Serologic Immunity to Tetanus in the United States, National Health and Nutrition Examination Survey, 2015-2016.

BACKGROUND: Tetanus, a life-threatening infection, has become rare in the United States since introduction of tetanus toxoid-containing vaccines (TTCVs), recommended as a childhood series followed by decennial boosters beginning at age 11-12 years; vaccination uptake is high in children but suboptimal in adults. The objective of this study was to estimate the prevalence of sero-immunity to tetanus among persons aged ≥6 years in the United States and to identify factors associated with tetanus sero-immunity. Understanding population protection against tetanus informs current and future vaccine recommendations. METHODS: Anti-tetanus toxoid antibody concentrations were measured for participants of the 2015-2016 National Health and Nutrition Examination Survey (NHANES) aged ≥6 years for whom surplus serum samples were available using a microsphere-based multiplex antibody capture assay. Prevalence of sero-immunity, defined as ≥0.10 IU/mL, was estimated overall and by demographic characteristics. Factors associated with tetanus sero-immunity were examined using multivariable regression. RESULTS: Overall, 93.8% of the US population aged ≥6 years had sero-protection against tetanus. Prevalence of sero-immunity was above 90% across racial/ethnic categories, sex, and poverty levels. By age, ≥ 90% had protective sero-immunity through age 69 years, but prevalence of sero-immunity declined thereafter, with 75.8% of those aged ≥80 years having protective sero-immunity. Older age (adjusted prevalence ratio [aPR]: 0.89, 95% confidence interval [CI]: .85-.92) and being born outside the United States (aPR: 0.96, 95% CI: .93-.98) were significantly associated with lower prevalence of sero-immunity. CONCLUSIONS: The majority of the US population has vaccine-induced sero-immunity to tetanus, demonstrating the success of the vaccination program.

Antibody Binding Captures High Energy State of an Antigen: The Case of Nsp1 SARS-CoV-2 as Revealed by Hydrogen-Deuterium Exchange Mass Spectrometry.

We describe an investigation using structural mass spectrometry (MS) of the impact of two antibodies, 15497 and 15498, binding the highly flexible SARS-CoV-2 Nsp1 protein. We determined the epitopes and paratopes involved in the antibody-protein interactions by using hydrogen-deuterium exchange MS (HDX-MS). Notably, the Fab (Fragment antigen binding) for antibody 15498 captured a high energy form of the antigen exhibiting significant conformational changes that added flexibility over most of the Nsp1 protein. The Fab for antibody 15497, however, showed usual antigen binding behavior, revealing local changes presumably including the binding site. These findings illustrate an unusual antibody effect on an antigen and are consistent with the dynamic nature of the Nsp1 protein. Our studies suggest that this interaction capitalizes on the high flexibility of Nsp1 to undergo conformational change and be trapped in a higher energy state by binding with a specific antibody.

Zika Virus IgM 25 Months after Symptom Onset, Miami-Dade County, Florida, USA.

We assessed IgM detection in Zika patients from the 2016 outbreak in Miami-Dade County, Florida, USA. Of those with positive or equivocal IgM after 12-19 months, 87% (26/30) had IgM 6 months later. In a survival analysis, ≈76% had IgM at 25 months. Zika virus IgM persists for years, complicating serologic diagnosis.

Development of luciferase-linked antibody capture assay based on luciferase immunoprecipitation systems for antibody detection of porcine reproductive and respiratory syndrome virus.

