ELISA Screening Research Articles

Active Surveillance in the investigation and analysis of RVFin Western and Central Uganda

ObjectiveTo detect presence of circulating Rift Valley Fever virus (RVFv) in animals of Western and Central Uganda following its confirmation in humans.To establish and communicate reliable information using the one health platformSignificnce:Although in E. Africa RVF was initially detected and known to be a disease endermic in Kenya, the people in Uganda were still hesitating wether the disease is already in existence. Following its first detection in 2016 in Humans there was need to carry out an investigation in the hot spot areas of the human infection to get the real picture and to inform the policy makers for informed decisions.IntroductionRift Valley fiver is viral zoonotic disease which was investigated and reported in Uganda in 20101. For some time now people are not aware whether the disease was still circulating or emerged in animals reared as a result of the inter country trade by the community of the cattle corridor in Uganda, since the last reports in 19682. The increase in the number of disease outbreaks in some parts of central and western Uganda from 2016 to date and the number of human patients investigated, diagnosed and confirmed with RVF by Ministry Of Health (MOH) under the one health program, has placed the disease to be among the top re-emerging diseases in the country3&4 and number 5 of the Multisectoral prioritization of zoonotic diseases in Uganda, 2017 under One Health perspective6.MethodsRift valley Fever was investigated in cattle, goats and sheep of Gomba,Mityana, Kiboga and Kiruhura in Central and Western Uganda. This followed 2 people that had been confirmed with RVF in 20161 Samples were aseptically collected from hot places from 543 victim’s animals including those of the neighbouring areas covering the victims routes of movement plus those areas where people were still sick and where death had reportedly occurred. Samples were then delivered to NADDEC laboratory from where tests were conducted.ResultsSamples were screened using a competition IgG ELISA, then IgM ELISA to capture the recently infected animals. The positive samples from the IgM ELISA were then confirmed using RT-PCR; 169/543 (31%) tested positive to IgG screening ELISA indicating exposure to RVF. The actual infection was found to be 13% (22/169) with IgM ELISA and 3/22 (13.6%) with RT-PCR.ConclusionsZoonotic diseases continue to be a public health burden to the people of Uganda. Considering some people’s behavior of eating the sick and dead animals, has posed a difficult situation to combat the ailment which has resulted in negative socioeconomic impacts, affecting the national policies that range from health security to control of diseases. Uganda has however developed capacity to investigate, test and confirm RVF disease. Since exposure was found in all animal species, detailed active surveillance plan and procedures have been set up to investigate any additional cases in animals to reduce chances of spread to humans and to cub international spread and also to determine the magnitude of exposure.References1 Nabukenya, Investigation and response to Rift Valley Fever and Yellow Fever outbreaks in humans in Uganda, 20162 Nyakarahuka L.prevalence and risk factors of Rift valley in humans and animals from kabale, 20163 Wang LF, Crameri G.Emerging zoonotic viral diseases.Rev Sci Tech Int Epiz.2014;33Institute of Medicine (U.S.), Committee on Achieving Sustainable Global Capacity for surveillance and4 Response to Emerging Diseases of Zoonotic Origin, Keusch G. Sustaining global surveillance and response to emerging zoonotic diseases, 20095 Musa Sekamatte, Vikram K.Multisectoral prioritization of zoonotic diseases in Uganda, 2017, A One Health perspective6 Munyua P, Bitek A, Osoro E, Pieracci EG, Muema J,Mwatondo A,et al, Prioritization of Zoonotic Diseases in Kenya,2015. PLOS ONE. 2016;11:e0161576. http://doi.org/10.1371/journal.pone.0161576 PMID:27557120

Management of a <i>Coxiella</i> <i>burnetii</i>-infected sheep flock after an outbreak of Q fever in humans

