Identification of Novel Genomic Regions for Bacterial Leaf Pustule (BLP) Resistance in Soybean (Glycine max L.) via Integrating Linkage Mapping and Association Analysis.

Bacterial leaf pustule (BLP), caused by Xanthornonas axonopodis pv. glycines (Xag), is a worldwide disease of soybean, particularly in warm and humid regions. To date, little is known about the underlying molecular mechanisms of BLP resistance. The only single recessive resistance gene rxp has not been functionally identified yet, even though the genotypes carrying the gene have been widely used for BLP resistance breeding. Using a linkage mapping in a recombinant inbred line (RIL) population against the Xag strain Chinese C5, we identified that quantitative trait locus (QTL) qrxp-17-2 accounted for 74.33% of the total phenotypic variations. We also identified two minor QTLs, qrxp-05-1 and qrxp-17-1, that accounted for 7.26% and 22.26% of the total phenotypic variations, respectively, for the first time. Using a genome-wide association study (GWAS) in 476 cultivars of a soybean breeding germplasm population, we identified a total of 38 quantitative trait nucleotides (QTNs) on chromosomes (Chr) 5, 7, 8, 9,15, 17, 19, and 20 under artificial infection with C5, and 34 QTNs on Chr 4, 5, 6, 9, 13, 16, 17, 18, and 20 under natural morbidity condition. Taken together, three QTLs and 11 stable QTNs were detected in both linkage mapping and GWAS analysis, and located in three genomic regions with the major genomic region containing qrxp_17_2. Real-time RT-PCR analysis of the relative expression levels of five potential candidate genes in the resistant soybean cultivar W82 following Xag treatment showed that of Glyma.17G086300, which is located in qrxp-17-2, significantly increased in W82 at 24 and 72 h post-inoculation (hpi) when compared to that in the susceptible cultivar Jack. These results indicate that Glyma.17G086300 is a potential candidate gene for rxp and the QTLs and QTNs identified in this study will be useful for marker development for the breeding of Xag-resistant soybean cultivars.

Exploring genetic architecture for pod-related traits in soybean using image-based phenotyping.

Mature pod color (PC) and pod size (PS) served as important characteristics are used in the soybean breeding programs. However, manual phenotyping of such complex traits is time-consuming, laborious, and expensive for breeders. Here, we collected pod images from two different populations, namely, a soybean association panel (SAP) consisting of 187 accessions and an inter-specific recombinant inbred line (RIL) population containing 284 RILs. An image-based phenotyping method was developed and used to extract the pod color- and size-related parameters from images. Genome-wide association study (GWAS) and linkage mapping were performed to decipher the genetic control of pod color- and size-related traits across 2 successive years. Both populations exhibited wide phenotypic variations and continuous distribution in pod color- and size-related traits, indicating quantitative polygenic inheritance of these traits. GWAS and linkage mapping approaches identified the two major quantitative trait loci (QTL) underlying the pod color parameters, i.e., qPC3 and qPC19, located to chromosomes 3 and 19, respectively, and 12 stable QTLs for pod size-related traits across nine chromosomes. Several genes residing within the genomic region of stable QTL were identified as potential candidates underlying these pod-related traits based on the gene annotation and expression profiling data. Our results provide the useful information for fine-mapping/map-based cloning of QTL and marker-assisted selection of elite varieties with desirable pod traits. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-021-01223-2.

Synergistic promotion of microalgal growth and copper removal from synthetic wastewater by nanoscale zero-valent iron particles.

The increasing concentrations of heavy metals in livestock wastewater pose a serious threat to the environmental safety and human health, limiting its resource utilisation. In the present study, microalgae and nanoscale zero-valent iron were selected to construct a coupled system for copper-containing wastewater treatment. The addition of 50 mg·L-1 nanoscale zero-valent iron (50 nm) was the optimal value for the experiment, which could significantly increase the biomass of microalgae. In addition, nanoscale zero-valent iron stimulated microalgal secretion of extracellular polymeric substances, increasing the contents of binding sites, organic ligands, and functional groups on the microalgal surfaces and ultimately promoting the settling of microalgae and binding of heavy metals. The coupled system could quickly adapt to copper-containing wastewater of 10 mg·L-1, and the copper removal rate reached 94.99%. Adsorption and uptake by organisms, together with the contribution of zero-valent iron nanoparticles, are the major copper removal pathways. Overall, this work offers a novel technical solution for enhanced treatment of copper-containing livestock wastewater, which will help improve the efficiency and quality of wastewater treatment.

