. However, these viruses exhibit dissimilar viremia and virulence phenotypes in birds: WNV is associated with high magnitude viremias that can result in mortality in certain species such as American crows (AMCRs, Corvus brachyrhynchos) whereas SLEV infection yields lower viremias that have not been associated with avian mortality. Cross-neutralization of these viruses in avian sera has been proposed to explain the reduced circulation of SLEV since the introduction of WNV in North America; however, in 2015, both viruses were the etiologic agents of concurrent human encephalitis outbreaks in Arizona, indicating the need to re-evaluate host factors and cross-neutralization responses as factors potentially affecting viral co-circulation. Reciprocal chimeric WNV and SLEV viruses were constructed by interchanging the pre-membrane (prM)-envelope (E) genes, and viruses subsequently generated were utilized herein for the inoculation of three different avian species: house sparrows (HOSPs; Passer domesticus), house finches (Haemorhous mexicanus) and AMCRs. Cross-protective immunity between parental and chimeric viruses were also assessed in HOSPs. Results indicated that the prM-E genes did not modulate avian replication or virulence differences between WNV and SLEV in any of the three avian species. However, WNV-prME proteins did dictate cross-protective immunity between these antigenically heterologous viruses. Our data provides further evidence of the important role that the WNV / SLEV viral non-structural genetic elements play in viral replication, avian host competence and virulence. [ABSTRACT FROM AUTHOR]

Dengue virus (DENV) infection imposes enormous health and economic burden worldwide with no approved treatment. Several small molecules, including lovastatin, celgosivir, balapiravir and chloroquine have been tested for potential anti-dengue activity in clinical trials; none of these have demonstrated a protective effect. Recently, based on identification and characterization of cross-serotype neutralizing antibodies, there is increasing attention on the potential for dengue immunotherapy. Here, we tested the ability of VIS513, an engineered cross-neutralizing humanized antibody targeting the DENV E protein domain III, to overcome antibody-enhanced infection and high but brief viremia, which are commonly encountered in dengue patients, in various in vitro and in vivo models. We observed that VIS513 efficiently neutralizes DENV at clinically relevant viral loads or in the presence of enhancing levels of DENV immune sera. Single therapeutic administration of VIS513 in mouse models of primary infection or lethal secondary antibody-enhanced infection, reduces DENV titers and protects from lethal infection. Finally, VIS513 administration does not readily lead to resistance, either in cell culture systems or in animal models of dengue infection. The findings suggest that rapid viral reduction during acute DENV infection with a monoclonal antibody is feasible. [ABSTRACT FROM AUTHOR]

Mosquito-borne Zika virus (ZIKV) typically causes a mild and self-limiting illness known as Zika fever. Since its recent emergence in 2014 in the American continent, ZIKV infection during pregnancy has been closely associated with a wide range of congenital abnormalities. To date, no vaccines or antivirals are publicly available. We developed Zika virus-like particles (VLPs) and evaluated their immunogenicity and protective efficacy in mouse models. ZIKV VLPs (ZIKVLPs) formulated with alum were injected into 6-8-week-old interferon deficient AG129 mice as well as wild type BALB/c mice. Control mice received PBS/alum. Animals were challenged with 200 PFU (>1000 AG129 LD50s) of ZIKV strain H/PF/2013. All vaccinated mice survived with no morbidity or weight loss while control animals either died at 9 days post challenge (AG129) or had increased viremia (BALB/c). Neutralizing antibodies were observed in all ZIKVLP vaccinated mice. The role of neutralizing antibodies in protecting mice was demonstrated by passive transfer. Our findings demonstrate the protective efficacy of the ZIKVLP vaccine and highlight the important role that neutralizing antibodies play in protection against ZIKV infection. [ABSTRACT FROM AUTHOR]

Background: Vibrio cholerae causes over 2 million cases of cholera and 90,000 deaths each year. Serosurveillance can be a useful tool for estimating the intensity of cholera transmission and prioritizing populations for cholera control interventions. Current methods involving venous blood draws and downstream specimen storage and transport methods pose logistical challenges in most settings where cholera strikes. To overcome these challenges, we developed methods for determining cholera-specific immune responses from dried blood spots (DBS). Methodology/principal findings: As conventional vibriocidal assay methods were unsuitable for DBS eluates from filter paper, we adopted a drop-plate culture method. We show that DBS collected from volunteers in South Sudan, and stored for prolonged periods in field conditions, retained functional vibriocidal antibodies, the titers of which correlated with paired serum titers determined by conventional spectrophotometric methods (r = 0.94, p = 0.00012). We also showed that eluates from DBS Serum Separator cards could be used with conventional spectrophotometric vibriocidal methods, and that they correlated with paired serum at a wide range of titers (r = 0.96, p<0.0001). Similarly, we used ELISA methods to show that V. cholerae O-specific polysaccharide antibody responses from DBS eluates correlated with results from paired serum for IgG (r = 0.85, p = 0.00006), IgM (r = 0.79, p = 0.00049) and IgA (r = 0.73, p = 0.0019), highlighting its potential for use in determination of isotype-specific responses. Storage of DBS cards at a range of temperatures did not change antibody responses. Conclusion: In conclusion, we have developed and demonstrated a proof-of-concept for assays utilizing DBS for assessing cholera-specific immune responses. [ABSTRACT FROM AUTHOR]

Background: Dengue is one of the fastest spreading vector-borne diseases, caused by four antigenically distinct dengue viruses (DENVs). Antibodies against DENVs are responsible for both protection as well as pathogenesis. A vaccine that is safe for and efficacious in all people irrespective of their age and domicile is still an unmet need. It is becoming increasingly apparent that vaccine design must eliminate epitopes implicated in the induction of infection-enhancing antibodies. Methodology/principal findings: We report a Pichia pastoris-expressed dengue immunogen, DSV4, based on DENV envelope protein domain III (EDIII), which contains well-characterized serotype-specific and cross-reactive epitopes. In natural infection, <10% of the total neutralizing antibody response is EDIII-directed. Yet, this is a functionally relevant domain which interacts with the host cell surface receptor. DSV4 was designed by in-frame fusion of EDIII of all four DENV serotypes and hepatitis B surface (S) antigen and co-expressed with unfused S antigen to form mosaic virus-like particles (VLPs). These VLPs displayed EDIIIs of all four DENV serotypes based on probing with a battery of serotype-specific anti-EDIII monoclonal antibodies. The DSV4 VLPs were highly immunogenic, inducing potent and durable neutralizing antibodies against all four DENV serotypes encompassing multiple genotypes, in mice and macaques. DSV4-induced murine antibodies suppressed viremia in AG129 mice and conferred protection against lethal DENV-4 virus challenge. Further, neither murine nor macaque anti-DSV4 antibodies promoted mortality or inflammatory cytokine production when passively transferred and tested in an in vivo dengue disease enhancement model of AG129 mice. Conclusions/significance: Directing the immune response to a non-immunodominant but functionally relevant serotype-specific dengue epitope of the four DENV serotypes, displayed on a VLP platform, can help minimize the risk of inducing disease-enhancing antibodies while eliciting effective tetravalent seroconversion. DSV4 has a significant potential to emerge as a safe, efficacious and inexpensive subunit dengue vaccine candidate. [ABSTRACT FROM AUTHOR]