Following an outbreak of Q fever in a group of students who contracted the infection during a training course on a sheep farm, a detailed investigation of the sheep flock involved was conducted. Of 478 flock animals, 60 Coxiella burnetii ELISA-positive and 60 ELISA-negative ewes were selected for the trial and divided into four groups. A month after the initial ELISA screening, all ewes in the flock (except the control group) were vaccinated. Sequentially collected blood samples were tested with ELISA and PCR; feces, milk, manure, bedding, and soil were tested with PCR. The immune response to the vaccination was 92.7%, while the overall C. burnetii seroprevalence in the flock after the human outbreak was 64.9%. PCR was positive for 0.2% of milk samples and 34.4% of fecal samples of animals from all four trial groups. C. burnetii DNA was not detected in any of the blood samples. Manure was PCR-positive for about 35 months; bedding from the stable was also positive while samples of pasture soil were negative. It appears that extensive cleaning and disinfection combined with vaccination could be regarded as an appropriate approach to control/prevent Q fever in farm settings even in the short term.

Next-generation sequencing-guided identification and reconstruction of antibody CDR combinations from phage selection outputs.

Next-generation sequencing (NGS) technologies have been employed in several phage display platforms for analyzing natural and synthetic antibody sequences and for identifying and reconstructing single-chain variable fragments (scFv) and antigen-binding fragments (Fab) not found by conventional ELISA screens. In this work, we developed an NGS-assisted antibody discovery platform by integrating phage-displayed, single-framework, synthetic Fab libraries. Due to limitations in attainable read and amplicon lengths, NGS analysis of Fab libraries and selection outputs is usually restricted to either VH or VL. Since this information alone is not sufficient for high-throughput reconstruction of Fabs, we developed a rapid and simple method for linking and sequencing all diversified CDRs in phage Fab pools. Our method resulted in a reliable and straightforward platform for converting NGS information into Fab clones. We used our NGS-assisted Fab reconstruction method to recover low-frequency rare clones from phage selection outputs. While previous studies chose rare clones for rescue based on their relative frequencies in sequencing outputs, we chose rare clones for reconstruction from less-frequent CDRH3 lengths. In some cases, reconstructed rare clones (frequency ∼0.1%) showed higher affinity and better specificity than high-frequency top clones identified by Sanger sequencing, highlighting the significance of NGS-based approaches in synthetic antibody discovery.

Protocol to Validate the Performance of the Mellitus Glycated CD59 ELISA for Gestational Diabetes Screening

Protocol to Validate the Performance of the Mellitus Glycated CD59 ELISA for Gestational Diabetes Screening

Design, synthesis and biological study of hybrid drug candidates of nitric oxide releasing cucurbitacin-inspired estrone analogs for treatment of hepatocellular carcinoma

Development of hybrid drug candidates is well known strategy for designing antitumor agents. Herein, a novel class of nitric oxide donating cucurbitacin inspired estrone analogs (NO-CIEAs) were designed and synthesized as multitarget agents. Synthesized analogs were initially evaluated for their anti-hepatocellular carcinoma activities. Among the tested analogs, NO-CIEAs 17 and 20a exhibited more potent activity against HepG2 cells (IC50 = 4.69 and 12.5 µM, respectively) than the reference drug Erlotinib (IC50 = 25 µM). Interestingly, NO-CIEA 17 exerted also a high potent activity against Erlotinib-resistant HepG2 cell line (HepG2-R) (IC50 = 8.21 µM) giving insight about its importance in drug resistance therapy. Intracellular measurements of NO revealed that NO-CIEAs 17 and 20a showed a significant increase in NO production in tumor cells after 1 h of incubation comparable to the reference prodrug JS-K. Flow cytometric analysis showed that both NO-CIEAs 17 and 20a mainly arrested the HepG2 cells in the G0/G1 phase. Also, In-Cell Based ELISA screening showed that NO-CIEA 17 resulted in a potential inhibitory activity towards the EGFR and MAPK (25% and 29% inhibition compared to untreated control cells, respectively). This data suggests the binding ability of NO-CIEA 17 to the EGFR and ERK to be well correlated along with the docking and cellular studies. Also, treatment of HepG2-R cells with NO-CIEA 17 showed a potential reduction of MRP2 expression in a dose dependent manner providing a significant impact on the chemotherapeutic resistance. Overall, the current study provides a potential new approach for the discovery of a novel antitumor agent against HCC.