BACKGROUND: Early detection of porcine reproductive and respiratory syndrome virus (PRRSV) infection of swine is necessary to control this devastating disease. By monitoring host serum antibodies to viral antigens, early virus detection within herds is feasible. In this study, recombinant antigens were generated using recombinant DNA techniques to fuse PRRSV structural protein (N) or nonstructural protein 1α (nsp1α) with the Rellina luciferase gene. Next, fused genes were cloned into plasmids and transfected into HEK-293 T cells for transient expression. Upon co-incubation of lysates with pig sera, antigen-antibody complexes formed that bound to Protein-G coated onto microplates. By further measurement of luminance value, a modified form of Luciferase Immunoprecipitation Systems, namely luciferase-linked antibody capture assay (LACA) was developed for detection of PRRSV-specific antibodies. RESULTS: Known anti-PRRSV antibody-positive or -negative serum samples (125 and 122 samples, respectively) were used to validate the LACA and compared it with IDEXX PRRS ×3 ELISA. Based on the result, N-Rluc and nsp1α-Rluc LACA results were 95.3 and 94.4% in agreement with IDEXX ELISA, suggested a similar specificity of LACA to IDEXX ELISA. Moreover, when both LACA and IDEXX ELISA were used to evaluate sequential serum samples obtained from PRRSV experimentally infected pigs, the PRRSV-specific antibody response was detectable as early as 3 days post-inoculation (dpi) using N-Rluc LACA, but undetectable until 7 dpi using IDEXX ELISA, suggesting an improved sensitivity of LACA. Meanwhile, antibodies specific for nsp1α were detected at higher levels overall, but were undetectable until 10 dpi. Furthermore,. Notably, one IDEXX ELISA positive result was not confirmed by LACA or IFA and was thus considered a false-positive result. CONCLUSION: The LACA exhibited similar specificity but improved sensitivity to that of the commercial IDEXX PRRS ×3 ELISA kit for detection of PRRSV-specific antibodies in pig serum. Importantly, LACA could be adapted for detecting antibodies against other PRRSV targets, such as nsp1α, to achieve earlier detection of PRRSV infection.

A universal genome sequencing method for rotavirus A from human fecal samples which identifies segment reassortment and multi-genotype mixed infection.

BACKGROUND: Genomic characterization of rotavirus (RoV) has not been adopted at large-scale due to the complexity of obtaining sequences for all 11 segments, particularly when feces are used as starting material. METHODS: To overcome these limitations, we developed a novel RoV capture and genome sequencing method combining commercial enzyme immunoassay plates and a set of routinely used reagents. RESULTS: Our approach had a 100% success rate, producing >90% genome coverage for diverse RoV present in fecal samples (Ct < 30). CONCLUSIONS: This method provides a novel, reproducible and comparatively simple approach for genomic RoV characterization and could be scaled-up for use in global RoV surveillance systems. TRIAL REGISTRATION (PROSPECTIVELY REGISTERED): Current Controlled Trials ISRCTN88101063 . Date of registration: 14/06/2012.

Laboratory Diagnosis of Chikungunya Virus Infections and Commercial Sources for Diagnostic Assays.

Detection of chikungunya virus (CHIKV) or viral RNA is the primary laboratory test used to diagnose infection in serum collected <6 days after onset of illness. Two real-time reverse transcription-polymerase chain reaction (RT-PCR) kits are available commercially, but validity data are limited. There are 2 commercial sources of inactivated positive-control CHIKV RNA to be used with purchased primers. The Centers for Disease Control and Prevention provides viral RNA-positive controls and primer and probe nucleotide sequences for real-time RT-PCR testing. Detection of CHIKV-specific immunoglobulin M (IgM) antibody becomes a sensitive test for samples collected approximately >5 days of illness. Commercially available CHIKV IgM-detection assays include lateral flow rapid tests, IgM antibody capture enzyme-linked immunosorbent assays (MAC-ELISAs), and indirect immunofluorescence tests. Nine commercial CHIKV IgM detection assays were evaluated at 3 reference laboratories to provide guidance to public health diagnostic laboratories on their performance parameters. Sensitivity of the rapid tests and 3 MAC-ELISAs was <50%, and thus these assays are not recommended. Three of the MAC-ELISA kits and 1 indirect immunofluorescence kit had comparable performance to the reference assays. In summary, commercial assays with performance comparable to reference assays are available for molecular and serological diagnosis of CHIKV infections.

Capsid-Specific Antibody Responses of Domestic Pigs Immunized with Low-Virulent African Swine Fever Virus.