Beta-variant recombinant booster vaccine elicits broad cross-reactive neutralization of SARS-CoV-2 including Omicron variants.

Background: SARS-CoV-2 Omicron lineage contains variants with multiple sequence mutations relative to the ancestral strain particularly in the viral spike gene. These mutations are associated inter alia with loss of neutralization sensitivity to sera generated by immunization with vaccines targeting ancestral strains or prior infection with circulating (non-Omicron) variants. Here we present a comparison of vaccine formulation elicited cross neutralization responses using two different assay readouts from a subpopulation of a Phase II/III clinical trial. Methods: Human sera from a Phase II/III trial (NCT04762680) was collected and evaluated for neutralizing responses to SARS-CoV-2 spike antigen protein vaccines formulated with AS03 adjuvant, following a primary series of two-doses of ancestral strain vaccine in individuals who were previously unvaccinated or as an ancestral or variant strain booster vaccine among individuals previously vaccinated with the mRNA BNT162b2 vaccine. Results: We report that a neutralizing response to Omicron BA.1 is induced by the two-dose primary series in 89% of SARS-CoV-2-seronegative individuals. A booster dose of each vaccine formulation raises neutralizing antibody titers that effectively neutralizes Omicron BA.1 and BA.4/5 variants. Responses are highest after the monovalent Beta variant booster and similar in magnitude to human convalescent plasma titers. Conclusion: The findings of this study suggest the possibility to generate greater breadth of cross-neutralization to more recently emerging viral variants through use of a diverged spike vaccine in the form of a Beta variant booster vaccine.

Haemagglutination inhibition and virus microneutralisation serology assays: use of harmonised protocols and biological standards in seasonal influenza serology testing and their impact on inter-laboratory variation and assay correlation: A FLUCOP collaborative study.

Introduction: The haemagglutination inhibition assay (HAI) and the virus microneutralisation assay (MN) are long-established methods for quantifying antibodies against influenza viruses. Despite their widespread use, both assays require standardisation to improve inter-laboratory agreement in testing. The FLUCOP consortium aims to develop a toolbox of standardised serology assays for seasonal influenza. Building upon previous collaborative studies to harmonise the HAI, in this study the FLUCOP consortium carried out a head-to-head comparison of harmonised HAI and MN protocols to better understand the relationship between HAI and MN titres, and the impact of assay harmonisation and standardisation on inter-laboratory variability and agreement between these methods. Methods: In this paper, we present two large international collaborative studies testing harmonised HAI and MN protocols across 10 participating laboratories. In the first, we expanded on previously published work, carrying out HAI testing using egg and cell isolated and propagated wild-type (WT) viruses in addition to high-growth reassortants typically used influenza vaccines strains using HAI. In the second we tested two MN protocols: an overnight ELISA-based format and a 3-5 day format, using reassortant viruses and a WT H3N2 cell isolated virus. As serum panels tested in both studies included many overlapping samples, we were able to look at the correlation of HAI and MN titres across different methods and for different influenza subtypes. Results: We showed that the overnight ELISA and 3-5 day MN formats are not comparable, with titre ratios varying across the dynamic range of the assay. However, the ELISA MN and HAI are comparable, and a conversion factor could possibly be calculated. In both studies, the impact of normalising using a study standard was investigated, and we showed that for almost every strain and assay format tested, normalisation significantly reduced inter-laboratory variation, supporting the continued development of antibody standards for seasonal influenza viruses. Normalisation had no impact on the correlation between overnight ELISA and 3-5 day MN formats.

Performance Evaluation of a Dengue IgG Rapid Diagnostic Test Designed to Determine Dengue Serostatus as Part of Prevaccination Screening.