АЛГОРИТМ ДІАГНОСТИКИ МІГРУЮЧОЇ ЕРИТЕМИ В ДІТЕЙ

Мета роботи – встановити передумови і частоту природного зараження B. burgdorferi sensu lato від кліщів, відібраних після укусу дітей у Тернопільській області в різні місяці року, а також визначити методи контролю за укусами кліщів у дітей.
 Матеріали і методи. Було обстежено 291 дитину, яка зазнала нападу кліщів. Батьки усіх обстежених відповіли на запитання уніфікованої міжнародної анкети. В лабораторії медичного університету протягом липня-жовтня 2017 р. було досліджено 169 кліщів, взятих із шкіри дітей. Використовували метод полімеразної ланцюгової реакції (ПЛР) в режимі реального часу, проводили двоступеневу серологічну експертизу: скринінговий тест ELISA на наявність антитіл IgM/IgG, Western blot.
 Результати. Хвороба Лайма проявляється у багатьох клінічних формах. Чим молодша дитина, тим складніше діагностувати хворобу Лайма.
 Висновки. Відзначено високу частоту реєстрації випадків захворювання на Лайм-бореліоз серед міських жителів. У 38,3 % досліджених від моменту укусу кліща до його видалення минуло більше 24 год. Випадки захворювання на Лайм-бореліоз реєструвалися протягом усього року, з піками в липні й жовтні. 29,0 % кліщів виявилися інфікованими. Наявності в анамнезі контакту з кліщем, ураженим B. burgdorferi, та виникнення у пацієнта мігруючої еритеми (МЕ) достатньо для того, щоб діагностувати хворобу Лайма та розпочати лікування. За допомогою двоступеневої серологічної діагностики вдалося виявити антитіла до Borrelia burgdorferi sensu lato у 52,0 % обстежених дітей.

Novel screening ELISA for sensitive detection of Mayaro virus infected patients

Trabalho apresentado no International Meeting on Emerging Diseases and Surveillance (IMED), 2018. Vienna, Austria.

Seasonal influence on TORCH infection and analysis of multi-positive samples with indirect immunofluorescence assay.

BackgroundTORCH including the pathogens of Toxoplasma gondii (TOX), rubella virus (RV), cytomegalovirus (CMV), and herpes simplex virus (HSV) causes intrauterine infections and poses a worldwide threat to women especially in pregnancy. In this study, we described the seasonal difference in TORCH infection and analyzed the anti‐TORCH IgM multipositive serum samples by the indirect immunofluorescence assays (IFA).MethodsTo observe the seasonal influence of the anti‐TORCH IgG and IgM antibodies, a retrospective study was conducted with 10 669 women (20–40 y old) before pregnancy from August 2016 to July 2017. Totally 199 ELISA anti‐TORCH IgM multipositive serum samples were further tested by IFAs for false‐positive analysis.ResultsThe prevalence of positive HSV1‐IgM, RV‐IgM, HSV2‐IgM, CMV‐IgM, and TOX‐IgM in the present population was 6.30%, 2.55%, 1.94%, 1.24%, and 0.67%, respectively. Additionally, the prevalence of positive RV‐IgM, CMV‐IgM, and HSV1‐IgM was statistically different among four seasons, with the highest positive rates of RV‐IgM (4.12%) in autumn, CMV‐IgM (1.75%) in summer, and HSV1‐IgM (7.53%) in winter. The confirmatory IFAs showed that the positive rates of RUV‐IgM, CMV‐IgM, and HSV2‐IgM were significantly different from those in ELISA screening experiments. Interestingly, only 32.7% (65/199) of the TORCH IgM multipositive results were consistent with those by the IFA, indicating that cross‐reaction caused false positives were common in ELISA IgM antibody screening.ConclusionThe TORCH infection displayed different prevalence among four seasons in our 12‐month retrospective study. The IgM multipositives by ELISA screening may need further confirmation analysis due to its relatively high cross‐reaction rate.