Objectives: We determined the seroprevalence of chikungunya virus (CHIKV) infection in the adult resident population in Singapore following local outbreaks of chikungunya fever (CHIKF) in 2008–2009. Methods: Our cross-sectional study involved residual sera from 3,293 adults aged 18–79 years who had participated in the National Health Survey in 2010. Sera were tested for IgG antibodies against CHIKV and dengue virus (DENV) and neutralizing antibodies against CHIKV. Results: The prevalence of CHIKV-neutralizing antibodies among Singapore residents aged 18–79 years was 1.9% (95% confidence interval: 1.4%– 2.3%). The CHIKV seroprevalence was highest in the elderly aged 70–79 years at 11.5%, followed by those aged 30–39 years at 3.1%. Men had significantly higher CHIKV seroprevalence than women (2.5% versus 1.3%, p = 0.01). Among the three main ethnic groups, Indians had the highest seroprevalence (3.5%) compared to Chinese (1.6%) and Malays (0.7%) (p = 0.02 and p = 0.01, respectively). Multivariable logistic regression identified adults aged 30–39 years and 70–79 years, men, those of Indian ethnicity and ethnic minor groups, and residence on ground floor of public and private housing apartments as factors that were significantly associated with a higher likelihood of exposure to CHIKV. The overall prevalence of anti-DENV IgG antibodies was 56.8% (95% CI: 55.1%– 58.5%), while 1.5% (95% CI: 1.1%– 2.0%) of adults possessed both neutralizing antibodies against CHIKV and IgG antibodies against DENV. Conclusions: Singapore remains highly susceptible to CHIKV infection. There is a need to maintain a high degree of vigilance through disease surveillance and vector control. Findings from such serological study, when conducted on a regular periodic basis, could supplement surveillance to provide insights on CHIKV circulation in at-risk population. [ABSTRACT FROM AUTHOR]

Zika virus (ZIKV) is a mosquito-borne flavivirus of significant public health concern. In the summer of 2016, ZIKV was first detected in the contiguous United States. Here we present one of the first cases of a locally acquired ZIKV infection in a dengue-naïve individual. We collected blood from a female with a maculopapular rash at day (D) 5 and D7 post onset of symptoms (POS) and we continued weekly blood draws out to D148 POS. To establish the ontogeny of the immune response against ZIKV, lymphocytes and plasma were analyzed in a longitudinal fashion. The plasmablast response peaked at D7 POS (19.6% of CD19+ B-cells) and was undetectable by D15 POS. ZIKV-specific IgM was present at D5 POS, peaked between D15 and D21 POS, and subsequently decreased. The ZIKV-specific IgG response, however, was not detected until D15 POS and continued to increase after that. Interestingly, even though the patient had never been infected with dengue virus (DENV), cross-reactive IgM and IgG binding against each of the four DENV serotypes could be detected. The highest plasma neutralization activity against ZIKV peaked between D15 and D21 POS, and even though DENV binding antibodies were present in the plasma of the patient, there was neither neutralization nor antibody dependent enhancement (ADE) of DENV. Interestingly, ADE against ZIKV arose at D48 POS and continued until the end of the study. CD4+ and CD8+ T-cells recognized ZIKV-NS2A and ZIKV-E, respectively. The tetramer positive CD8+ T-cell response peaked at D21 POS with elevated levels persisting for months. In summary, this is the first study to establish the timing of the ontogeny of the immune response against ZIKV. [ABSTRACT FROM AUTHOR]

Background: Rabies is a major public health problem with a fatality rate close to 100%; however, complete prevention can be achieved through pre- or post-exposure prophylaxis. The rapid fluorescent focus inhibition test (RFFIT) is one of the recommended testing methods to determine the production of neutralizing antibodies after vaccination. Here, we report the development of a new monoclonal antibody (mAb) designed to react specifically with Rabies virus (RABV) phosphoprotein (P protein), and the evaluation of its applicability to the RFFIT and its effectiveness as a diagnostic reagent for human rabies. Methodology/principal findings: The mAb KGH P 16B8 was produced to target the P protein of the Korean KGH RABV strain. An indirect immunofluorescence assay (IFA) was conducted to detect various strains of RABV in various cell lines. Alexa-conjugated KGH P 16B8 (16B8-Alexa) was developed for the RFFIT. The IFA test could detect RABV up to a 1:2,500 dilution, with a detection limit comparable to that of a commercial diagnostic reagent. The sensitivity, specificity, positive predictive value, and negative predictive value of the RFFIT using 16B8-Alexa in 414 clinical specimens were 98.67%, 99.47%, 99.55%, and 98.42%, respectively. The results of the RFFIT with 16B8-Alexa were strongly correlated with those obtained using an existing commercial diagnostic reagent (r = 0.995, p<0.001). Conclusions/significance: The mAb developed in this study shows high sensitivity and specificity, confirming its clinical utility with the RFFIT to measure the rabies neutralizing antibody titer and establish a diagnosis in human. Thus, 16B8-Alexa is expected to serve as an alternative diagnostic reagent that is widely accessible, with potentially broad applications beyond those of the RFFIT in Korea. Further studies with 16B8-Alexa should provide insight into the immunological mechanism of the P protein of Korean RABV. [ABSTRACT FROM AUTHOR]

Rabies is a neglected zoonotic disease that is preventable in humans by appropriate post-exposure prophylaxis (PEP). However, current PEP relies on polyclonal immune globulin products purified from pooled human (HRIG) or equine (ERIG) plasma that are either in chronic shortage or in association with safety concerns. Here, we present the development of an antibody cocktail, SYN023, made of two novel monoclonal antibodies (MAb) CTB011 and CTB012 that could serve as safer and more cost-effective alternatives to the current RIG products. Both CTB011 and CTB012 are humanized MAbs that bind to non-overlapping epitopes on the rabies virus (RABV) glycoprotein (G) with sub-nanomolar affinities. Sequence analysis revealed that many of the critical residues in binding are highly conserved across different species of lyssaviruses. When combined at a 1:1 ratio, CTB011/CTB012 exhibited neutralization capabilities equivalent or superior to HRIG against 10 North American street RABV isolates in vitro and 15 prevalent Chinese RABV strains in animal models. Finally, SYN023, at a dosage of 0.03 mg/kg, was able to offer the same degree of protection as standard HRIG administration (20 IU/kg) in Syrian hamsters challenged with a highly virulent bat (Tadarida brasiliensis) RABV variant. Taken together, the high-potency and broad-spectrum neutralization demonstrated by SYN023 make it an effective candidate for human rabies PEP consideration. [ABSTRACT FROM AUTHOR]