African swine fever (ASF) is a lethal disease in pigs that has grave socio-economic implications worldwide. For the development of vaccines against the African swine fever virus (ASFV), immunogenic antigens that generate protective immune responses need to be identified. There are over 150 viral proteins-many of which are uncharacterized-and humoral immunity to ASFV has not been closely examined. To profile antigen-specific antibody responses, we developed luciferase-linked antibody capture assays (LACAs) for a panel of ASFV capsid proteins and screened sera from inbred and outbred animals that were previously immunized with low-virulent ASFV before challenge with virulent ASFV. Antibodies to B646L/p72, D117L/p17, M1249L, and E120R/p14.5 were detected in this study; however, we were unable to detect B438L-specific antibodies. Anti-B646L/p72 and B602L antibodies were associated with recovery from disease after challenges with genotype I OUR T88/1 but not genotype II Georgia 2007/1. Antibody responses against M1249L and E120R/p14.5 were observed in animals with reduced clinical signs and viremia. Here, we present LACAs as a tool for the targeted profiling of antigen-specific antibody responses to inform vaccine development.

Diagnostic value of recombinant nanoluciferase fused Toxoplasma gondii antigens in Luciferase-linked Antibody Capture Assay (LACA) for Toxoplasma infection in pigs.

Toxoplasmosis is a widespread protozoan zoonosis. Since ingesting undercooked meat harboring Toxoplasma gondii cyst is considered one of the major transmission routes to humans, the screening of T. gondii in meat-producing animals can reduce the risk of food-borne toxoplasmosis in humans. Among serological diagnostic methods, Luciferase-linked Antibody Capture Assay (LACA) has been found to be a promising platform with high sensitivity and specificity. In this study, we aimed to evaluate recombinant nanoluciferase fused-T. gondii antigens (rNluc-GRA6, rNluc-GRA7, rNluc-GRA8 and rNluc-BAG1) for their potential use in LACA for pigs. As a result, the sensitivity of GRA6-, GRA7-, GRA8- and BAG1-LACA were 70.0%, 80.0%, 80.0% and 30.0% with specificity 87.0%, 81.5%, 74.1% and 50.0%, respectively. The cocktail LACA using a mixture of rNluc-GRA6, rNluc-GRA7 and rNluc-GRA8 indicated higher sensitivity (90.0%) and a similar specificity (96.3%) in comparison with the commercial ELISA kit. Compared to the Dye-Test as a reference test, cocktail LACA showed strong agreement (kappa value=0.811) when we assessed pig sera collected at the slaughterhouse. In addition, we also successfully established the rapid LACA format for the detection of Toxoplasma infection in pigs (called Rapid-LACA) in which the test could be performed within 30 min. In Rapid-LACA, the protein A pre-coated/blocked plates could be preserved at -30°C, 4°C or room temperature conditions for at least two months without compromising on the quality of assay.

A novel luciferase-linked antibody capture assay (LACA) for the diagnosis of Toxoplasma gondii infection in chickens.

Toxoplasma gondii is an obligate intracellular protozoan parasite that causes the most common parasitic zoonosis worldwide in multiples species of mammals and birds. Although free-range chickens may play a role as an important reservoir for T. gondii, there is no reliable and commercially available diagnostic test for this disease in chickens. In this study, we aimed to develop a novel Luciferase-linked Antibody Capture Assay (LACA) for the serodiagnosis of Toxoplasma infection in chickens. Recombinant nanoluciferase fused-T. gondii dense granule antigen 8 (rNluc-GRA8) was produced and applied to LACA assay as a diagnostic antigen. GRA8-LACA was tested with the sera from uninfected and experimentally infected chickens with T. gondii and other parasitic pathogens and showed unexpectedly high sensitivity (90.5%) and specificity (95.4%). Interestingly, E. coli lysate expressing rNluc-GRA8 could be applied in GRA8-LACA with 85.7% sensitivity and an increased specificity (96.9%) that gave better diagnostic performance compared to conventional ELISA. We applied our diagnostic system to examine 267 free-range chicken sera collected from 12 farms and 100 closed-house broiler chicken sera from local poultry abattoirs. The overall seroprevalence of toxoplasmosis in free-range chickens was 10.9% (95% CI: 10.6%-11.1%), while no positive case was found in broiler chickens. GRA8-LACA could be a useful diagnostic technique for T. gondii infection in chickens. The detection of T. gondii seropositive chickens in this study warns a potential risk of Toxoplasma transmission by the consumption of raw or undercooked chicken meat.