Approved drugs screening against the nsP1 capping enzyme of Venezuelan equine encephalitis virus using an immuno-based assay

Alphaviruses such as the Venezuelan equine encephalitis virus (VEEV) are important human emerging pathogens transmitted by mosquitoes. They possess a unique viral mRNA capping mechanism catalyzed by the viral non-structural protein nsP1, which is essential for virus replication. The alphaviruses capping starts by the methylation of a GTP molecule by the N7-guanine methyltransferase (MTase) activity; nsP1 then forms a covalent link with m7GMP releasing pyrophosphate (GT reaction) and the m7GMP is next transferred onto the 5′-diphosphate end of the viral mRNA to form a cap-0 structure. The cap-0 structure decreases the detection of foreign viral RNAs, prevents RNA degradation by cellular exonucleases, and promotes viral RNA translation into proteins. Additionally, reverse-genetic studies have demonstrated that viruses mutated in nsP1 catalytic residues are both impaired towards replication and attenuated. The nsP1 protein is thus considered an attractive antiviral target for drug discovery. We have previously demonstrated that the guanylylation of VEEV nsP1 can be monitored by Western blot analysis using an antibody recognizing the cap structure. In this study, we developed a high throughput ELISA screening assay to monitor the GT reaction through m7GMP-nsP1 adduct quantitation. This assay was validated using known nsP1 inhibitors before screening 1220 approved compounds. 18 compounds inhibiting the nsP1 guanylylation were identified, and their IC50 determined. Compounds from two series were further characterized and shown to inhibit the nsP1 MTase activity. Conversely, these compounds barely inhibited a cellular MTase demonstrating their specificity towards nsP1. Analogues search and SAR were also initiated to identify the active pharmacophore features. Altogether the results show that this HT enzyme-based assay is a convenient way to select potent and specific hit compounds targeting the viral mRNA capping of Alphaviruses.

A RAGE-Targeted Antibody-Drug Conjugate: Surface Plasmon Resonance as a Platform for Accelerating Effective ADC Design and Development.

Antibodies, antibody-like molecules, and therapeutics incorporating antibodies as a targeting moiety, such as antibody-drug conjugates, offer significant potential for the development of highly efficacious drugs against a wide range of disorders. Despite some success, truly harnessing the superior targeting properties of these molecules requires a platform from which to effectively identify the best candidates for drug development. To streamline the development of antibody-drug conjugates targeting gynecological cancers within our laboratory, we incorporated surface plasmon resonance analysis (Biacore™ T200) into our development toolkit. Antibodies, selected based on positive ELISA screens as suitable for development as antibody-drug conjugates, were evaluated using surface plasmon resonance to determine a wide range of characteristics including specificity, kinetics/affinity, the effect of linker binding, the impact of the drug to antibody ratio, and the effect of endosomal pH on antibody-antigen binding. Analysis revealed important kinetics data and information regarding the effect of conjugation and endosomal pH on our antibody candidates that correlated with cell toxicity and antibody internalization data. As well as explaining observations from cell-based assays regarding antibody-drug conjugate efficacies, these data also provide important information regarding intelligent antibody selection and antibody-drug conjugate design. This study demonstrates the application of surface plasmon resonance technology as a platform, where detailed information can be obtained, supporting the requirements for rapid and high-throughput screening that will enable enhanced antibody-drug conjugate development.

New approaches for post-vaccination surveillance of foot-and-mouth disease.

Foot-and-mouth disease (FMD) is a contagious and important transboundary disease of cloven-hoofed animals and ruminants. In ruminants, an animal is considered as a foot-and-mouth disease virus (FMDV) carrier if a live FMDV/FMDV RNA is obtained from the oro-pharyngeal fluid (OPF) beyond 28 days after infection. These carrier animals may pose a risk for causing outbreaks in healthy animals. Moreover, it is important to conduct serosurveillance to know the virus circulation. In the present study, an ELISA was developed using field samples to detect FMDV specific secretory IgA antibodies. These samples were also tested for the presence of FMDV RNA using quantitative real-time PCR (qRT-PCR). It was found that more carrier animals were detected by IgA ELISA in comparison to qRT-PCR. Thus, IgA ELISA is an important tool to detect FMD carriers. An ELISA based on detection of antibodies against FMDV 2B non-structural protein (NSP) was also used to confirm the results obtained from screening of 3AB3 NSP ELISA. These two new approaches (IgA ELISA and 2B ELISA) form important tools for detection of carriers and virus circulation, respectively, during FMD eradication program. Keywords: foot-and-mouth disease virus; carriers; IgA; 2B non-structural protein; 3AB3 non-structural protein.