B-cells, in addition to antibody secretion, have emerged increasingly as effector and immunoregulatory cells in several chronic inflammatory diseases. Although Erythema Nodosum Leprosum (ENL) is an inflammatory complication of leprosy, the role of B- cell subsets has never been studied in this patient group. Therefore, it would be interesting to examine the contribution of B-cells in the pathogenesis of ENL. A case-control study design was used to recruit 30 untreated patients with ENL and 30 non-reactional lepromatous leprosy (LL) patient controls at ALERT Hospital, Ethiopia. Peripheral blood samples were obtained before, during and after treatment from each patient. Peripheral blood mononuclear cells (PBMCs) were isolated and used for immunophenotyping of B- cell subsets by flow cytometry. The kinetics of B-cells in patients with ENL before, during and after Prednisolone treatment of ENL was compared with LL patient controls as well as within ENL group. Total B-cells, mature B-cells and resting memory B-cells were not significantly different between patients with ENL reactions and LL controls before treatment. Interestingly, while the percentage of naive B-cells was significantly lower in untreated ENL patients than in LL patient controls, the percentage of activated memory B-cells was significantly higher in these untreated ENL patients than in LL controls. On the other hand, the percentage of tissue-like memory B-cells was considerably low in untreated ENL patients compared to LL controls. It appears that the lower frequency of tissue-like memory B-cells in untreated ENL could promote the B-cell/T-cell interaction in these patients through downregulation of inhibitory molecules unlike in LL patients. Conversely, the increased production of activated memory B-cells in ENL patients could imply the scale up of immune activation through antigen presentation to T-cells. However, the generation and differential function of these memory B-cells need further investigation. The finding of increased percentage of activated memory B-cells in untreated patients with ENL reactions suggests the association of these cells with the ENL pathology. The mechanism by which inflammatory reactions like ENL affecting these memory cells and contributing to the disease pathology is an interesting area to be explored for and could lead to the development of novel and highly efficacious drug for ENL treatment. [ABSTRACT FROM AUTHOR]

Background: Cases of acute respiratory tract infection caused by Pteropine orthoreovirus (PRV) of the genus Orthoreovirus (family: Reoviridae) have been reported in Southeast Asia, where it was isolated from humans and bats. It is possible that PRV-associated respiratory infections might be prevalent in Southeast Asia. The clinical course of PRV is not fully elucidated. Methods: The virulence, pathology, and pathogenesis of two PRV strains, a human-borne PRV strain (isolated from a patient, who returned to Japan from Bali, Indonesia in 2007) and a bat-borne PRV (isolated from a bat [Eonycteris spelaea] in the Philippines in 2013) were investigated in BALB/c mice using virological, pathological, and immunological study methods. Results: The intranasal inoculation of BALB/c mice with human-borne PRV caused respiratory infection. In addition, all mice with immunity induced by pre-inoculation with a non-lethal dose of PRV were completely protected against lethal PRV infection. Mice treated with antiserum with neutralizing antibody activity after inoculation with a lethal dose of PRV showed a reduced fatality rate. In this mouse model, bat-borne PRV caused respiratory infection similar to human-borne PRV. PRV caused lethal respiratory disease in an animal model of PRV infection, in which BALB/c mice were used. Conclusions: The BALB/c mouse model might help to accelerate research on the virulence of PRV and be useful for evaluating the efficacy of therapeutic agents and vaccines for the treatment and prevention of PRV infection. PRV was shown for the first time to be a causative virus of respiratory disease on the basis of Koch’s postulations by the additional demonstration that PRV caused respiratory disease in mice through their intranasal inoculation with PRV. [ABSTRACT FROM AUTHOR]

Although effective rabies virus vaccines have been existing for decades, each year, rabies virus infections still cause around 50.000 fatalities worldwide. Most of these cases occur in developing countries, where these vaccines are not available. The reasons for this are the prohibitive high costs of cell culture or egg grown rabies virus vaccines and the lack of a functional cold chain in many regions in which rabies virus is endemic. Here, we describe the excellent temperature resistance of a non-replicating mRNA based rabies virus vaccine encoding the rabies virus glycoprotein (RABV-G). Prolonged storage of the vaccine from -80°C to up to +70°C for several months did not impact the protective capacity of the mRNA vaccine. Efficacy after storage was demonstrated by the induction of rabies specific virus neutralizing antibodies and protection in mice against lethal rabies infection. Moreover, storing the vaccine at oscillating temperatures between +4° and +56°C for 20 cycles in order to simulate interruptions of the cold chain during vaccine transport, did not affect the vaccine’s immunogenicity and protective characteristics, indicating that maintenance of a cold chain is not essential for this vaccine. [ABSTRACT FROM AUTHOR]

Background: With few studies conducted to date, very little is known about the epidemiology of rickettsioses in Bhutan. Due to two previous outbreaks and increasing clinical cases, scrub typhus is better recognized than other rickettsial infections and Q fever. Methodology: A descriptive cross-sectional serosurvey was conducted from January to March 2015 in eight districts of Bhutan. Participants were 864 healthy individuals from an urban (30%) and a rural (70%) sampling unit in each of the eight districts. Serum samples were tested by microimmunofluorescence assay for rickettsial antibodies at the Australian Rickettsial Reference Laboratory. Results: Of the 864 participants, 345 (39.9%) were males and the mean age of participants was 41.1 (range 13–98) years. An overall seroprevalence of 49% against rickettsioses was detected. Seroprevalence was highest against scrub typhus group (STG) (22.6%) followed by spotted fever group (SFG) rickettsia (15.7%), Q fever (QF) (6.9%) and typhus group (TG) rickettsia (3.5%). Evidence of exposure to multiple agents was also noted; the commonest being dual exposure to STG and SFG at 5%. A person’s likelihood of exposure to STG and SFG rickettsia significantly increased with age and farmers were twice as likely to have evidence of STG exposure as other occupations. Trongsa district appeared to be a hotspot for STG exposure while Punakha district had the lowest STG exposure risk. Zhemgang had the lowest exposure risk to SFG rickettsia compared to other districts. People living at altitudes above 2000 meters were relatively protected from STG infections but this was not observed for SFG, TG or QF exposure. Conclusion: This seroprevalence study highlights the endemicity of STG and SFG rickettsia in Bhutan. The high seroprevalence warrants appropriate public health interventions, such as diagnostic improvements and clinical treatment guidelines. Future studies should focus on vector profiles, geospatial, bio-social and environmental risk assessment and preventive and control strategies. [ABSTRACT FROM AUTHOR]