Bell-shaped agonist activation of 5-HT1A receptor-coupled Gαi3 G-proteins: Receptor density-dependent switch in receptor signaling.

A previous study observed bell-shaped concentration-response isotherms for activation of Gαi3 G-protein subunits by high efficacy 5-HT1A receptor agonists in a Chinese hamster ovary (CHO) cell line expressing high levels of these receptors. This suggested that a signaling switch took place in that cell line (from Gαi3 to activation of other G-proteins) but it was unclear if such effects are observed for 5-HT1A receptors in other cellular environments. Here, using an antibody capture-based [35S]GTPγS binding assay for Gαi3 activation, we investigated whether efficacious 5-HT1A receptor agonists (5-HT, F13714, befiradol, NLX-101), prototypical agonists ((+) and (-)8-OH-DPAT), and partial agonist, antagonists, inverse agonists (pindolol, WAY100635, spiperone) produced similar effects on 5 cell lines expressing different levels of human 5-HT1A receptors. In membranes from cell lines (HeLa, C6-glia and CHO-low) expressing moderate receptor levels (between 1 and 4 pmol/mg of protein), 5-HT, F13714, befiradol and NLX-101 elicited classical sigmoid concentration-response isotherms. In contrast, in cell lines (CHO-high, HEK-293F) expressing high receptor levels (>9 pmol/mg) these agonists elicited bell-shaped concentration-response isotherms that peaked at nanomolar-range concentrations and then returned to baseline or below. Spiperone elicited inverse agonist inhibitory sigmoid isotherms in all membrane preparations while WAY100635 was mostly 'silent' for Gαi3 activation. The other compounds elicited diverse responses in the different cell lines suggesting that other factors, in addition to receptor expression levels, could be influencing Gαi3 activation. These data indicate that Gαi3 G-protein activation by 5-HT1A receptor ligands is highly dependent on receptor expression levels and on cellular background. Moreover, the induction of bell-shape concentration-response isotherms by 5-HT and other high-efficacy agonists is consistent with a switch in signaling to other G-protein-mediated signaling cascades, possibly elicited by receptor conformational changes.

A descriptive study on prevalence pattern of Japanese encephalitis in State of Manipur.

Background and Objective: Japanese encephalitis (JE) surveillance is not well established in many countries, and laboratory confirmation is challenging, the true extent and prevalence of the virus and burden of disease are not well understood. It is estimated that 67,900 clinical cases of JE occur annually despite the widespread availability of vaccine, with approximately 13,600-20,400 deaths and an overall incidence rate of 1.8/100,000 in the 24 countries with JE risk. The present study aimed at determining the prevalence rate (PR) and distribution (time, place and person) of JE cases in Manipur. This descriptive study was conducted over 24-month period (2016-2017). Materials and Methods: A total of 1770 cases of acute encephalitis syndrome tested for JE including 251 confirmed JE were diagnosed by IgM antibody-capture enzyme-linked immunosorbent assay. Results: The JE cases were most commonly reported in the age group of >15 years. Most of JE prevalence was seen in rural distribution in our study. There is a strong seasonal pattern of JE occurrence in Manipur which peaked in July-August and declined by October each year, which corresponds to the monsoon season. The JE cases were reported in all the districts of the state expanding in the plains and hill regions. Conclusions: The changing pattern of JE cases among different age groups was also observed in our study. The present study reveals the changing pattern of the prevalence of JE in the State of Manipur and initiated a systematic approach of JE surveillance also highlights the need for further expanding of surveillance across the state.

Designing affinity chromatographic processes for the capture of antibodies. Part I: A simplified approach.

