Development of a duplex Fluorescent Microsphere Immunoassay (FMIA) for the detection of antibody responses to influenza A and newcastle disease viruses.

Highly pathogenic avian influenza virus (HPAI) and virulent forms of avian paramyxovirus-1 (APMV-1) cause serious illnesses in domestic poultry, both of which are reportable to the World Organization of Animal Health (OIE). The clinical presentation of avian influenza (AI) and APMV-1 infections are difficult to differentiate, emphasizing the importance of rapid and sensitive serologic assays that are able to distinguish them. Currently, a variety of serological assays are used for the serologic diagnosis of both diseases, but these assays are not used in multiplex formats. In this study, development of a duplex fluorescent microsphere immunoassay (FMIA) based on Luminex xMAP Technology is described. The assay employs MagPlex magnetic microspheres that are covalently coated with recombinant avian influenza virus nucleoprotein and APMV-1 nucleocapsid antigens produced in a baculovirus insect cell expression system. The assay is able to detect AIV antibodies against all existing hemagglutinin (H1-H16) subtypes and simultaneously detect antibodies against APMV-1. In the process of this assay development different bead coupling conditions were compared. The assay has the capability of detecting serum antibodies from chickens and turkeys and optimization was accomplished by using 2462 chicken and 446 turkey field and experimental sera and had a comparable detection capability with currently used assays in the laboratory. Assay threshold values were calculated with Receiver Operating Characteristic Analysis (ROC) in non-parametric analysis due to a highly skewed data distribution; this analysis resulted in AIV nucleoprotein relative diagnostic sensitivity and specificity of 99.7%, and 97.3% respectively. The APMV-1 nucleocapsid relative diagnostic sensitivity and specificity were 95.4%, and 98.5% respectively.

Pre-exposing Canada Geese (Branta canadensis) to a low-pathogenic H1N1 avian influenza virus protects them against H5N1 HPAI virus challenge.

In previous studies we examined the role of Canada Geese (Branta canadensis) in the epidemiology of Eurasian highly pathogenic avian influenza (HPAI) H5N1. To expand on this and better understand how pre-exposure to heterosubtypic low-pathogenic avian influenza (LPAI) viruses might influence the outcome of H5N1 HPAI infection, we pre-exposed naïve juvenile Canada Geese to different North American wild-bird-origin LPAI viruses. We selected H1, H2, and H6 hemagglutinin subtype viruses based on their higher-order evolutionary relatedness to the H5 hemagglutinin. Pre-exposing Canada Geese to either H2N3 or H6N5 viruses did not protect them against a lethal H5N1 HPAI virus challenge. In addition, H5N1 was transmitted to naïve control birds that were placed among both groups resulting in death by 5 days postcontact. In contrast, Canada Geese that were pre-exposed to H1N1 were protected against a lethal H5N1 challenge, shed minimal amounts of the virus into the environment, and did not transmit the infection to naïve contact birds. None of the H1N1, H2N3, or H6N5 pre-exposure sera neutralized H5N1 in vitro; however, sera from H1N1-infected birds reduced virus plaque size but not number when compared with H2N3, H6N5, or negative sera, suggesting that antibodies directed against the neuraminidase may have had a role in the protective effects observed.

Development of neutralizing monoclonal antibodies against the pandemic H1N1 virus (2009) using plasmid DNA immunogen.

Reported here is the development and characterization of eighteen mouse monoclonal antibodies (MAbs) against the 2009 pandemic H1N1 influenza virus (A/Mexico/InDRE4487/2009). To our knowledge, this is the first report on pandemic (pH1) H1N1 MAbs developed using plasmid DNA encoding the viral surface glycoprotein, hemagglutinin (HA). All eighteen MAbs were specific for A/Mexico/InDRE4487/2009 HA. Ten MAbs were found to cross-react with A/Swine/Indiana/81 using a dot blot assay. However, there was no cross-reactivity detected against any other strains of influenza A viruses despite screening against all 16 hemagglutinin subtypes. Examination of these MAbs identified individual antibodies suitable for use in several practical applications including ELISA, immunoblot and immunofluorescence assays. Analysis of the kinetics of each MAb revealed significant binding affinities (K(D)<10(-8) M) confirming the antibodies are highly specific for A/Mexico/InDRE4487/2009 HA. Functional analysis demonstrated the panel of MAbs included antibodies with HA inhibition and virus neutralization activities. Not all MAbs inhibited hemagglutination or neutralized the virus. Furthermore, the panel of MAbs was not found to be cross-reactive against additional strains tested in hemagglutination inhibition assays. Finally, the MAbs were tested in competitive ELISA (cELISA) using reference serum antibodies developed against different clusters of H1 (pH1, α, ß, γ, and δ). The developed MAbs outcompeted serum antibodies of pH1 in 16/18, 15/18 (γ), 3/18 (α), 2/18 (δ1) and 1/18 (ß) samples. Overall, this panel of MAbs proved specific and highly sensitive for A/Mexico/InDRE4487/2009 HA and could potentially serve as immunodiagnostic tools for the rapid detection of this specific strain of influenza virus.