This study describes two longitudinal serological surveys of European Bat Lyssavirus type 1 (EBLV-1) antibodies in serotine bat (Eptesicus serotinus) maternity colonies located in the North-East of France. This species is currently considered as the main EBLV-1 reservoir. Multievent capture-recapture models were used to determine the factors influencing bat rabies transmission as this method accounts for imperfect detection and uncertainty in disease states. Considering the period of study, analyses revealed that survival and recapture probabilities were not affected by the serological status of individuals, confirming the capacity of bats to be exposed to lyssaviruses without dying. Five bats have been found with EBLV-1 RNA in the saliva at the start of the study, suggesting they were caught during virus excretion period. Among these bats, one was interestingly recaptured one year later and harbored a seropositive status. Along the survey, some others bats have been observed to both seroconvert (i.e. move from a negative to a positive serological status) and serorevert (i.e. move from a positive to a negative serological status). Peak of seroprevalence reached 34% and 70% in site A and B respectively. On one of the 2 sites, global decrease of seroprevalence was observed all along the study period nuanced by oscillation intervals of approximately 2–3 years supporting the oscillation infection dynamics hypothesized during a previous EBLV-1 study in a Myotis myotis colony. Seroprevalence were affected by significantly higher seroprevalence in summer than in spring. The maximum time observed between successive positive serological statuses of a bat demonstrated the potential persistence of neutralizing antibodies for at least 4 years. At last, EBLV-1 serological status transitions have been shown driven by age category with higher seroreversion frequencies in adults than in juvenile. Juveniles and female adults seemed indeed acting as distinct drivers of the rabies virus dynamics, hypothesis have been addressed but their exact role in the EBLV-1 transmission still need to be specified. [ABSTRACT FROM AUTHOR]

Rabies continues to pose a significant threat to human and animal health in regions of Indonesia. Indonesia has an extensive network of veterinary diagnostic laboratories and the 8 National laboratories are equipped to undertake diagnostic testing for rabies using the commercially-procured direct fluorescent antibody test (FAT), which is considered the reference (gold standard) test. However, many of the Indonesian Provincial diagnostic laboratories do not have a fluorescence microscope required to undertake the FAT. Instead, certain Provincial laboratories continue to screen samples using a chemical stain-based test (Seller’s stain test, SST). This test has low diagnostic sensitivity, with negative SST-tested samples being forwarded to the nearest National laboratory resulting in significant delays for completion of testing and considerable additional costs. This study sought to develop a cost-effective and diagnostically-accurate immunoperoxidase antigen detection (RIAD) test for rabies that can be readily and quickly performed by the resource-constrained Provincial laboratories. This would reduce the burden on the National laboratories and allow more rapid diagnoses and implementation of post-exposure prophylaxis. The RIAD test was evaluated using brain smears fixed with acetone or formalin and its performance was validated by comparison with established rabies diagnostic tests used in Indonesia, including the SST and FAT. A proficiency testing panel was distributed between Provincial laboratories to assess the reproducibility of the test. The performance of the RIAD test was improved by using acetone fixation of brain smears rather than formalin fixation such that it was of equivalent accuracy to that of the World Organisation for Animal Health (OIE)-recommended FAT, with both tests returning median diagnostic sensitivity and specificity values of 0.989 and 0.993, respectively. The RIAD test and FAT had higher diagnostic sensitivity than the SST (median = 0.562). Proficiency testing using a panel of 6 coded samples distributed to 16 laboratories showed that the RIAD test had good reproducibility with an overall agreement of 97%. This study describes the successful development, characterisation and use of a novel RIAD test and its fitness for purpose as a screening test for use in provincial Indonesian veterinary laboratories. [ABSTRACT FROM AUTHOR]

Background: Detection of Trypanosoma cruzi antigens in clinical samples is considered an important diagnostic tool for Chagas disease. The production and use of polyclonal antibodies may contribute to an increase in the sensitivity of immunodiagnosis of Chagas disease. Methodology/Principal findings: Polyclonal antibodies were raised in alpacas, rabbits, and hens immunized with trypomastigote excreted-secreted antigen, membrane proteins, trypomastigote lysate antigen and recombinant 1F8 to produce polyclonal antibodies. Western blot analysis was performed to determine specificity of the developed antibodies. An antigen capture ELISA of circulating antigens in serum, plasma and urine samples was developed using IgY polyclonal antibodies against T. cruzi membrane antigens (capture antibody) and IgG from alpaca raised against TESA. A total of 33 serum, 23 plasma and 9 urine samples were analyzed using the developed test. Among serum samples, compared to serology, the antigen capture ELISA tested positive in 55% of samples. All plasma samples from serology positive subjects were positive in the antigen capture ELISA. All urine positive samples had corresponding plasma samples that were also positive when tested by the antigen capture ELISA. Conclusions: Polyclonal antibodies are useful for detection of circulating antigens in both the plasma and urine of infected individuals. Detection of antigens is direct evidence of the presence of the parasite, and could be a better surrogate of current infection status. [ABSTRACT FROM AUTHOR]

Background: Several arboviruses, including dengue virus (DENV), Zika virus (ZIKV) and chikungunya virus (CHIKV), transmitted by Aedes mosquitoes, circulate in northeast Brazil. Diseases caused by these viruses are of great public health relevance, however, their epidemiological features in areas where the three viruses co-circulate are scarce. Here, we present analyses of molecular and serological diagnostics in a prospective study of acute febrile patients recruited from May 2015 to May 2016 in Recife, Brazil. Methods: Two hundred sixty-three acute febrile patients with symptoms suggestive of an arboviral disease who attended an urgent heath care clinic in the Recife Metropolitan Region in northeast Brazil were enrolled. Acute and convalescent blood samples were collected and tested using molecular and serological assays for infection with DENV, ZIKV and CHIKV. Results: Quantitative real-time reverse-transcriptase polymerase chain reactions (qRTPCR) performed on acute phase sera detected no patients positive for DENV, but 26 (9.9%) positive for ZIKV and 132 (50.2%) positive for CHIKV. There were a few suspected and only one confirmed dengue case. Specific serological assays for ZIKV and CHIKV confirmed the qRTPCR data. Analyses of DENV IgM and IgG ELISAs in the context of qRTPCR results suggested high levels of cross reactive antibodies in ZIKV-positive samples. Results from neutralization assays highly corroborated those from qRTPCR and ZIKV ELISA, indicating very few positive DENV cases. ZIKV infections were temporally clustered in the first months of the study and started to decrease concomitantly with an increase in CHIKV infections in August 2015. The proportion of CHIKV infections increased significantly in September 2015 and remained high until the end of the study period, with an average of 84.7% of recruited patients being diagnosed from August 2015 to May 2016. ZIKV infections exhibited a female bias and the cases were spread over the study site, while CHIKV cases had a male bias and were spatially clustered in each month. Conclusions: In 2015–2016 in the Recife Metropolitan Region, we detected the tail end of a Zika epidemic, which was displaced by a chikungunya epidemic. Few dengue cases were identified despite a high number of official dengue notifications in the area during this period. We show here important epidemiological features of these cases. [ABSTRACT FROM AUTHOR]