The World Health Organization has recommended prevaccination screening for prior dengue infection as the preferred approach prior to vaccination with the dengue vaccine CYD-TDV. These screening tests need to be highly specific and sensitive, and deliverable at the point-of-care. We evaluate here the sensitivity and specificity of the newly developed OnSite Dengue IgG rapid diagnostic test (RDT). A retrospective double-blind study of the sensitivity and specificity of the OnSite Dengue IgG RDT was performed using a sample panel consisting of archived serum specimens collected during CYD-TDV clinical trials in Latin American and Asia, with the reference serostatus for each sample determined by an algorithm using measured dengue PRNT90, PRNT50, and NS1 IgG ELISA. An additional panel of dengue seronegative samples positive for other flaviviruses and infections was used to assess cross-reactivity. Samples were included from 579 participants; 346 in the specificity panel and 233 in the sensitivity panel. The OnSite dengue IgG RDT exhibited a specificity of 98.0% (95% CI = 95.9 to 99.2) and sensitivity of 95.3% (95% CI = 91.7 to 97.6). The sensitivity for samples exhibiting a multitypic immune profile (PRNT90-positive to >1 dengue serotype) was 98.8% while for monotypic immune samples (PRNT90-positive to a single dengue serotype) it was 88.1%. The OnSite dengue IgG RDT showed minimal to no cross-reactivity to related flaviviruses. These findings support the use of the OnSite dengue IgG RDT to determine dengue serostatus in CYD-TDV prevaccination screening. IMPORTANCE Dengue remains a significant public health issue, with over 5.2 million cases reported to the World Health Organization (WHO) in 2019. The tetravalent dengue vaccine (CYD-TDV) is currently licensed for use in those aged ≥9 years; however, vaccinees with no previous exposure to dengue experience an increased risk of hospitalized and severe dengue upon subsequent heterotypic infection. Consequently, WHO recommends screening for prior dengue infection before vaccination. Screening tests for previous infection need to be highly specific and sensitive, and deliverable at the point-of-care. High sensitivity ensures that the largest number of individuals with previous infection can be identified and vaccinated, while high specificity prevents the inadvertent vaccination of those without previous infection. This study of the OnSite Dengue IgG Rapid Test, which was explicitly developed to meet this need, found that it had both high specificity (98.0% [95% CI = 95.9 to 99.2]) and sensitivity (95.3% [95% CI = 91.7 to 97.6]).

A New Electrochemiluminescence-Based Multiplex Assay for the Assessment of Human Antibody Responses to Bordetella pertussis Vaccines.

INTRODUCTION: Commercially available enzyme-linked immunosorbent assay (ELISA) kits designed for pertussis diagnostic purposes are frequently used to assess antibody responses to pertussis vaccines in clinical trials, but have limited accuracy and are not calibrated against international standards. We developed a new electrochemiluminescence (ECL)-based multiplexed assay and compared its performance to two commercial Bordetella pertussis ELISA kits and to historical in-house ELISAs. METHODS: The ECL assay quantifies serum concentrations of antibodies against four B. pertussis antigens: pertussis toxin (PT), filamentous hemagglutinin (FHA), pertactin (PRN), and fimbrial agglutinogen (FIM). The assay was validated for precision, accuracy, dilutability, lower limit of quantification, and specificity. Sera from a clinical trial (CTRI/2016/11/007434) were used to compare the ECL assay to two commercial ELISA kits available from GenWay BioTech and Demeditec Diagnostics for accuracy, linearity, specificity, and concordance to both internal (WWO-2-043) and international (NIBSC 06/140) references. Sera from four clinical trials (NCT02587520, NCT00255047, NCT00347958, NCT01346293) were used to compare the concordance to clinical ELISAs. Informed consent was ensured prior to using any sera. RESULTS: Precision, accuracy, dilutability, lower limit of quantification, and specificity were demonstrated for the ECL assay. Concordance between the ECL assay and established clinical ELISAs was met for antibody responses to PT, FIM, and PRN, but not for FHA. The ECL assay demonstrated higher accuracy and linearity than the ELISA kits. While concordance between the ECL and commercial kits was low, the ECL assay better distinguished between pre- and post-vaccination clinical samples. CONCLUSION: The new ECL assay was validated for the quantitative evaluation of anti-PT, anti-FHA, anti-FIM, and anti-PRN IgG antibodies in samples from clinical trials, and demonstrated equivalent or better performance than two commercially available ELISA kits.

Assay Harmonization and Use of Biological Standards To Improve the Reproducibility of the Hemagglutination Inhibition Assay: a FLUCOP Collaborative Study.