Protein A affinity chromatography is a standard technique for the purification of therapeutic antibodies. Recently, multi-column chromatographic processes have emerged to turn classical capture processes into more efficient and continuous systems. The design of such chromatographic processes, be they single-column or multi-column systems, is described in this work. A rational method to conceive, scale-up and compare processes is proposed and illustrated with different examples. All along this article, the results of the equilibrium theory, i.e. obtained with an infinitely efficient column, are used to normalize the system and propose a frame for a rigorous comparison between the different configurations considered in this work. Then, the impact of the fluid velocity, the column length, the refresh time, the sequence organization and the number of columns on the yield, productivity and fluid requirement is investigated. It is found that the optimal process, in terms of number of columns and residence time, depends in the targeted protein recovery. For instance, when considering a titer of 1g/L, 2 columns connected in series in the loading zone are necessary to reach 90% recovery, 3 columns to reach 95% and 4 columns to obtain 99% recovery.

Kinetics characterization of c-Src binding to lipid membranes: Switching from labile to persistent binding.

Cell signaling by the c-Src proto-oncogen requires the attachment of the protein to the inner side of the plasma membrane through the myristoylated N-terminal region, known as the SH4 domain. Additional binding regions of lower affinity are located in the neighbor intrinsically disordered Unique domain and the structured SH3 domain. Here we present a surface plasmon resonance study of the binding of a myristoylated protein including the SH4, Unique and SH3 domains of c-Src to immobilized liposomes. Two distinct binding processes were observed: a fast and a slow one. The second process lead to a persistently bound form (PB) with a slower binding and a much slower dissociation rate than the first one. The association and dissociation of the PB form could be detected using an anti-SH4 antibody. The kinetic analysis revealed that binding of the PB form follows a second order rate law suggesting that it involves the formation of c-Src dimers on the membrane surface. A kinetically equivalent PB form is observed in a myristoylated peptide containing only the SH4 domain but not in a construct including the three domains but with a 12-carbon lauroyl substituent instead of the 14-carbon myristoyl group. The PB form is observed with neutral lipids but its population increases when the immobilized liposomes contain negatively charged lipids. We suggest that the PB form may represent the active signaling form of c-Src while the labile form provides the capacity for fast 2D search of the target signaling site on the membrane surface.

Application of Oral Fluid Assays in Support of Mumps, Rubella and Varicella Control Programs.

Detection of specific viral antibody or nucleic acid produced by infection or immunization, using oral fluid samples, offers increased potential for wider population uptake compared to blood sampling. This methodology is well established for the control of HIV and measles infections, but can also be applied to the control of other vaccine preventable infections, and this review describes the application of oral fluid assays in support of mumps, rubella and varicella national immunization programs. In England and Wales individuals with suspected mumps or rubella, based on clinical presentation, can have an oral fluid swab sample taken for case confirmation. Universal varicella immunization of children has led to a drastic reduction of chickenpox in those countries where it is used; however, in England and Wales such a policy has not been instigated. Consequently, in England and Wales most children have had chickenpox by age 10 years; however, small, but significant, numbers of adults remain susceptible. Targeted varicella zoster virus (VZV) immunization of susceptible adolescents offers the potential to reduce the pool of susceptible adults and oral fluid determination of VZV immunity in adolescents is a potential means of identifying susceptible individuals in need of VZV vaccination. The main application of oral fluid testing is in those circumstances where blood sampling is deemed not necessary, or is undesirable, and when the documented sensitivity and specificity of the oral fluid assay methodology to be used is considered sufficient for the purpose intended.

Production of a Sindbis/Eastern Equine Encephalitis chimeric virus inactivated cell culture antigen.