The Pantanal is a hotspot for arbovirus studies in South America. Various medically important flaviviruses and alphaviruses have been reported in domestic and wild animals in the region. To expand the knowledge of local arbovirus circulation, a serosurvey for 14 Brazilian orthobunyaviruses was conducted with equines, sheep and free-ranging caimans. Sera were tested for specific viral antibodies using plaque-reduction neutralization test (PRNT). Monotypic reactions were detected for Maguari, Xingu, Apeu, Guaroa, Murutucu, Oriboca, Oropouche and Nepuyo viruses. Despite the low titers for most of the orthobunyaviruses tested, the detection of monotypic reactions for eight orthobunyaviruses suggests the Pantanal as a region of great orthobunyavirus diversity. The present data, in conjunction with previous studies that detected a high diversity of other arboviruses, ratify the Pantanal as an important natural reservoir for sylvatic and medically important arboviruses in Brazil. [ABSTRACT FROM AUTHOR]

Rabies is an ancient neglected tropical disease that causes tens of thousands of human deaths and millions of cattle deaths annually. In order to develop a new vaccine for potential use in bats, a reservoir of rabies infection for humans and animals alike, an in silico antigen designer tool was used to create a mosaic glycoprotein (MoG) gene using available sequences from the rabies Phylogroup I glycoprotein. This sequence, which represents strains more likely to occur in bats, was cloned into raccoonpox virus (RCN) and the efficacy of this novel RCN-MoG vaccine was compared to RCN-G that expresses the glycoprotein gene from CVS-11 rabies or luciferase (RCN-luc, negative control) in mice and big brown bats (Eptesicus fuscus). Mice vaccinated and boosted intradermally with 1 x 107 plaque forming units (PFU) of each RCN-rabies vaccine construct developed neutralizing antibodies and survived at significantly higher rates than controls. No significant difference in antibody titers or survival was noted between rabies-vaccinated groups. Bats were vaccinated either oronasally (RCN-G, RCN-MoG) with 5x107 PFU or by topical application in glycerin jelly (RCN-MoG, dose 2x108 PFU), boosted (same dose and route) at 46 days post vaccination (dpv), and then challenged with wild-type big brown variant RABV at 65 dpv. Prior to challenge, 90% of RCN-G and 75% of RCN-MoG oronasally vaccinated bats had detectable levels of serum rabies neutralizing antibodies. Bats from the RCN-luc and topically vaccinated RCN-MoG groups did not have measurable antibody responses. The RCN-rabies constructs were highly protective and not significantly different from each other. RCN-MoG provided 100% protection (n = 9) when delivered oronasally and 83% protection (n = 6) when delivered topically; protection provided by the RCN-G construct was 70% (n = 10). All rabies-vaccinated bats survived at a significantly (P ≤ 0.02) higher rate than control bats (12%; n = 8). We have demonstrated the efficacy of a novel, in silico designed rabies MoG antigen that conferred protection from rabies challenge in mice and big brown bats in laboratory studies. With further development, topical or oronasal administration of the RCN-MoG vaccine could potentially mitigate rabies in wild bat populations, reducing spillover of this deadly disease into humans, domestic mammals, and other wildlife. [ABSTRACT FROM AUTHOR]

The article presents a case study of a 37-year-old Brazilian man who was receiving treatment with infliximab and has been using adalimumab for ankylosin spondylitis. It cites that the patients was complaining of rhinorrhea and nasal obstruction for 3 months. The article also notes that he was treated with a liposomal amphotericin B, but after seven months after treatment, he returned with complaints of rhinorrhea, nose bleeding and congestion.

West Nile Fever is a zoonotic disease caused by a mosquito-borne flavivirus, WNV. By its clinical sensitivity to the disease, the horse is a useful sentinel of infection. Because of the virus’ low-level, short-term viraemia in horses, the primary tools used to diagnose WNV are serological tests. Inter-laboratory proficiency tests (ILPTs) were held in 2010 and 2013 to evaluate WNV serological diagnostic tools suited for the European network of National Reference Laboratories (NRLs) for equine diseases. These ILPTs were designed to evaluate the laboratories’ and methods’ performances in detecting WNV infection in horses through serology. The detection of WNV immunoglobulin G (IgG) antibodies by ELISA is widely used in Europe, with 17 NRLs in 2010 and 20 NRLs in 2013 using IgG WNV assays. Thanks to the development of new commercial IgM capture kits, WNV IgM capture ELISAs were rapidly implemented in NRLs between 2010 (4 NRLs) and 2013 (13 NRLs). The use of kits allowed the quick standardisation of WNV IgG and IgM detection assays in NRLs with more than 95% (20/21) and 100% (13/13) of satisfactory results respectively in 2013. Conversely, virus neutralisation tests (VNTs) were implemented in 33% (7/21) of NRLs in 2013 and their low sensitivity was evidenced in 29% (2/7) of NRLs during this ILPT. A comparison of serological diagnostic methods highlighted the higher sensitivity of IgG ELISAs compared to WNV VNTs. They also revealed that the low specificity of IgG ELISA kits meant that it could detect animals infected with other flaviviruses. In contrast VNT and IgM ELISA assays were highly specific and did not detect antibodies against related flaviviruses. These results argue in favour of the need for and development of new, specific serological diagnostic assays that could be easily transferred to partner laboratories. [ABSTRACT FROM AUTHOR]