The hemagglutination inhibition (HAI) assay is an established technique for assessing influenza immunity, through measurement of antihemagglutinin antibodies. Improved reproducibility of this assay is required to provide meaningful data across different testing laboratories. This study assessed the impact of harmonizing the HAI assay protocol/reagents and using standards on interlaboratory variability. Human pre- and postvaccination sera from individuals (n = 30) vaccinated against influenza were tested across six laboratories. We used a design of experiment (DOE) method to evaluate the impact of assay parameters on interlaboratory HAI assay variability. Statistical and mathematical approaches were used for data analysis. We developed a consensus protocol and assessed its performance against in-house HAI testing. We additionally tested the performance of several potential biological standards. In-house testing with four reassortant viruses showed considerable interlaboratory variation (geometric coefficient of variation [GCV] range of 50% to 117%). The age, concentration of turkey red blood cells, incubation duration, and temperature were key assay parameters affecting variability. Use of a consensus protocol with common reagents, including viruses, significantly reduced GCV between laboratories to 22% to 54%. Pooled postvaccination human sera from different vaccination campaigns were effective as biological standards. Our results demonstrate that the harmonization of protocols and critical reagents is effective in reducing interlaboratory variability in HAI assay results and that pools of postvaccination human sera have potential as biological standards that can be used over multiple vaccination campaigns. Moreover, the use of standards together with in-house protocols is as potent as the use of common protocols and reagents in reducing interlaboratory variability. IMPORTANCE The hemagglutination inhibition (HAI) assay is the most commonly used serology assay to detect antibodies from influenza vaccination or influenza virus infection. This assay has been used for decades but requires improved standardization of procedures to provide meaningful data. We designed a large study to assess selected parameters for their contribution to assay variability and developed a standard protocol to promote consistent HAI testing methods across laboratories. The use of this protocol and common reagents resulted in lower levels of variability in results between participating laboratories than achieved using in-house HAI testing. Human sera sourced from vaccination campaigns over several years, and thus including antibody to different influenza vaccine strains, served as effective assay standards. Based on our findings, we recommend the use of a common protocol and/or human serum standards, if available, for testing human sera for the presence of antibodies against seasonal influenza using turkey red blood cells.

Immunological Distinctions between Acellular and Whole-Cell Pertussis Immunizations of Baboons Persist for at Least One Year after Acellular Vaccine Boosting.

While both whole-cell (wP) and acellular pertussis (aP) vaccines have been highly effective at reducing the global pertussis disease burden, there are concerns that compared to wP vaccination, the immune responses to aP vaccination may wane more rapidly. To gain insights into the vaccine elicited immune responses, pre-adult baboons were immunized with either aP or wP vaccines, boosted with an aP vaccine, and observed over a nearly two-year period. Priming with a wP vaccine elicited a more Th17-biased response than priming with aP, whereas priming with an aP vaccine led to a more Th2-biased response than priming with wP. These differences were maintained after aP vaccine boost immunizations. Compared to aP, animals primed with a wP vaccine exhibited greater numbers of pertussis specific memory B cells. While aP and wP vaccine priming initially elicited similar levels of anti-pertussis toxin antibody, titers declined more rapidly in aP vaccine primed animals leading to a 4-fold difference. Both wP and aP vaccine immunization could induce serum bactericidal activity (SBA); however, only one wP vaccine immunization was required to elicit SBA while multiple aP vaccine immunizations were required to elicit lower, less durable SBA titers. In conclusion, when compared to aP vaccine, priming with wP vaccine elicits distinct cellular and humoral immune responses that persist after aP vaccine boosting.

Preparation and qualification of internal rabies reference standards for use in the rabies rapid fluorescent focus inhibition test.

The World Health Organization (WHO) international standard rabies immune globulins (SRIGs) allow the standardisation of the cell-based rapid fluorescent-focus inhibition test (RFFIT) for rabies virus neutralising antibody measurement. SRIG stocks have been depleted. We describe the preparation and qualification of two internal rabies reference standards (IRRSs), calibrated against WHO SRIGs. Candidate IRRSs IMORAB2, from human rabies immunoglobulin; and GCIRAB1, from pooled serum samples from healthy adults immunised with licensed rabies vaccine, were generated. IRRSs were qualified for use in RFFIT based on pre-determined acceptance criteria. Unitage (IU/mL) was assigned using WHO-1 and WHO-2 SRIGs as calibrators. Geometric mean concentrations (GMCs) (% geometric coefficient of variation), calibrated against WHO-1 and WHO-2 SRIGs, were: 1.8 IU/mL (18.7%) and 1.5 IU/mL (17.8%) for IMORAB2; and 2.9 IU/mL (17.5%) and 2.5 IU/mL (16.7%), respectively, for GCIRAB1. We demonstrated IRRS specificity in competition studies using homologous (inactivated Pitman Moore rabies virus) and heterologous (inactivated vesicular stomatitis virus) antigens and acceptable accuracy/linearity of WHO SRIGs using IRRSs as calibrators. Concordance between IRRS and the WHO-1 SRIG was demonstrated using (non-)clinical human serum samples. The candidate reference standards are suitable for use as IRRS in the in-house rabies RFFIT. Funding:Sanofi Pasteur.