Development of a competitive indirect ELISA for high-throughput screening of hydrocortisone in cosmetic sample

ABSTRACT Hydrocortisone (HYD) is one of the corticosteroids that illegally used in cosmetic products, which could lead to a potential health problem. In this work, HYD was conjugated to bovine serum albumin via a succinic anhydride spacer arm to yield an immunogen. New Zealand rabbits were immunized to obtain a polyclonal antibody against HYD. Based on the pAb, a heterologous competitive indirect enzyme-linked immunosorbent assay (ciELISA) by using progesterone carboxylic acid as a competitive hapten was developed. The assay showed an IC50 of 0.50 ng mL−1, detection limit of 0.04 ng mL−1 and a linear range of 0.10–2.00 ng mL−1. The assay was applied to determine HYD in cosmetic samples. Recoveries from 82.5% to 118.9% and CV from 1.3% to 9.5% were obtained using a simple pretreatment. Good correlation between the results of ciELISA and standard LC–MS/MS was obtained, suggesting its potential usage in HYD monitoring in cosmetic samples.

Multiplexed ELISA screening assay for nine paralytic shellfish toxins in human plasma.

Paralytic shellfish poisoning is a lethal syndrome that can develop in humans who consume shellfish contaminated with paralytic shellfish toxins. These toxins have a short half-life in the human body, so a rapid diagnostic assessment of the poisoning is necessary. In this paper, we have developed and validated a rapid ELISA screening assay using anti-saxitoxin antibodies to screen nine toxins: saxitoxin; decarbamoyl saxitoxin; gonyautoxin 2,3; decarbamoyl GTX 2,3; neosaxitoxin; and gonyautoxin 1,4, in human plasma with lower limits of detection of 0.02, 0.08, 0.12, 1.2, 5.0, and 25 ng mL-1, respectively. Intra-day and inter-day precision experiments showed good reproducibility with a percent coefficient of variation less than 15%. The assay was 100% accurate in determining the presence or absence of these toxins in human plasma specimens. Blank specimens were assessed as negative for toxin content indicating that the method has excellent analyte specificity. This rapid screening assay can be used to quickly diagnose exposure to paralytic shellfish toxins, though an additional confirmatory method will be necessary to identify and quantitate the specific toxin in an exposure.

Investigation of syphilis coinfection and performance of the Architect Syphilis Tp ELISA screening test in HIV positive patients

Limited data on syphilis coinfection in human immunodeficiency virus (HIV) positive cases exist in Turkey. Our aim is to investigate syphilis coinfection and to evaluate the compatibility of the screening Architect Syphilis Tp ELISA with the fluorescent treponemal antibody absorption (FTA-abs) confirmation test in HIV positive cases. Totally 519 HIV positive patients were included in the study. Enzyme linked fluorescent assay (ELFA) was used as a screening test and positive samples were confirmed by line immunassay (LIA). In order to discriminate acute HIV infection and false ELISA positivity, HIV-1 RNA PCR was performed in ELFA positive and LIA negative samples. Architect Syphilis TP ELISA was used for the detection of total antibodies against Treponema pallidum in HIV positive patients. Positive results were confirmed by the FTA-abs test. Out of 519 HIV-1 positive patients, IgG and IgM positivity, and only IgG positivity was detected as 1.9% and 11.4% in all the samples, respectively. A total of 79 (15.2%) sera were positive with Architect Syphilis Tp ELISA test and 69 (13.3%) were positive with FTA-abs test. Statistically significant, almost perfect agreement was found between Architect Syphilis Tp ELISA and FTA-abs tests (kappa = 0.921 and P < 0.001). Implementation of syphilis and HIV screening tests together among risk groups is considered to be appropriate.