Objective: In some Pacific Island countries, such as Solomon Islands and Fiji, active trachoma is common, but ocular Chlamydia trachomatis (Ct) infection and trachomatous trichiasis (TT) are rare. On Tarawa, the most populous Kiribati island, both the active trachoma sign “trachomatous inflammation—follicular” (TF) and TT are present at prevalences warranting intervention. We sought to estimate prevalences of TF, TT, ocular Ct infection, and anti-Ct antibodies on Kiritimati Island, Kiribati, to assess local relationships between these parameters, and to help determine the need for interventions against trachoma on Kiribati islands other than Tarawa. Methods: As part of the Global Trachoma Mapping Project (GTMP), on Kiritimati, we examined 406 children aged 1–9 years for active trachoma. We collected conjunctival swabs (for droplet digital PCR against Ct plasmid targets) from 1–9-year-olds with active trachoma, and a systematic selection of 1–9-year-olds without active trachoma. We collected dried blood spots (for anti-Pgp3 ELISA) from all 1–9-year-old children. We also examined 416 adults aged ≥15 years for TT. Prevalence of TF and TT was adjusted for age (TF) or age and gender (TT) in five-year age bands. Results: The age-adjusted prevalence of TF in 1–9-year-olds was 28% (95% confidence interval [CI]: 24–35). The age- and gender-adjusted prevalence of TT in those aged ≥15 years was 0.2% (95% CI: 0.1–0.3%). Twenty-six (13.5%) of 193 swabs from children without active trachoma, and 58 (49.2%) of 118 swabs from children with active trachoma were positive for Ct DNA. Two hundred and ten (53%) of 397 children had anti-Pgp3 antibodies. Both infection (p<0.0001) and seropositivity (p<0.0001) were strongly associated with active trachoma. In 1–9-year-olds, the prevalence of anti-Pgp3 antibodies rose steeply with age. Conclusion: Trachoma presents a public health problem on Kiritimati, where the high prevalence of ocular Ct infection and rapid increase in seropositivity with age suggest intense Ct transmission amongst young children. Interventions are required here to prevent future blindness. [ABSTRACT FROM AUTHOR]

The article discusses the authors' views on dengue diagnostics and their application for dengue surveillance in countries where the CYD-TDV vaccine has been implemented. The authors discuss the limitations of dengue diagnostics, techniques for flavivirus antibody detection, and an algorithm of dengue diagnostic testing after CYD-TDV dengue vaccine implementation in countries with a high burden of dengue.

In the last decade, the number of emerging Flaviviruses described worldwide has increased considerably. Among them Zika virus (ZIKV) and Usutu virus (USUV) are African mosquito-borne viruses that recently emerged. Recently, ZIKV has been intensely studied due to major outbreaks associated with neonatal death and birth defects, as well as neurological symptoms. USUV pathogenesis remains largely unexplored, despite significant human and veterinary associated disorders. Circulation of USUV in Africa was documented more than 50 years ago, and it emerged in Europe two decades ago, causing massive bird mortality. More recently, USUV has been described to be associated with neurological disorders in humans such as encephalitis and meningoencephalitis, highlighting USUV as a potential health threat. The aim of this study was to evaluate the ability of USUV to infect neuronal cells. Our results indicate that USUV efficiently infects neurons, astrocytes, microglia and IPSc-derived human neuronal stem cells. When compared to ZIKV, USUV led to a higher infection rate, viral production, as well as stronger cell death and anti-viral response. Our results highlight the need to better characterize the physiopathology related to USUV infection in order to anticipate the potential threat of USUV emergence. [ABSTRACT FROM AUTHOR]

In mid-2015, Salvador, Brazil, reported an outbreak of Guillain-Barré syndrome (GBS), coinciding with the introduction and spread of Zika virus (ZIKV). We found that GBS incidence during April–July 2015 among those ≥12 years of age was 5.6 cases/100,000 population/year and increased markedly with increasing age to 14.7 among those ≥60 years of age. We conducted interviews with 41 case-patients and 85 neighborhood controls and found no differences in demographics or exposures prior to GBS-symptom onset. A higher proportion of case-patients (83%) compared to controls (21%) reported an antecedent illness (OR 18.1, CI 6.9–47.5), most commonly characterized by rash, headache, fever, and myalgias, within a median of 8 days prior to GBS onset. Our investigation confirmed an outbreak of GBS, particularly in older adults, that was strongly associated with Zika-like illness and geo-temporally associated with ZIKV transmission, suggesting that ZIKV may result in severe neurologic complications. [ABSTRACT FROM AUTHOR]

Monkeypox (MPX) is a zoonotic disease endemic in Central and West Africa and is caused by Monkeypox virus (MPXV), the most virulent orthopoxvirus affecting humans since the eradication of Variola virus (VARV). Many aspects of the MPXV transmission cycle, including the natural host of the virus, remain unknown. African rope squirrels (Funisciurus spp.) are considered potential reservoirs of MPXV, as serosurveillance data in Central Africa has confirmed the circulation of the virus in these rodent species [,]. In order to understand the tissue tropism and clinical signs associated with infection with MPXV in these species, wild-caught rope squirrels were experimentally infected via intranasal and intradermal exposure with a recombinant MPXV strain from Central Africa engineered to express the luciferase gene. After infection, we monitored viral replication and shedding via in vivo bioluminescent imaging, viral culture and real time PCR. MPXV infection in African rope squirrels caused mortality and moderate to severe morbidity, with clinical signs including pox lesions in the skin, eyes, mouth and nose, dyspnea, and profuse nasal discharge. Both intranasal and intradermal exposures induced high levels of viremia, fast systemic spread, and long periods of viral shedding. Shedding and luminescence peaked at day 6 post infection and was still detectable after 15 days. Interestingly, one sentinel animal, housed in the same room but in a separate cage, also developed severe MPX disease and was euthanized. This study indicates that MPXV causes significant pathology in African rope squirrels and infected rope squirrels shed large quantities of virus, supporting their role as a potential source of MPXV transmission to humans and other animals in endemic MPX regions. [ABSTRACT FROM AUTHOR]

Background: Bothrops, Crotalus and Lachesis represent the most medically relevant genera of pitvipers in Central and South America. Similarity in venom phenotype and physiopathological profile of envenomings caused by the four nominal Lachesis species led us to hypothesize that an antivenom prepared against venom from any of them may exhibit paraspecificity against all the other congeneric taxa. Methods: To assess this hypothesis, in this work we have applied antivenomics and immunochemical methods to investigate the immunoreactivity of three monovalent antivenoms and two polyvalent antivenoms towards the venoms from different geographic populations of three different Lachesis species. The ability of the antivenoms to neutralize the proteolytic, hemorrhagic, coagulant, and lethal activities of the seven Lachesis venoms was also investigated. Results: A conspicuous pattern of immunorecognition and cross-neutralization for all effects was evident by the polyspecific antivenoms, indicating large immunoreactive epitope conservation across the genus during more than 10 million years since the Central and South American bushmasters diverged. Conclusions: Despite the broad geographic distribution of Lachesis, antivenoms against venoms of different species are effective in the neutralization of congeneric venoms not used in the immunization mixture, indicating that they can be used equivalently for the clinical treatment of any lachesic envenoming. General significance: This study demonstrates that antivenoms raised against venom of different Lachesis species are indistinctly effective in the neutralization of congeneric venoms not used in the immunization mixture, indicating that antivenoms against conspecific venoms may be used equivalently for the clinical treatment of envenomings caused by any bushmaster species. [ABSTRACT FROM AUTHOR]