Evaluation of dengue serological tests available in Puerto Rico for identification of prior dengue infection for prevaccination screening.

We assessed a dengue IgG rapid diagnostic test (RDT) and IgG enzyme-linked immunosorbent assay (ELISA) to determine their suitability for dengue prevaccination screening. Each exhibited ≥99% specificity. The RDT demonstrated no Zika cross-reactivity, while the ELISA displayed greater sensitivity. Both could safely guide vaccination in Puerto Rico pending availability of improved serotests.

Microneutralization assay titer correlates analysis in two phase 3 trials of the CYD-TDV tetravalent dengue vaccine in Asia and Latin America.

We previously showed that Month 13 50% plaque reduction neutralization test (PRNT50) neutralizing antibody (nAb) titers against dengue virus (DENV) correlated with vaccine efficacy (VE) of CYD-TDV against symptomatic, virologically-confirmed dengue (VCD) in the CYD14 and CYD15 Phase 3 trials. While PRNT is the gold standard nAb assay, it is time-consuming and costly. We developed a next-generation high-throughput microneutralization (MN) assay and assessed its suitability for immune-correlates analyses and immuno-bridging applications. We analyzed MN and PRNT50 titers measured at baseline and Month 13 in a randomly sampled immunogenicity subset, and at Month 13 in nearly all VCD cases through Month 25. For each serotype, MN and PRNT50 titers showed high correlations, at both baseline and Month 13, with MN yielding a higher frequency of baseline-seronegatives. For both assays, Month 13 titer correlated inversely with VCD risk. Like PRNT50, high Month 13 MN titers were associated with high VE, and estimated VE increased with average Month 13 MN titer. We also studied each assay as a valid surrogate endpoint based on the Prentice criteria, which supported each assay as a valid surrogate for DENV-1 but only partially valid for DENV-2, -3, and -4. In addition, we applied Super-Learner to assess how well demographic, Month 13 MN, and/or Month 13 PRNT50 titers could predict Month 13-25 VCD outcome status; prediction was best when using demographic, MN, and PRNT50 information. We conclude that Month 13 MN titer performs comparably to Month 13 PRNT50 titer as a correlate of risk, correlate of vaccine efficacy, and surrogate endpoint. The MN assay could potentially be used to assess nAb titers in immunogenicity studies, immune-correlates studies, and immuno-bridging applications. Additional research would be needed for assessing the utility of MN titer in correlates analyses of other DENV endpoints and over longer follow-up periods.

Evaluation of rapid diagnostic tests and conventional enzyme-linked immunosorbent assays to determine prior dengue infection.

BACKGROUND: In September 2018, the World Health Organization recommended that prevaccination screening be used with the tetravalent dengue vaccine (CYD-TDV), to ensure that only individuals with evidence of prior dengue infection (PDI) are vaccinated. Dengue rapid diagnostic tests (RDTs) would offer a potential solution for prevaccination screening at the point-of-care, but data on performance of available RDTs for identifying PDI are limited. We determined the suitability of four dengue RDTs and two conventional enzyme-linked immunosorbent assays (ELISAs) to identify PDI and evaluated cross-reactivity with co-circulating flaviviruses.Methods: Specificity was assessed using 534 dengue-negative [determined by 50% plaque reduction neutralization test (PRNT50)] serum samples from USA (n = 229) and dengue-endemic regions (n = 305). Sensitivity was assessed using 270 samples from recent (n = 90) or remote (n = 90) virologically confirmed prior dengue cases, and dengue PRNT50-positive samples (n = 90). Cross-reactivity was assessed in dengue-seronegative samples that were seropositive for yellow fever (n = 57), Japanese encephalitis (n = 37), West Nile (n = 59) or Zika (n = 41).Results: Dengue IgG RDTs and the Panbio ELISA exhibited favourable specificities (99-100%), higher than the Focus ELISA (95%). The RDTs had variable sensitivities (40-70%) that were lower than those of the ELISAs (≥90%). Cross-reactivity to other flaviviruses was low with RDTs (≤7%), but more significant with ELISAs (up to 51% for West Nile and 34% for Zika). No cross-reactivity to any of the four closely related flaviviruses was observed with the CTK Biotech RDT. For each SeroTest, sensitivity appeared similar in samples from individuals with recent (<13 months) vs remote (3-4 years) virologically confirmed PDI.Conclusions: In general, dengue IgG RDTs were found to be more specific and less cross-reactive than the ELISAs, but the latter were more sensitive for identifying PDI cases. Currently available RDTs could be temporizing tools for rapid and safe prevaccination screening until improved RDTs with increased sensitivity become available.