Development and characterization of neutralizing monoclonal antibodies against the pandemic H1N1 virus (2009).

The 2009 H1N1 influenza pandemic was a major international public health crisis which caused considerable morbidity and mortality worldwide. The goal of this study was to produce anti-H1 monoclonal antibodies (MAbs) for improving diagnostic immunological assays and to develop potential immunotherapeutics. Nine MAbs were produced after immunizing mice with recombinant hemagglutinin (HA) protein from A/California/06/09. Two spleenocyte myeloma fusions yielded 1588 hybridoma cultures. After screening the hybridoma culture supernatants for antibody reactivity to rHA, nine clones were selected for further characterization. Cross-reactivity studies of the anti-rHA antibodies against a panel of influenza viruses (H1-H16) revealed eight out of nine MAbs were specific to the pandemic H1 subtype, except for MAb F256G2sc1 which also cross-reacted with H5 subtype virus. All MAbs were of the IgG1κ isotype, except F256G2sc1 which was IgG2aκ. The anti-rHA MAbs had binding affinities to rHA that ranged from a K(D) (disassociation constant) of 1.34×10(-9)M (F255G7sc1) to the weakest affinity of 4.60×10(-8)M (F255G4sc1). Interestingly, in a plaque reduction neutralization assay, all MAbs except F255G3sc1 demonstrated neutralizing ability. Furthermore, all MAbs except F255G3sc1 and F255G9sc1 exhibited anti-hemagglutinin activity against pandemic H1N1 viruses, but not against classical North American swine influenza viruses of the same subtype. Immunofluorescence assay (IFA) demonstrated that all MAbs except F255G1sc1 and F255G3sc1 were able to detect 2009 pandemic H1N1 (2009) virus- infected MDCK cells. The MAbs were also evaluated for potential use in competitive ELISA (cELISA), and with the exception of F255G3sc1, all MAbs showed competitive activity with serum collected from pigs infected with pandemic H1N1 virus (2009). The developed MAbs have demonstrated utility as immunodiagnostic and research reagents, and their neutralizing capabilities also hold potential for designing antiviral drugs against pandemic influenza.

Molecular characterization of pandemic H1N1 influenza viruses isolated from turkeys and pathogenicity of a human pH1N1 isolate in turkeys.

Suspected human-to-animal transmission of the 2009 pandemic H1N1 (pH1N1) virus has been reported in several animal species, including pigs, dogs, cats, ferrets, and turkeys. In this study we describe the genetic characterization of pH1N1 viruses isolated from breeder turkeys that was associated with a progressive drop in egg production. Sequence analysis of all eight gene segments from three viruses isolated from this outbreak demonstrated homology with other human and swine pH1N1 isolates. The susceptibility of turkeys to a human pH1N1 isolate was further evaluated experimentally. The 50% turkey infectious dose (TID50) for the human isolate A/Mexico/LnDRE/4487/2009 was determined by inoculating groups of 8-10-week-old turkeys with serial 10-fold dilutions of virus by oronasal and cloacal routes. We estimated the TID50 to be between 1 x 10(5) and 1 x 10(6) TCID50. The pathogenesis of pH1N1 in oronasally or cloacally inoculated juvenile turkeys was also examined. None of the turkeys exhibited clinical signs, and no significant difference in virus shedding or seroconversion was observed between the two inoculation groups. More than 50% of the turkeys in both oronasal and cloacal groups shed virus beginning at 2 days postinoculation (dpi). All birds that actively shed virus seroconverted by 14 dpi. Virus antigen was demonstrated by immunohistochemistry in the cecal tonsils and bursa of Fabricius in two of the birds that were infected by the cloacal route. Virus transmission to naive contact turkeys was at best doubtful. This report provides additional evidence that pH1N1 can cross the species barrier and cause disease outbreaks in domestic turkeys. However, it appears that the reproductive status of the host as well as environmental factors such as concurrent infections, stress, the presence or absence of litter, and stocking density may also contribute to efficient infection and transmission of this agent.

