Nanoluciferase Reporter Zika Viruses as Tools for Assessing Infection Kinetics and Antibody Potency.

Zika virus (ZIKV) has become endemic in multiple tropical and subtropical regions and has the potential to become widespread in countries with limited prior exposure to this infection. One of the most concerning sequelae of ZIKV infection is the teratogenic effect on the developing fetus, with the mechanisms of viral spread to and across the placenta remaining largely unknown. Although vaccine trials and prophylactic or therapeutic treatments are being studied, there are no approved treatments or vaccines for ZIKV. Appropriate tests, including potency and in vivo assays to assess the safety and efficacy of these modalities, can greatly aid both the research of the pathophysiology of the infection and the development of anti-ZIKV therapeutics. Building on previous work, we tested reporter ZIKV variants that express nanoluciferase in cell culture and in vivo assays. We found that these variants can propagate in cells shown to be susceptible to the widely used clinical isolate PRVABC59, including Vero and human placenta cell lines. When used in neutralization assays with bioluminescence as readout, these variants gave rise to neutralization curves similar to those produced by PRVABC59, while being better suited for performing high-throughput assays. In addition, the engineered reporter variants can be useful research tools when used in other in vitro and in vivo assays, as we illustrated in transcytosis experiments and a pilot study in guinea pigs.

Interchangeability of the Assays Used to Assess the Activity of Anti-SARS-CoV-2 Monoclonal Antibodies.

The recent global COVID-19 pandemic caused by SARS-CoV-2 lasted for over three years. A key measure in combatting this pandemic involved the measurement of the monoclonal antibody (mAb)-mediated inhibition of binding between the spike receptor-binding domain (RBD) and hACE2 receptor. Potency assessments of therapeutic anti-SARS-CoV-2 mAbs typically include binding or cell-based neutralization assays. We assessed the inhibitory activity of five anti-SARS-CoV-2 mAbs using ELISA, surface plasmon resonance (SPR), and four cell-based neutralization assays using different pseudovirus particles and 293T or A549 cells expressing hACE2 with or without TMPRSS2. We assessed the interchangeability between cell-based and binding assays by applying the Bland-Altman method under certain assumptions. Our data demonstrated that the IC50 [nM] values determined by eight neutralization assays are independent of the cell line, presence of TMPRSS2 enzyme on the cell surface, and pseudovirus backbone used. Moreover, the Bland-Altman analysis showed that the IC50 [nM] and KD [nM] values determined by neutralization/ELISA or by SPR are equivalent and that the anti-spike mAb activity can be attributed to one variable directly related to its tertiary conformational structure conformation, rate dissociation constant Koff. This parameter is independent from the concentrations of the components of the mAb:RBD:hACE2 complexes and can be used for a comparison between the activities of the different mAbs.

Method for screening influenza neutralizing antibodies in crude human plasma and its derivatives using SPR.

We applied Surface Plasmon Resonance (SPR) technology to develop a method for potency screening and quantification of anti-influenza antibodies in minimally processed human plasma samples and intravenous immunoglobulin (IGIV) products. We found that specific antibodies in human plasma or IGIV capable of inhibiting binding of influenza hemagglutinin to receptor-analogous glycans do so in concentration-dependent manner. We ranked the inhibitory activity of plasma samples from multiple donors and found a good correlation (r = 0.87) of SPR assay measurements and conventional hemagglutination inhibition (HAI) assay results. This method was also applied to screen for specific anti-influenza antibodies in IGIV lots manufactured pre- and post-2009 H1N1 pandemic. The SPR method was also applied to study binding inhibition of the intact A/California/04/2009 H1N1 and B/Victoria/504/2000 influenza viruses to α2,6 or α2,3-linked synthetic glycans. In contrast to recombinant H1 hemagglutinin, which was found to interact primarily with α2,6-linked terminal sialic acids, intact H1N1 or influenza B virus recognized both types of receptor analogs with different observed dissociation rates and the inhibitory activity of plasma antibodies was dependent on the type of sialic acid link. The SPR method can provide a high-throughput, time-saving and semi-automated alternative to conventional assays such as HAI or microneutralization in situations where screening of large numbers of plasma donations to identify high titer units is needed to product highly potent immunoglobulins.

Thrombogenic potential of picomolar coagulation factor XIa is mediated by thrombin wave propagation.