Use of a Blockade-of-Binding ELISA and Microneutralization Assay to Evaluate Zika Virus Serostatus in Dengue-Endemic Areas.

Zika virus (ZIKV) serological diagnostics are compromised in areas where dengue viruses (DENV) co-circulate because of their high levels of protein sequence homology. Here, we describe the characterization of a Zika blockade-of-binding ELISA (Zika BOB) and a Zika microneutralization assay (Zika MN) for the detection of ZIKV nonstructural protein 1 (NS1)-specific antibodies and ZIKV neutralizing antibodies, respectively. Zika BOB and Zika MN cutoffs were established as 10 and 100 endpoint titers, respectively, using samples collected pre- and post-virologically confirmed ZIKV infection from subjects living in DENV-endemic areas. Specificity of the assays was equally high, whereas sensitivity of Zika BOB was lower than that of Zika MN, especially in samples collected > 6 months post-infection. Immunosurveillance analysis, using combined results from both Zika BOB and Zika MN, carried out also in DENV-endemic regions in Colombia, Honduras, Mexico, and Puerto Rico before (2013-2014) and after (2017-2018) ZIKV introduction in the Americas suggests unapparent ZIKV seroprevalence rates ranged from 25% to 80% over the specified period of time in the regions investigated.

Rapid fluorescent focus inhibition test optimization and validation: Improved detection of neutralizing antibodies to rabies virus.

The rabies rapid fluorescent focus inhibition test (RFFIT) is the most widely used cell-based assay for detecting and quantitating rabies virus neutralizing antibodies (RVNA) in human serum. However, it is a complex, labor intensive, and somewhat subjective manual assay, the performance of which may be affected by a number of factors including the quality of cells and virus, variability of assay reagents and the skill and expertise of analysts. This study sought to identify and evaluate conditions that may impact RFFIT performance and RVNA detection by evaluating assay parameters including: different serial dilution scheme of serum samples in a 96-well microplate using semi-automated pipetting systems, the range of dose of challenge virus standard (CVS-11) strain of rabies virus, the effect of complement (C'), the effect of cell seeding density and passage number, the effect of diethylaminoethyl (DEAE) dextran concentration on virus infectivity, and the assay incubation period prior to immunostaining. In addition the evaluation of counting fluorescent foci using a microscope versus using scanned images from a cell imaging reader was performed in an effort to ease the reading of slides and have permanent records of the raw data. The results from optimization of each parameter are presented along with subsequent assay validation in accordance with the International Conference on Harmonization (ICH) guidelines. The improved and optimized RFFIT accuracy, linearity and sensitivity was demonstrated by testing World Health Organization (WHO)-1 and WHO-2 Standard Rabies Immune Globulins (SRIGs) and complete assay development and validation was performed in compliance with Good Clinical Laboratory Practice (GCLP) guidelines.

Comparison of adjuvants to optimize influenza neutralizing antibody responses.

Seasonal influenza vaccines represent a positive intervention to limit the spread of the virus and protect public health. Yet continual influenza evolution and its ability to evade immunity pose a constant threat. For these reasons, vaccines with improved potency and breadth of protection remain an important need. We previously developed a next-generation influenza vaccine that displays the trimeric influenza hemagglutinin (HA) on a ferritin nanoparticle (NP) to optimize its presentation. Similar to other vaccines, HA-nanoparticle vaccine efficacy is increased by the inclusion of adjuvants during immunization. To identify the optimal adjuvants to enhance influenza immunity, we systematically analyzed TLR agonists for their ability to elicit immune responses. HA-NPs were compatible with nearly all adjuvants tested, including TLR2, TLR4, TLR7/8, and TLR9 agonists, squalene oil-in-water mixtures, and STING agonists. In addition, we chemically conjugated TLR7/8 and TLR9 ligands directly to the HA-ferritin nanoparticle. These TLR agonist-conjugated nanoparticles induced stronger antibody responses than nanoparticles alone, which allowed the use of a 5000-fold-lower dose of adjuvant than traditional admixtures. One candidate, the oil-in-water adjuvant AF03, was also tested in non-human primates and showed strong induction of neutralizing responses against both matched and heterologous H1N1 viruses. These data suggest that AF03, along with certain TLR agonists, enhance strong neutralizing antibody responses following influenza vaccination and may improve the breadth, potency, and ultimately vaccine protection in humans.