Eastern Equine Encephalitis virus (EEEV) is a medically important pathogen that can cause severe encephalitis in humans, with mortality rates ranging from 30 to 80%. Unfortunately there are no antivirals or licensed vaccines available for human use, and laboratory diagnosis is essential to differentiate EEEV infection from other pathogens with similar clinical manifestations. The Arboviral Diseases Branch (ADB) reference laboratory at the CDC Division of Vector-Borne Diseases (DVBD) produces reference antigens used in serological assays such as the EEEV immunoglobulin M antibody-capture enzyme-linked immunosorbent assay (MAC-ELISA). However, EEEV is classified as a HHS select agent and requires biosafety level (BSL) three containment, limiting EEEV antigen production in non-select agent and BSL-2 laboratories. A recombinant Sindbis virus (SINV)/EEEV has been constructed for use under BSL-2 conditions and is not regulated as a select agent. Cell culture production of inactivated EEEV antigen from SINV/EEEV for use in the EEEV MAC-ELISA is reported here. Cell culture conditions and inactivation procedures were analyzed for SINV/EEEV using a recently developed antigen production algorithm, with the MAC-ELISA as the performance indicator.

Development of an algorithm for production of inactivated arbovirus antigens in cell culture.

Arboviruses are medically important pathogens that cause human disease ranging from a mild fever to encephalitis. Laboratory diagnosis is essential to differentiate arbovirus infections from other pathogens with similar clinical manifestations. The Arboviral Diseases Branch (ADB) reference laboratory at the CDC Division of Vector-Borne Diseases (DVBD) produces reference antigens used in serological assays such as the virus-specific immunoglobulin M antibody-capture enzyme-linked immunosorbent assay (MAC-ELISA). Antigen production in cell culture has largely replaced the use of suckling mice; however, the methods are not directly transferable. The development of a cell culture antigen production algorithm for nine arboviruses from the three main arbovirus families, Flaviviridae, Togaviridae, and Bunyaviridae, is described here. Virus cell culture growth and harvest conditions were optimized, inactivation methods were evaluated, and concentration procedures were compared for each virus. Antigen performance was evaluated by the MAC-ELISA at each step of the procedure. The antigen production algorithm is a framework for standardization of methodology and quality control; however, a single antigen production protocol was not applicable to all arboviruses and needed to be optimized for each virus.

Preliminary application of antibody-capture ELISA detection the antibody of Japanese encephalitis virus / 中国地方病学杂志

Objective To set up an antibody-capture ELISA method to detect the Japanese encephalitis virus(JEV)antibody.Methods ELISA plate was coated with the monoclonal antibody which was specific to the envelope protein epitope E39 of JEV,JEV SA14-14-2 strain as the source of antigen was used to absorb the monoclonal antibody,the absorbed virus used to capture the JEV'S antibody.The antibody that captured ELISA was established.The indirect ELISA method using the virus particles from cell culture was compared with coating ELISA plate,105 clinical serum were checked.Results The background in indirect ELISA assay could not be abscised,positive and negative serum diluted in a ratio of 1:10,1:100,1:1000,the relative value of A posative/A negative were 1.02,0.99,1.13,all<2.1.But the antibody-captured ELISA method when the serum dilution was 1:10,1:100,the A posative/A negative were 3.57,2.94,all>2.1;when the dilution was 1:1000,the A posative/A negative was 1.42,<2.1,it meant the method could distinguish the positive and negative serum efficiently when the dilution Was 1:100,the background problem in indirect ELISA assay could be solved.Antibody-capture method was used to check 105 serum samples,the A posative/A negative over a range of 0.257～0.321(0.262±0.050),all<2.1,no positive sample found.Conclusion The antibody-capture ELISA method has been preliminary set up with a high specificity,capable of quickly identifying JEV from other virus.

SEQUENTIAL DETERMINATION OF SPECIFIC IGM ANTIBODIES IN SERUM SAMPLES FROM PATIENTS WITH EPIDEMIC HEMORRHAGIC FEVER / 西安交通大学学报(医学版)

The specific IgM (SIgM) antibodies in serum samples from 32 patients with epidemic hemorrhagic fever (EHF) were sequentially determined by IgM antibody capture ELISA (MacELISA) and indirect immunofluorescence technique (IFA) sitnultaneously. Of the samples detected by the two methods, 99.46% had the same positive or negative results. The response curves of SIgM titres separately determined by MacELISA or IFA were parellel, whereas, the SIgM titres detected by MacELISA were comparatively higher than those detected by IFA. During the course of the disease, no significant defference was found between the SIgM titres of defferent illness types at the same illness day.