Ensuring the adequacy of response to rabies vaccination in dogs is important, particularly in the context of pet travel. Few studies have examined the factors associated with dogs’ failure to achieve an adequate antibody titer after vaccination (0.5 IU/ml). This study evaluated rabies antibody titers in dogs after primary vaccination. Dogs under one year of age whose serum was submitted to a reference laboratory for routine diagnostics, and which had no prior documented history of vaccination were enrolled (n = 8,011). Geometric mean titers (GMT) were calculated and univariate analysis was performed to assess factors associated with failure to achieve 0.5 IU/mL. Dogs vaccinated at >16 weeks of age had a significantly higher GMT compared to dogs vaccinated at a younger age (1.64 IU/ml, 1.57–1.72, ANOVA p < 0.01). There was no statistical difference in GMT between dogs vaccinated <12 weeks and dogs vaccinated 12–16 weeks (1.22 IU/ml and 1.21 IU/ml). The majority of dogs failed to reach an adequate titer within the first 3 days of primary vaccination; failure rates were also high if the interval from vaccination to titer check was greater than 90 days. Over 90% of dogs that failed primary vaccination were able to achieve adequate titers after booster vaccination. The ideal timing for blood draw is 8–30 days after primary vaccination. In the event of a failure, most dogs will achieve an adequate serologic response upon a repeat titer (in the absence of booster vaccination). Booster vaccination after failure provided the highest probability of an acceptable titer. [ABSTRACT FROM AUTHOR]

Fiji, an upper-middle income state in the Pacific Ocean, has experienced an increase in confirmed case notifications of enteric fever caused by Salmonella enterica serovar Typhi (S. Typhi). To characterize the epidemiology of typhoid exposure, we conducted a cross-sectional sero-epidemiological survey measuring IgG against the Vi antigen of S. Typhi to estimate the effect of age, ethnicity, and other variables on seroprevalence. Epidemiologically relevant cut-off titres were established using a mixed model analysis of data from recovering culture-confirmed typhoid cases. We enrolled and assayed plasma of 1787 participants for anti-Vi IgG; 1,531 of these were resident in mainland areas that had not been previously vaccinated against S. Typhi (seropositivity 32.3% (95%CI 28.2 to 36.3%)), 256 were resident on Taveuni island, which had been previously vaccinated (seropositivity 71.5% (95%CI 62.1 to 80.9%)). The seroprevalence on the Fijian mainland is one to two orders of magnitude higher than expected from confirmed case surveillance incidence, suggesting substantial subclinical or otherwise unreported typhoid. We found no significant differences in seropositivity prevalences by ethnicity, which is in contrast to disease surveillance data in which the indigenous iTaukei Fijian population are disproportionately affected. Using multivariable logistic regression, seropositivity was associated with increased age (odds ratio 1.3 (95% CI 1.2 to 1.4) per 10 years), the presence of a pit latrine (OR 1.6, 95%CI 1.1 to 2.3) as opposed to a septic tank or piped sewer, and residence in settlements rather than residential housing or villages (OR 1.6, 95% CI 1.0 to 2.7). Increasing seropositivity with age is suggestive of low-level endemic transmission in Fiji. Improved sanitation where pit latrines are used and addressing potential transmission routes in settlements may reduce exposure to S. Typhi. Widespread unreported infection suggests there may be a role for typhoid vaccination in Fiji, in addition to public health management of cases and outbreaks. [ABSTRACT FROM AUTHOR]

Few live attenuated vaccines protect against multiple serotypes of bacterial pathogen because host serotype-specific immune responses are limited to the serotype present in the vaccine strain. Here, immunization with a mutant of Shigella flexneri 2a protected guinea pigs against subsequent infection by S. dysenteriae type 1 and S. sonnei strains. This deletion mutant lacked the RNA-binding protein Hfq leading to increased expression of the type III secretion system via loss of regulation, resulting in attenuation of cell viability through repression of stress response sigma factors. Such increased antigen production and simultaneous attenuation were expected to elicit protective immunity against Shigella strains of heterologous serotypes. Thus, the vaccine potential of this mutant was tested in two guinea pig models of shigellosis. Animals vaccinated in the left eye showed fewer symptoms upon subsequent challenge via the right eye, and even survived subsequent intestinal challenge. In addition, oral vaccination effectively induced production of immunoglobulins without severe side effects, again protecting all animals against subsequent intestinal challenge with S. dysenteriae type 1 or S. sonnei strains. Antibodies against common virulence proteins and the O-antigen of S. flexneri 2a were detected by immunofluorescence microscopy. Reaction of antibodies with various strains, including enteroinvasive Escherichia coli, suggested that common virulence proteins induced protective immunity against a range of serotypes. Therefore, vaccination is expected to cover not only the most prevalent serotypes of S. sonnei and S. flexneri 2a, but also various Shigella strains, including S. dysenteriae type 1, which produces Shiga toxin. [ABSTRACT FROM AUTHOR]

Chagas disease is one of the most important endemic infections in Latin America affecting around 6–7 million people. About 30–50% of patients develop the cardiac form of the disease, which can lead to severe cardiac dysfunction and death. In this scenario, the identification of immunological markers of disease progression would be a valuable tool for early treatment and reduction of death rates. In this observational study, the production of anti-Trypanosoma cruzi antibodies through a retrospective longitudinal follow-up in chronic Chagas disease patients´ cohort and its correlation with disease progression and heart commitment was evaluated. Strong inverse correlation (ρ = -0.6375, p = 0.0005) between anti-T. cruzi IgG1 titers and left ventricular ejection fraction (LVEF) in chronic Chagas cardiomyopathy (CCC) patients were observed after disease progression. Elevated levels of anti-T. cruzi IgG3 titers were detected in all T. cruzi-infected patients, indicating a lack of correlation of this IgG isotype with disease progression. Furthermore, low levels of anti-T. cruzi IgG2, IgG4, and IgA were detected in all patients through the follow-up. Although without statistical significance anti-T. cruzi IgE tends to be more reactive in patients with the indeterminate form (IND) of the disease (p = 0.0637). As this study was conducted in patients with many years of chronic disease no anti-T. cruzi IgM was detected. Taken together, these results indicate that the levels of anti-T. cruzi IgG1 could be considered to seek for promising biomarkers to predict the severity of chronic Chagas disease cardiomyopathy. [ABSTRACT FROM AUTHOR]