Inhibitors of coagulation factor XIa (FXIa) are currently being investigated as potential anticoagulant therapies. We hypothesize that circulating FXIa could be a potential target for these therapies. Using previous analyses of FXIa impurities in immune globulin products involved in thrombotic adverse events, we estimated that picomolar levels of FXIa can be thrombogenic. In an in vitro clot-growth assay, 0.1-3 pM of FXIa did not, by itself, activate clotting but increased the size of growing clots. Spatio-temporal reconstruction of thrombin activity inside the clot revealed that FXIa's effect was limited to the clot-plasma interface, in which FXIa produced a taller than standard wave of thrombin. Factor-depleted plasma and a panel of selective anti-FXIa antibodies showed that exogenous FXIa effects are (1) blocked by anti-FXIa antibodies, (2) independent of FXI activation inside the clot, and (3) larger than the contribution of in situ FXIa. In a thrombin generation (TG) assay, picomolar FXIa did not initiate TG but rather promoted TG triggered by tissue factor or thrombin, suggesting that the effect of FXIa on the thrombin wave is mediated by the elevation of thrombin-triggered TG. In circulating bovine blood, low doses of human FXIa did not initiate clotting but increased the size of stenosis-triggered thrombi. FXIa injection in mice enhanced TG in plasma for at least 6 hours ex vivo, confirming the persistence of circulating FXIa. Our findings suggest that picomolar levels of circulating FXIa may not be able to initiate thrombosis but can facilitate thrombus growth through the facilitation of TG inside the clot.

An optimized microplate-based method to evaluate complement-dependent hemolysis mediated by intravenous immunoglobulins (IVIG).

Hemolytic reactions can cause serious complications after administration of Intravenous Immunoglobulin (IVIG), due to passive transfer of anti-A and anti-B IgG antibodies (isoagglutinins). A maximum allowable amount of isoagglutinins is established in the US and EU for licensed IVIG, as measured by a specified direct hemagglutination test (DHAT). Despite this limit, reports of hemolysis have increased over time, raising the question of how well the DHAT predicts clinically significant hemolysis. This study was undertaken to develop a microplate-based complement-dependent hemolysis assay (CDHA) that reproducibly measures functional hemolytic activity of IVIG, for assessment of IVIG products. An IVIG working reference reagent (NIBSC 14/160) was qualified as an assay control and for quantitation purposes. Hemolytic activities of 36 IVIG product lots encompassing seven brands and including 6 clinically hemolytic lots were measured. Hemolytic activity varied among IVIG product brands, and to a lesser extent, from lot-to-lot for individual brands. Correlation between the CDHA and DHAT was not robust which may reflect imprecision of the DHAT method or additional variables that influence complement-dependent hemolysis after opsonization. In conclusion, the CDHA provides a simple, specific, and sensitive tool for IVIG product characterization and investigation of hemolytic events by manufacturers, researchers, and regulatory authorities.

Thrombin generation test based on a 96-channel pipettor for evaluation of FXIa procoagulant activity in pharmaceuticals.

Thrombin generation (TG) assays are used widely to investigate both diseases and drugs that impact thrombosis and bleeding. TG assays were also instrumental in the identification of thrombogenic impurities in immune globulin products, which were associated with thrombotic adverse events in patients. TG assays are therefore now used by quality control laboratories of plasma derivative drug manufacturers and regulatory agencies responsible for the safety testing and release of immune globulin products. In this protocol, we describe a robust and sensitive version of the TG assay for quantitative measurement of thrombogenic activity in immune globulin products. Compared with the version of the assay commonly used in clinical laboratories that compares individual patient plasma samples with normal donor samples, our TG assay is suitable for quick (170-260 min) semiautomated analysis of multiple drug samples against the World Health Organization international standard for factor XIa. Commercially available reagents can be used for the assay, and it does not require specialized equipment. The protocol can be easily adapted for the measurement of the procoagulant activity of other biopharmaceuticals, e.g., coagulation factors.

Detecting factor XIa in immune globulin products: Commutability of international reference materials for traditional and global hemostasis assays.