Development of an anti-dengue NS1 IgG ELISA to evaluate exposure to dengue virus.

Dengue virus infection elicits immune responses to multiple viral antigens including antibodies to dengue non-structural protein 1 (NS1) which are rapidly induced and detected within days of infection. The recombinant, live, attenuated, tetravalent dengue vaccine (CYD-TDV; Sanofi Pasteur) uses the yellow fever vaccine virus as a back-bone but expresses dengue virus pre-membrane and envelop proteins. Since CYD-TDV does not express dengue NS1, we evaluated the utility of dengue NS1-specific IgG antibodies as biomarkers of dengue exposure in CYD-TDV recipients and controls. We optimized and evaluated a quantitative anti-dengue NS1 IgG enzyme-linked immunosorbent assay (ELISA). Parameters assessed included: accuracy, dilutability/linearity, precision, limit of quantitation and specificity. The assay specificity was further evaluated using Japanese Encephalitis virus, West Nile virus, Yellow Fever virus or Zika virus positive sera samples collected following confirmed infection or vaccination. Receiver-operating-characteristics (ROC) curves as well as sensitivity and specificity for discriminating previous dengue exposure were assessed using 1250 reference samples. Overall, the anti-dengue NS1 IgG ELISA was able to discriminate previous dengue exposure from non-exposure before vaccination with CYD-TDV (ROC area under the curve > 0.9). Assessment of paired samples from 2511 vaccinated participants showed high overall agreement (93%) between pre-vaccination and post-vaccination dengue serostatus classification based on the anti-dengue NS1 IgG ELISA. However, misclassification of dengue serostatus was observed after vaccination likely due to a combination of asymptomatic dengue infections, assay variability and a modest effect of CYD-TDV on the anti-dengue NS1 IgG ELISA readout.

Automated Imaging and Analysis of the Hemagglutination Inhibition Assay.

The hemagglutination inhibition (HAI) assay quantifies the level of strain-specific influenza virus antibody present in serum and is the standard by which influenza vaccine immunogenicity is measured. The HAI assay endpoint requires real-time monitoring of rapidly evolving red blood cell (RBC) patterns for signs of agglutination at a rate of potentially thousands of patterns per day to meet the throughput needs for clinical testing. This analysis is typically performed manually through visual inspection by highly trained individuals. However, concordant HAI results across different labs are challenging to demonstrate due to analyst bias and variability in analysis methods. To address these issues, we have developed a bench-top, standalone, high-throughput imaging solution that automatically determines the agglutination states of up to 9600 HAI assay wells per hour and assigns HAI titers to 400 samples in a single unattended 30-min run. Images of the tilted plates are acquired as a function of time and analyzed using algorithms that were developed through comprehensive examination of manual classifications. Concordance testing of the imaging system with eight different influenza antigens demonstrates 100% agreement between automated and manual titer determination with a percent difference of ≤3.4% for all cases.

Study on the interaction between carbonyl-fused N-confused porphyrin and bovine serum albumin by spectroscopic techniques.

The interaction between carbonyl-fused N-confused porphyrin (CF-NCP) and bovine serum albumin (BSA) was investigated by fluorescence and ultraviolet-visible (UV-Vis) spectroscopy. The results indicated that CF-NCP has strong ability to quench the intrinsic fluorescence of BSA by forming complexes. The binding constants (Ka), binding sites (n) were obtained. The corresponding thermodynamic parameters (ΔH, ΔS and ΔG) of the interaction system were calculated at three different temperatures. The results revealed that the binding process is spontaneous, and the acting force between CF-NCP and BSA were mainly electrostatic forces. According to Förster non-radiation energy transfer theory, the binding distance between CF-NCP and BSA was calculated to be 4.37nm. What is more, the conformation of BSA was observed from synchronous fluorescence spectroscopy.