Background: Helminth infections are among the most prevalent neglected tropical diseases, causing an enormous impact in global health and the socioeconomic growth of developing countries. In this context, the study of helminth biology, with emphasis on host-parasite interactions, appears as a promising approach for developing new tools to prevent and control these infections. Methods/Principal findings: The role that antibody responses have on helminth infections is still not well understood. To go in depth into this issue, work on the intestinal helminth Echinostoma caproni (Trematoda: Echinostomatidae) has been undertaken. Adult parasites were recovered from infected mice and cultured in vitro. Double indirect immunofluorescence at increasing culture times was done to show that in vivo-bound surface antibodies become trapped within a layer of excretory/secretory products that covers the parasite. Entrapped antibodies are then degraded by parasite-derived proteases, since protease inhibitors prevent for antibody loss in culture. Electron microscopy and immunogold-labelling of secreted proteins provide evidence that this mechanism is consistent with tegument dynamics and ultrastructure, hence it is feasible to occur in vivo. Secretory vesicles discharge their content to the outside and released products are deposited over the parasite surface enabling antibody trapping. Conclusion/Significance: At the site of infection, both parasite secretion and antibody binding occur simultaneously and constantly. The continuous entrapment of bound antibodies with newly secreted products may serve to minimize the deleterious effects of the antibody-mediated attack. This mechanism of immune evasion may aid to understand the limited effect that antibody responses have in helminth infections, and may contribute to the basis for vaccine development against these highly prevalent diseases. [ABSTRACT FROM AUTHOR]

Snakebite antivenom is a 120 years old invention based on polyclonal mixtures of antibodies purified from the blood of hyper-immunized animals. Knowledge on antibody recognition sites (epitopes) on snake venom proteins is limited, but may be used to provide molecular level explanations for antivenom cross-reactivity. In turn, this may help guide antivenom development by elucidating immunological biases in existing antivenoms. In this study, we have identified and characterized linear elements of B-cell epitopes from 870 pit viper venom protein sequences by employing a high-throughput methodology based on custom designed high-density peptide microarrays. By combining data on antibody-peptide interactions with multiple sequence alignments of homologous toxin sequences and protein modelling, we have determined linear elements of antibody binding sites for snake venom metalloproteases (SVMPs), phospholipases A2s (PLA2s), and snake venom serine proteases (SVSPs). The studied antivenom antibodies were found to recognize linear elements in each of the three enzymatic toxin families. In contrast to a similar study of elapid (non-enzymatic) neurotoxins, these enzymatic toxins were generally not recognized at the catalytic active site responsible for toxicity, but instead at other sites, of which some are known for allosteric inhibition or for interaction with the tissue target. Antibody recognition was found to be preserved for several minor variations in the protein sequences, although the antibody-toxin interactions could often be eliminated completely by substitution of a single residue. This finding is likely to have large implications for the cross-reactivity of the antivenom and indicate that multiple different antibodies are likely to be needed for targeting an entire group of toxins in these recognized sites. [ABSTRACT FROM AUTHOR]

Background: Leishmaniases are parasitic diseases present worldwide that are transmitted to the vertebrate host by the bite of an infected sand fly during a blood feeding. Phlebotomine sand flies inoculate into the mammalian host Leishmania parasites embedded in promastigote secretory gel (PSG) with saliva, which is composed of a diverse group of molecules with pharmacological and immunomodulatory properties. Methods and findings: In this review, we focus on 3 main aspects of sand fly salivary molecules: (1) structure and composition of salivary glands, including the properties of salivary molecules related to hemostasis and blood feeding, (2) immunomodulatory properties of salivary molecules and the diverse impacts of these molecules on leishmaniasis, ranging from disease exacerbation to vaccine development, and (3) use of salivary molecules for field applications, including monitoring host exposure to sand flies and the risk of Leishmania transmission. Studies showed interesting differences between salivary proteins of Phlebotomus and Lutzomyia species, however, no data were ever published on salivary proteins of Sergentomyia species. Conclusions: In the last 15 years, numerous studies have characterized sand fly salivary proteins and, in parallel, have addressed the impact of such molecules on the biology of the host–sand fly–parasite interaction. The results obtained shall pave the way for the development of field-application tools that could contribute to the management of leishmaniasis in endemic areas. [ABSTRACT FROM AUTHOR]

Dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) are severe disease manifestations that can occur following sequential infection with different dengue virus serotypes (DENV1-4). At present, there are no licensed therapies to treat DENV-induced disease. DHF and DSS are thought to be mediated by serotype cross-reactive antibodies that facilitate antibody-dependent enhancement (ADE) by binding to viral antigens and then Fcγ receptors (FcγR) on target myeloid cells. Using genetically engineered DENV-specific antibodies, it has been shown that the interaction between the Fc portion of serotype cross-reactive antibodies and FcγR is required to induce ADE. Additionally, it was demonstrated that these antibodies were as neutralizing as their non-modified variants, were incapable of inducing ADE, and were therapeutic following a lethal, antibody-enhanced infection. Therefore, we hypothesized that avian IgY, which do not interact with mammalian FcγR, would provide a novel therapy for DENV-induced disease. We demonstrate here that goose-derived anti-DENV2 IgY neutralized DENV2 and did not induce ADE in vitro. Anti-DENV2 IgY was also protective in vivo when administered 24 hours following a lethal DENV2 infection. We were also able to demonstrate via epitope mapping that both full-length and alternatively spliced anti-DENV2 IgY recognized different epitopes, including epitopes that have not been previously identified. These observations provide evidence for the potential therapeutic applications of goose-derived anti-DENV2 IgY. [ABSTRACT FROM AUTHOR]

Chikungunya virus (CHIKV) is a mosquito-borne virus that causes a febrile syndrome in humans associated with acute and chronic debilitating joint and muscle pain. Currently no licensed vaccines or therapeutics are available to prevent or treat CHIKV infections. We recently isolated a panel of potently neutralizing human monoclonal antibodies (mAbs), one (4N12) of which exhibited prophylactic and post-exposure therapeutic activity against CHIKV in immunocompromised mice. Here, we describe the development of an engineered CHIKV mAb, designated SVIR001, that has similar antigen binding and neutralization profiles to its parent, 4N12. Because therapeutic administration of SVIR001 in immunocompetent mice significantly reduced viral load in joint tissues, we evaluated its efficacy in a rhesus macaque model of CHIKV infection. Rhesus macaques that were treated after infection with SVIR001 showed rapid elimination of viremia and less severe joint infiltration and disease compared to animals treated with SVIR002, an isotype control mAb. SVIR001 reduced viral burden at the site of infection and at distant sites and also diminished the numbers of activated innate immune cells and levels of pro-inflammatory cytokines and chemokines. SVIR001 therapy; however, did not substantively reduce the induction of CHIKV-specific B or T cell responses. Collectively, these results show promising therapeutic activity of a human anti-CHIKV mAb in rhesus macaques and provide proof-of-principle for its possible use in humans to treat active CHIKV infections. [ABSTRACT FROM AUTHOR]