A Model for NAD(P)H:Quinoneoxidoreductase 1 (NQO1) Targeted Individualized Cancer Chemotherapy.

NQO1 (NAD(P)H:quinoneoxidoreductase 1) is a reductive enzyme that is an important activator of bioreductive antitumor agents. NQO1 activity varies in individual tumors but is generally higher in tumor cells than in normal cells. NQO1 has been used as a target for tumor specific drug development. We investigated a series of bioreductive benzoquinone mustard analogs as a model for NQO1 targeted individualized cancer chemotherapy. We compared the tumor cell growth inhibitory activity of benzoquinone mustard analogs with sterically bulky groups of different size and placed at different positions on the benzoquinone ring, using tumor cell lines with different levels of NQO1. We demonstrated that functional groups of different steric size could be used to produce a series of bioreductive antitumor agents that were activated by different levels of NQO1 in tumor cells. This series of drugs could then be used to target cells with specific levels of NQO1 for growth inhibition and to avoid damage to normal cells, like bone marrow cells, that have low levels of NQO1. This approach could be used to develop new bioreductive antitumor agents for NQO1 targeted individualized cancer chemotherapy.

Delayed sodium thiosulphate administration reduces cisplatin efficacy on mouse EMT6 tumour cells in vitro.

OBJECTIVE: To determine whether simultaneous and/or delayed administration of sodium thiosulphate (STS) affects the oncologic effect of cisplatin or cisdiaminedichloroplatinum (CDDP) in EMT6 tumour cells in vitro. SETTING: Cell biology research laboratory. METHODS: Clonogenic assays of EMT6 tumour cells with CDDP alone, CDDP plus simultaneous STS, and CDDP plus a 4-hour delay of STS were performed. Growth fractions under these three conditions were compared. RESULTS: Tumour growth was statistically significantly increased when CDDP and STS were administered compared with CDDP alone. There was no statistically significant difference between simultaneous and 4-hour delay of STS administration. We conclude that in EMT6 cells, either simultaneous administration or a 4-hour delay of STS administration significantly decreases CDDP efficacy. CONCLUSION: STS adversely affects CDDP's oncologic efficacy in EMT6 cell cultures in vitro.

Role of NADPH cytochrome P450 reductase in activation of RH1.

PURPOSE: RH1 is a new bioreductive agent that is an excellent substrate for the two-electron reducing enzyme, NAD(P)H quinone oxidoreductase 1 (NQO1). RH1 may be an effective NQO1-directed antitumor agent for treatment of cancer cells having elevated NQO1 activity. As some studies have indicated that RH1 may also be a substrate for the one-electron reducing enzyme, NADPH cytochrome P450 reductase (P450 Red), P450 Red may contribute to the activation of RH1 where NQO1 activities are low and P450 Red activities are high. The mean P450 Red activity in the human tumor cell line panel used by NCI for evaluation of new anticancer agents is 14.8 nmol min(-1) mg prot(-1), while the mean NQO1 activity in these cell lines is 199.5 nmol min(-1) mg prot(-1). Thus, we investigated whether P450 Red could play a role in activating RH1. METHODS: Reduction of RH1 by purified human P450 Red was investigated using electron paramagnetic resonance and spectroscopic assays. The ability of RH1 to produce DNA damage following reduction by P450 Red was studied using gel assays. To determine the role of P450 Red in activation of RH1 in cells, cell growth inhibition studies with inhibitors of P450 Red and NQO1 were carried out in T47D human breast cancer cells and T47D cells transfected with the human P450 Red gene (T47D-P450) that have P450 Red activities of 11.5 and 311.8 nmol min(-1) mg prot(-1), respectively. RESULTS: Reduction studies using purified P450 Red and NQO1 confirmed that RH1 can be reduced by both enzymes, but redox cycling was slower following reduction by NQO1. RH1 produced DNA strand breaks and crosslinks in isolated DNA after reduction by either P450 Red or NQO1. DPIC, an inhibitor of P450 Red, had no effect on cell growth inhibition by RH1 in T47D cells, and had only a small effect on cell growth inhibition by RH1 in the presence of the NQO1 inhibitor, dicoumarol, in T47D-P450 cells. CONCLUSIONS: These results demonstrated that P450 Red does not contribute to the activation of RH1 in cells with normal P450 Red activity and plays only a minor role in activating this agent in cells with high levels of this enzyme. These studies confirmed that P450 Red could activate RH1 and provided the first direct evidence that RH1 could produce both DNA strand breaks and DNA crosslinks after reduction by P450 Red. However, the results strongly suggest that P450 Red does not play a significant role in activating RH1 in cells with normal P450 Red activity.