BACKGROUND: Activated coagulation factor XIa (FXIa) is an impurity and primary source of procoagulant activity in thrombosis-implicated immune globulin (IG) products. Several assays, of varying quality and precision are used to assess FXIa-like procoagulant activity in units relevant to their respective principles. OBJECTIVES: To advance unified reporting, we sought to employ the World Health Organization reference reagents (RRs) to present the results of differing methodologies in units of FXIa activity and rank the sensitivity and robustness of these methodologies. METHODS: RR 11/236 served as a calibrator in several FXIa-sensitive blood coagulation tests: two commercial chromogenic FXIa assays (CAs); a nonactivated partial thromboplastin time (NaPTT); an in-house fibrin generation (FG) assay; an in-house thrombin generation (TG) assay; and an assay for FXIa- and kallikrein-like proteolytic activities based on cleavage of substrate SN13a. Some assays were tested in either normal or FXI-deficient plasma. RESULTS: Each method demonstrated a sigmoidal dose-response to RRs. NaPTT was the least sensitive to FXIa and the least precise; our in-house TG was the most sensitive; and the two CAs were the most precise. All methods, except for SN13a, which is less specific for thrombotic impurities, gave comparable (within 20% difference) FXIa activity assignments for IG lots. CONCLUSIONS: Purified FXIa reference standards support quantitation of FXIa levels in IG products in all tested assay methodologies. This should help to standardize the measurement of thrombotic potentials in IG products and prevent products exhibiting high procoagulant activity from distribution for patient use. Further research is needed to address the effect of IG product-specific matrixes on assay performance.

Characterization of source plasma from self-identified vaccinated or convalescent donors during the 2009 H1N1 pandemic.

BACKGROUND: Influenza immune globulin, manufactured from plasma of convalescent or vaccinated donors has been proposed as a potential therapy for severe influenza. In 2009, a program was initiated to collect plasma from donors who self-identified as having had H1N1 influenza or having received the H1N1 pandemic vaccine. The goal of this study was to determine the efficiency of donor screening by self-identification without antibody testing, and to evaluate demographic predictors of high-titer donations. STUDY DESIGN AND METHODS: Plasma samples from self-identified or control donors were randomly selected to evaluate hemagglutination inhibition (HAI) antibody responses. HAI titers were correlated with donor age, gender, location, and influenza exposure history. RESULTS: Both self-identified vaccinated and convalescent donor groups had higher geometric mean titers (GMTs) against A/California/07/2009 (H1N1) virus compared to the control donors (39.9, 24, and 8.5, respectively). The proportion of samples with titers ≥64 in vaccinated, convalescent, and control donors were 54%, 37%, and 10%, respectively. Donations with titers ≤16 were predominant in control donors (80%) and substantial in convalescent (47%) and vaccinated (40%) donors. Titers did not correlate with donor age, gender, or geographical location. GMTs for vaccinated donors were significantly higher than for convalescent donors and in both groups significantly higher than in the control. CONCLUSION: Targeted collection of plasma containing high levels of anti-influenza antibodies from self-identified donors was effective, but could be further improved by reducing the number of low-titer donations. More selective donor screening and/or testing for influenza antibodies could increase the potency of an influenza antibody-rich immune globulin (FLUIGIV).

Vitamin K5 is an efficient photosensitizer for ultraviolet A light inactivation of bacteria.

Photodynamic treatment combining light and a photosensitizer molecule can be an effective method to inactivate pathogenic bacteria. This study identified vitamin K5 as an efficient photosensitizer for ultraviolet light A (UVA)-induced bacterial inactivation. Six bacterial species, Bacillus cereus (vegetative form), Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus epidermidis, Klebsiella pneumoniae, and two species of antibiotic-resistant bacteria, Pseudomonas aeruginosa* and Staphylococcus aureus*, were suspended in aqueous solutions with or without vitamin K5 and exposed to UVA irradiation. UVA irradiation (5.8 J cm-2) with vitamin K5 (1600 µmol l-1) reduced the colony forming units (CFU) of these bacteria by three to seven logs. Antibiotic resistant bacteria were also susceptible to the bactericidal effects of UVA and vitamin K5 combination treatment. Inactivation of bacteria in human plasma required higher doses of UVA light and vitamin K5. UVA irradiation (30 J cm-2) with vitamin K5 (2000 µmol l-1) reduced E. coli and S. aureus spiked into human plasma by seven logs CFU/ml. Reactive oxygen species, such as superoxide anion radicals and hydroxyl radicals, were found to be generated in vitamin K5 aqueous solution after UVA irradiation, suggesting these oxygen species may mediate the inactivation of the bacteria.

Binding and neutralizing anti-cytomegalovirus activities in immune globulin products.

Congenital infection as well as infection of immunocompromised individuals by cytomegalovirus (CMV) can be associated with significant morbidity, mortality, and long-term adverse health outcomes. Assessment of anti-viral activity using appropriate assays is essential for ensuring safe and efficacious use of therapeutic CMV immune globulin (IG) products. In this study, we used commercial ELISA kits to compare anti-CMV antibody binding activity and avidity for lots of CMV-specific and normal IG products available in the US market. Additionally, neutralizing activity of IG products was measured against CMV strains (AD169wt131 or TB40E-GFP) in MRC-5 human fibroblasts and ARPE-19 human epithelial cells. Our data revealed that, regardless of the method, anti-CMV activity was higher in CMV IG lots we tested compared with normal IG lots; CMV binding activity was at least 4-fold higher, and neutralizing activity at least 2- and 3-fold higher for epithelial and fibroblast cells, respectively, in CMV IG lots compared with normal IG lots. Furthermore, anti-CMV activity values from all three methods (ELISA, neutralization in MRC-5 cells, and neutralization in ARPE-19 cells) were highly correlated, whereas avidity, although higher in CMV IG lots, did not correlate well with either binding or neutralizing activities.