Seroprevalence of Hepatitis B and Hepatitis C Virus among Healthy Blood Donors in Tertiary Care Hospital

Hepatitis B virus (HBV) &amp; Hepatitis C virus (HCV) are transfusion transmissible infections. A study was conducted from September, 2015 to March, 2016 at the Transfusion Medicine Department of Rajshahi Medical College Hospital to determine the prevalence of asymptomatic hepatitis B and C virus in apparently healthy blood donors, were subjected to ELISA for screening of hepatitis B surface antigen and Hepatitis C virus. A total of 200 blood donors were screened, the seroprevalence of HBV &amp; HCV infection was 5.0% &amp; 2.0% respectively. The HBV seroprevalence in male donors was 182/8 (4.4%) and in female donors was 18/2 (11.1%). Four donors were found seropositive for HCV giving a seroprevalence of 2%. HCV seroprevalence in male was 182/4 (2.2%) and no female. Seroprevalence of HCV and HBV were higher in younger donors, service holders, married respondents and middle class donors. To reduce the prevalence of post-transfusion hepatitis and stringent donor screening procedure, routine screening for HBV &amp; HCV in blood banks should be performed using more sensitive methods (ELISA).TAJ 2015; 28(1): 26-31

Combination of ELISA screening and seroneutralisation tests to expedite Zika virus seroprevalence studies

Here we propose a strategy allowing implementing efficient and practicable large-scale seroepidemiological studies for Zika Virus (ZIKV). It combines screening by a commercial NS1 protein-based Zika IgG ELISA, and confirmation by a cytopathic effect-based virus neutralization test (CPE-based VNT). In post-epidemic samples from Martinique Island blood donors (a population with a dengue seroprevalence above 90%), this strategy allowed reaching specificity and sensitivity values over 98%. The CPE-based VNT consists of recording CPE directly under the optical microscope, which is easy to identify with ZIKV strain H/PF/2013 at day 5 pi. Overall, considered that CPE-based VNT is cost effective and widely automatable, the NS1 protein-based Zika IgG ELISA+CPE-based VNT combination strategy represents a convenient tool to expedite ZIKV seroprevalence studies.

Presentation Mode of Glycans Affect Recognition of Human Serum anti-Neu5Gc IgG Antibodies.

Recognition of carbohydrates by antibodies can be affected by antigen composition and density. This had been investigated in a variety of controllable multivalent systems using synthetic carbohydrate antigens, yet such effects on anticarbohydrate antibodies in circulating human serum have not been fully addressed thus far. All humans develop a polyclonal and diverse response against carbohydrates containing a nonhuman sialic acid form, N-glycolylneuraminic acid (Neu5Gc). This red meat-derived monosaccharide is incorporated into a diverse collection of human glycans resulting in circulating anti-Neu5Gc antibodies in human sera. Such antibodies can cause exacerbation of diseases mediated by chronic inflammation such as cancer and atherosclerosis. We aimed to evaluate how different presentation modes of Neu5Gc-glycans can affect the detection of anti-Neu5Gc IgGs in human serum. Here, we compare serum IgG recognition of Neu5Gc-containing glycoproteins, glycopeptides, and synthetic glycans. First, Neu5Gc-positive or Neu5Gc-deficient mouse strains were used to generate glycopeptides from serum glycoproteins. Then we developed a reproducible ELISA to screen human sera against Neu5Gc-positive glycopeptides for detection of human serum anti-Neu5Gc IgGs. Finally, we evaluated ELISA screens against glycopeptides in comparison with glycoproteins, as well as against elaborated arrays displaying synthetic Neu5Gc-glycans. Our results demonstrate that the presentation mode and diversity of Neu5Gc-glycans are critical for detection of the full collection of human serum anti-Neu5Gc IgGs.

Development and Evaluation of CART Targeting Bcma with Humanized Alpaca-Derived Single-Domain Antibody As Antigen Recognition Domain