Sodium thiosulphate impairs the cytotoxic effects of cisplatin on FADU cells in culture.

OBJECTIVES: To study the effect of cisplatin (CDDP) on FADU human squamous cell carcinoma cells and to determine if sodium thiosulphate (STS), an agent protective against CDDP ototoxicity, affects the tumoricidal activity of CDDP. METHOD: FADU tumour cells were grown in culture. Cells were exposed to varying concentrations of CDDP with and without concomitant STS, and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) assays were then performed to determine the effect of treatment on FADU cell growth. RESULTS: Dose-response curves were generated for the FADU cells exposed to CDDP with and without concomitant STS. Concomitant STS was found to inhibit the tumoricidal activity of CDDP in vitro. CONCLUSIONS: Simultaneous administration of STS to in vitro culture of FADU tumor cells leads to inhibition of CDDP's tumoricidal activity.

Effect of sodium thiosulphate and cis-diamminedichloroplatinum on FADU tumour cells in nude mice.

OBJECTIVES: To study the effect of cis-diamminedichloroplatinum (CDDP) on FADU squamous cell carcinoma cells in a nude mouse model and to determine the effect of sodium thiosulphate (STS) on CDDP activity. METHODS: CD1 nude mice were inoculated with FADU tumour cells to both flanks. They were then randomized to four treatment groups: control, CDDP only, STS only, or CDDP and STS. Tumour growth was measured using calipers and charted at 3-day intervals. RESULTS: Tumour volumes were calculated as an ellipsoid and charted against time. CONCLUSIONS: CDDP inhibited FADU tumour cell growth compared with saline controls (p < .005). The addition of STS did not inhibit the CDDP activity when compared with CDDP-alone activity (p = .989). Compared with saline control solution, STS alone also inhibited tumour growth significantly (p < .005).

Effect of NQO1 induction on the antitumor activity of RH1 in human tumors in vitro and in vivo.

NQO1 is a reductive enzyme that is important for the activation of many bioreductive agents and is a target for an enzyme-directed approach to cancer therapy. It can be selectively induced in many tumor types by a number of compounds including dimethyl fumarate and sulforaphane. Mitomycin C is a bioreductive agent that is used clinically for treatment of solid tumors. RH1 (2,5-diaziridinyl-3-(hydroxymethyl)- 6-methyl-1,4-benzoquinone) is a new bioreductive agent currently in clinical trials. We have shown previously that induction of NQO1 can enhance the antitumor activity of mitomycin C in tumor cells in vitro and in vivo. As RH1 is activated selectively by NQO1 while mitomycin C is activated by many reductive enzymes, we investigated whether induction of NQO1 would produce a greater enhancement of the antitumor activity of RH1 compared with mitomycin C. HCT116 human colon cancer cells and T47D human breast cancer cells were incubated with or without dimethyl fumarate or sulforaphane followed by mitomycin C or RH1 treatment, and cytotoxic activity was measured by a clonogenic (HCT116) or MTT assay (T47D). Dimethyl fumarate and sulforaphane treatment increased NQO1 activity by 1.4- to 2.8-fold and resulted in a significant enhancement of the antitumor activity of mitomycin C, but not of RH1. This appeared to be due to the presence of a sufficient constitutive level of NQO1 activity in the tumor cells to fully activate the RH1. Mice were implanted with HL60 human promyelocytic leukemia cells, which have low levels of NQO1 activity. The mice were fed control or dimethyl fumarate-containing diet and were treated with RH1. NQO1 activity in the tumors increased but RH1 produced no antitumor activity in mice fed control or dimethyl fumarate diet. This is consistent with a narrow window of NQO1 activity between no RH1 activation and maximum RH1 activation. This study suggests that selective induction of NQO1 in tumor cells is not likely to be an effective strategy for enhancing the antitumor activity of RH1. In addition, we found that RH1 treatment produced significant leukopenia in mice that may be of concern in the clinic. These results suggest that the ease of reduction of RH1 by NQO1 makes it a poor candidate for an enzyme-directed approach to cancer therapy.