Mucosal antibody responses are directed by viral burden in children with acute influenza infection.

BACKGROUND: Influenza infection causes excess hospitalizations and deaths in younger patients, but susceptibility to severe disease is poorly understood. While mucosal antibodies can limit influenza-associated infection and disease, little is known about acute mucosal antibody responses to influenza infection. OBJECTIVES: These studies characterize mucosal antiviral antibody production in children during lower respiratory infection (LRI) with H1N1 influenza versus other viral LRI and examine the relationship between mucosal antiviral antibodies and protection against severe disease. METHODS: B lymphocytes were assessed by immunohistochemistry in lung tissue from infants with fatal acute seasonal influenza infection. Nasopharyngeal secretions (NPS) were obtained at presentation from children with acute respiratory illness, including H1N1 (2009) influenza infection. Total and antiviral antibodies, and inflammatory and immune mediators, were quantified by ELISA. Neutralizing activity in NPS was detected using a pseudotyped virus assay. Viral burden was assessed by qPCR. RESULTS AND CONCLUSIONS: B lymphocytes were abundant in lung tissue of infants with fatal acute influenza LRI. Among surviving children with H1N1 infection, only a small subset (11%) demonstrated H1N1 neutralizing activity in NPS. H1N1 neutralizing activity coincided with high local levels of antiviral IgM, IgG and IgA, greater detection of inflammatory mediators, and higher viral burden (P = 0·016). Patients with mucosal antiviral antibody responses demonstrated more severe respiratory symptoms including greater hypoxia (P = 0·0018) and pneumonia (P = 0·038). These patients also trended toward younger age, longer duration of illness and longer hospital stays. Prophylaxis strategies that heighten neutralizing antibody production in the mucosa are likely to benefit both older and younger children.

Cytosolic, autocrine alpha-1 proteinase inhibitor (A1PI) inhibits caspase-1 and blocks IL-1ß dependent cytokine release in monocytes.

RATIONALE: Activation state-dependent secretion of alpha-1 proteinase inhibitor (A1PI) by monocytes and macrophages was first reported in 1985. Since then, monocytes and tissue macrophages have emerged as key sentinels of infection and tissue damage via activation of self-assembling pattern recognition receptors (inflammasomes), which trigger inflammation and cell death in a caspase-1 dependent process. These studies examine the relationship between A1PI expression in primary monocytes and monocytic cell lines, and inflammatory cytokine expression in response to inflammasome directed stimuli. METHODS: IL-1 ß expression was examined in lung macrophages expressing wild type A1PI (A1PI-M) or disease-associated Z isoform A1PI (A1PI-Z). Inflammatory cytokine release was evaluated in THP-1 monocytic cells or THP-1 cells lacking the inflammasome adaptor ASC, transfected with expression vectors encoding A1PI-M or A1PI-Z. A1PI-M was localized within monocytes by immunoprecipitation in hypotonic cell fractions. Cell-free titration of A1PI-M was performed against recombinant active caspase-1 in vitro. RESULTS: IL-1 ß expression was elevated in lung macrophages expressing A1PI-Z. Overexpression of A1PI-M in THP-1 monocytes reduced secretion of IL-1ß and TNF-α. In contrast, overexpression of A1PI-Z enhanced IL-1ß and TNF- α secretion in an ASC dependent manner. A1PI-Z-enhanced cytokine release was inhibited by a small molecule caspase-1 inhibitor but not by high levels of exogenous wtA1PI. Cytosolic localization of A1PI-M in monocytes was not diminished with microtubule-inhibiting agents. A1PI-M co-localized with caspase-1 in gel-filtered cytoplasmic THP-1 preparations, and was co-immunoprecipitated with caspase 1 from nigericin-stimulated THP-1 cell lysate. Plasma-derived A1PI inhibited recombinant caspase-1 mediated conversion of a peptide substrate in a dose dependent manner. CONCLUSIONS: Our results suggest that monocyte/macrophage-expressed A1PI-M antagonizes IL-1ß secretion possibly via caspase-1 inhibition, a function which disease-associated A1PI-Z may lack. Therapeutic approaches which limit inflammasome responses in patients with A1PI deficiency, in combination with A1PI augmentation, may provide additional respiratory tissue-sparing benefits.