Chimeric antigen receptor T cells (CART) targeting CD19 have shown substantial activity against leukemia and lymphoma, which motivated developing CART cell therapy for Multiple myeloma (MM). B cell maturation antigen (BCMA) is the target molecule in MM. Several kinds of CART targeting BCMA have been created from 2016. Among these, the Bluebird Bio uses the humanized murine BCMA scFv to make CART, the Nanjing Legend company uses the single-domain antibody targeting BCMA to make CART.The single-domain antibody refers to the heavy chain antibody naturally present in camel species, which has no light chain. All VHh (Variable region of Heavy chain in heavy chain antibody) domains from the immunized alpaca were cloned into the phage-display library, then specific VHhs were screened out by solid-phase ELISA. Not like scFv using the linker region to connect the VH and VL to obtain the single chain region, the VHh itself is one single chain, which make it very suitable for making the recognition domain of CART. In order to increase the avidity, the Nanjing Legend company uses two tandem VHhs for binding BCMA. However, the single domain antibody has the capacity to bind the target molecule with very high affinity, one VHh should be able to bind BCMA tightly. To this aim, using our platform of single-domain antibody discovery and CART development, we successfully obtain the CART targeting BCMA (CART-BCMA) just using one VHh for recognizing the BCMA on tumor cells.Firstly, we immunized the alpaca using the extracellular domain of BCMA, then the B cell RNA were extracted, and the VHh phage-display library were constructed and screened. Finally we got the 6 single-domain antibodies through solid-phase ELISA screening. After gene sequencing and humanization of VHh, we constructed 6 lentiviral vectors with CAR gene including different VHh sequence connecting with CD8 hinge and transmembrane region, CD137 intracellular region, and CD3ζ intracellular region. We used 293T to package the lentivirus, and infected the healthy T cells to make 6 kinds of CART-BCMA cells. With in vitro killing assay, we found the #3 CART-BCMA is the best (83% killing rate at the E:T ratio of 4:1). Simultaneously, we used the Biocore to test the binding affinity of single-domain antibodies, the #3 antibody's affinity is 1.14nM, which indicated only one #3 VHh domain already have enough binding ability for CART to recognize the BCMA positive tumor cells. Next, we did further experiment to demonstrate the function of #3 CART-BCMA.We used the MM tumor cell line MM.1S (expressing high BCMA), myeloid tumor cell line K562 (no BCMA expression) as the target, the results show that CART-BCMA kill the MM.1S cells in 42% rate at the low E:T ratio of 0.5:1, but the CART-BCMA cells can't kill K562 cells even in high 4:1 E:T ratio. Accordingly, the cytokine level of IFN-γ or TNF-α is elevated dramatically in CART-BCMA wells. In animal experiment, the 1.5x106 MM.1S-Luc were injected into one NSG mouse. 40 mice were separated into 5 groups for PBS, 10x106 Mock T, 1.0x106 CART, 5.0x106 CART, 10x106 CART treatment. The results show that three CART groups of 1.0x106, 5.0x106, 10x106 CART are effective.Using the purified lentivirus, we found the lentivirus can infect the T cells at the MOI of 3, the CAR positive rate is over 50% by FACS, this implied that the CAR gene can be easily expressed on T cell membrane. For T cell infection, CAR-BCMA lentivirus need less amount compared to traditional CAR virus derived from scFv.We performed the clinical pilot study to investigate CART-BCMA's safety and effect on MM patients pre-conditioned with Fludarabine/cyclophosphamide. Until the time of abstract submission, 4 patients were enrolled, and got the dose of 5x106/kg (Pt1), dose of 10x106/kg (Pt2 and Pt3, split dose of 50% and 50% in two days), dose of 10x106/kg (Pt4) CART-BCMA cells. Pt1 has the CR response, and has the fever after 14 days. Pt2 has the PR response with no CRS. Pt3 has the PR response with grade 2 CRS. Pt4 has the PR response with grade 3 CRS and neurotoxicity (see table).In summary, we developed one CART-BCMA for MM treatment, which has the BCMA antigen recognition domain derived from the alpaca. Especially, the lentivirus carrying the CAR-BCMA gene can infect the T cells at the low MOI of 3, which will save the amount of lentivirus. The clinical pilot study showed that 4 patients received the CART-BCMA has the objective response (1 CR, 3 PR), further follow-up and enrollment is being performed. [Display omitted] DisclosuresZhou:Health and Family Planning Commission of Henan Province: Patents & Royalties: Scientific and technological innovative talents “51282” Project leaders; Affiliated Cancer Hospital of Zhengzhou University: Employment; Natural Science Foundation of China: Research Funding; Henan Cancer Hospital: Consultancy, Employment. Zhang:The Shenzhen Pregene Biopharma Company, Ltd.: Employment. Du:The Shenzhen Pregene Biopharma Company, Ltd.: Employment. Li:The Shenzhen Pregene Biopharma Company, Ltd.: Employment.

2286. Revisiting Immune Interference: Evaluation of Immune Response to Yellow Fever Vaccine at Various Time Points Following Live-Attenuated Influenza Vaccination

BackgroundDue to concerns for immune interference, current recommendations are to avoid other live virus vaccines for 30 days pre- and post-mass vaccination campaigns leading to interruptions in routine vaccinations. During rapid preparations for Operation United Assistance (OUA) which supplied humanitarian assistance during the Ebola epidemic, mass yellow fever vaccine (YFV) administration to deploying personnel was needed during ongoing live-attenuated influenza vaccine (LAIV) administration. This study is the first to compare seroconversion rates for YFV when given per guidelines (VBG) to rates when YFV is given 1–29 days post-LAIV (NVBG).MethodsAll personnel who received LAIV concurrently or before YFV for OUA and had pre- and post-vaccination archived serum at the Department of Defense Serum Repository were included. VBG was defined as YFV given concurrently or ≥30 days after LAIV and NVBG as YFV given 1–29 days post-LAIV. YFV seroresponse was determined by screening ELISA followed by confirmation with plaque reduction neutralization testing (PRNT) on all positive samples. YFV PRNT ≥1:20 was considered positive. Exclusion criteria were prior YFV and pre-vaccination positive PRNT. Statistical analysis was performed using SPSS v22.ResultsDuring OUA preparations, 676 personnel were vaccinated with LAIV concurrently or before YFV. Sixteen were excluded due to positive pre-vaccination PRNT. Of the 660 who met inclusion criteria, 507 were VBG (482 concurrently and 25 vaccinated ≥30 days post-LAIV) and 153 were NVBG. Median age was 25 (IQR 22, 29) for both groups. Pre-vaccination serum was drawn 280 and 345 days for VBG and NVBG respectively (P = 0.05). Post-YFV serum was drawn a median of 154 days following YFV in both groups. Seroconversion rates were 98% for VBG and 95% for NVBG (P = 0.15). Median yellow fever titers were 320 (IQR 160, 640) in both groups post-vaccination. Seroconversion rates were 98% for those with LAIV and YFV concurrently (n = 471), 100%, 95%, 92%, 100%, and 100% for those with YFV on days 1–6 (n = 18), days 7–13 (n = 42), days 14–21 (n = 66), days 22–27 (n = 8), and ≥28 days (n = 44) post-LAIV respectively (P = 0.12).ConclusionIn this healthy, adult population, YFV provided high levels of protection regardless of timing following LAIV.Disclosures All authors: No reported disclosures.

Variables associated with the prevalence of anti-Leishmania spp. antibodies in dogs on the tri-border of Foz do Iguaçu, Paraná, Brazil.

The aim of this study was to investigate the occurrence of anti-Leishmania spp. antibodies in dogs from localities in the city of Foz do Iguaçu, Paraná state, Brazil, on the border with Argentina and Paraguay. Blood samples dogs were collected to perform the following serologic tests: immunochromatographic DPP® rapid test, indirect immunoenzymatic assay (ELISA) and indirect immunofluorescence assay (IFA). In 2012, 285 dogs were analyzed on Argentina border, and in 2013, serum samples from 396 dogs on the border of Paraguay were collected. Using ELISA for screening and IFA for the confirmatory test, the results showed that the antibody prevalence was 1.8% (5/285) on the border of Argentina and 3.0% (12/396) on Paraguay border. When using the DPP® for screening and ELISA as a confirmatory analysis, we observed a seroreagent prevalence in dogs of 2.5% (7/285) on Argentina border and 5.1% (20/396) on Paraguay border. The non-public collection of domestic waste (p= 0.0004) was shown to be associated with leishmaniasis. This study shows the presence of leishmaniasis and suggest the emergence of canine visceral leishmaniasis in state of Paraná due to the confirmed occurrence of seroreactive dogs on Argentina and Paraguay border, which has environmental and geographical characteristics that favor the spread of the parasite.