Epitope mapping by random peptide phage display reveals essential residues for vaccinia extracellular enveloped virion spread.

BACKGROUND: A33 is a type II integral membrane protein expressed on the extracellular enveloped form of vaccinia virus (VACV). Passive transfer of A33-directed monoclonal antibodies or vaccination with an A33 subunit vaccine confers protection against lethal poxvirus challenge in animal models. Homologs of A33 are highly conserved among members of the Orthopoxvirus genus and are potential candidates for inclusion in vaccines or assays targeting extracellular enveloped virus activity. One monoclonal antibody directed against VACV A33, MAb-1G10, has been shown to target a conformation-dependent epitope. Interestingly, while it recognizes VACV A33 as well as the corresponding variola homolog, it does not bind to the monkeypox homolog. In this study, we utilized a random phage display library to investigate the epitope recognized by MAb-1G10 that is critical for facilitating cell-to-cell spread of the vaccinia virus. RESULTS: By screening with linear or conformational random phage libraries, we found that phages binding to MAb-1G10 display the consensus motif CEPLC, with a disulfide bond formed between two cysteine residues required for MAb-1G10 binding. Although the phage motif contained no linear sequences homologous to VACV A33, structure modeling and analysis suggested that residue D115 is important to form the minimal epitope core. A panel of point mutants expressing the ectodomain of A33 protein was generated and analyzed by either binding assays such as ELISA and immunoprecipitation or a functional assessment by blocking MAb-1G10 mediated comet inhibition in cell culture. CONCLUSIONS: These results confirm L118 as a component of the MAb-1G10 binding epitope, and further identify D115 as an essential residue. By defining the minimum conformational structure, as well as the conformational arrangement of a short peptide sequence recognized by MAb-1G10, these results introduce the possibility of designing small molecule mimetics that may interfere with the function of A33 in vivo. This information will also be useful for designing improved assays to evaluate the potency of monoclonal and polyclonal products that target A33 or A33-modulated EV dissemination.

Eczema vaccinatum.

Eczema vaccinatum (EV) is a complication of smallpox vaccination that can occur in persons with eczema/atopic dermatitis (AD), in which vaccinia virus disseminates to cause an extensive rash and systemic illness. Because persons with eczema are deferred from vaccination, only a single, accidentally transmitted case of EV has been described in the medical literature since military vaccination was resumed in the United States in 2002. To enhance understanding of EV, we review its history during the era of universal vaccination and discuss its relationship to complications in persons with other diseases or injuries of the skin. We then discuss current concepts of the pathophysiology of AD, noting how defective skin barrier function, epidermal hyperplasia, and abnormal immune responses favor the spread of poxviral infection, and identify a number of unanswered questions about EV. We conclude by considering how its occurrence might be minimized in the event of a return to universal vaccination.

Pandemic Influenza Detection by Electrically Active Magnetic Nanoparticles and Surface Plasmon Resonance.

Influenza A virus (FLUAV), the causative agent of influenza infection, has received extensive attention due to the recent swine-origin H1N1 pandemic. FLUAV has long been the cause of annual epidemics as well as less frequent but more severe global pandemics. Here, we describe a biosensor utilizing electrically active magnetic (EAM) polyaniline-coated nanoparticles as the transducer in an electrochemical biosensor for rapidly identifying FLUAV strains based on receptor specificity, which will be useful to monitor animal influenza infections and to characterize pandemic potential of strains that have transmitted from animals to humans. Pandemic potential requires human-to-human transmissibility, which is dependent upon FLUAV hemagglutinin (HA) specificity for host glycan receptors. Avian FLUAV preferentially bind to α2,3-linked receptors, while human FLUAV bind to α2,6-linked receptors. EAM nanoparticles were prepared by synthesizing aniline monomer around gamma iron (III) oxide (γ-Fe2O3) cores, yielding 25-100-nm diameter nanoparticles that were structurally characterized by transmission electron microscopy and electron diffraction. The EAM nanoparticles were coated with monoclonal antibodies specific to H5N1 (A/Vietnam/1203/04). Specificity of binding between glycans and H5 was demonstrated. The biosensor results were correlative to supporting data from a surface plasmon resonance assay that characterized HA/glycan binding and α-H5 antibody activity. This novel study applies EAM nanoparticles as the transducer in a specific, portable, easy-to-use biosensor with great potential for disease monitoring and biosecurity applications.