MAB8257 Sigma-AldrichAnti-Influenza A Antibody, nucleoprotein, clone A1

We investigated immune determinants of antibody responses and B-cell memory to pH1N1 vaccine in HIV-infected children.Ninety subjects 4 to less than 25 years of age received two double doses of pH1N1 vaccine. Serum and cells were frozen at baseline, after each vaccination, and at 28 weeks post-immunization. Hemagglutination inhibition (HAI) titers, avidity indices (AI), B-cell subsets, and pH1N1 IgG and IgA antigen secreting cells (ASC) were measured at baseline and after each vaccination. Neutralizing antibodies and pH1N1-specific Th1, Th2 and Tfh cytokines were measured at baseline and post-dose 1.At entry, 26 (29%) subjects had pH1N1 protective HAI titers (≥1:40). pH1N1-specific HAI, neutralizing titers, AI, IgG ASC, IL-2 and IL-4 increased in response to vaccination (pless than 0.05), but IgA ASC, IL-5, IL-13, IL-21, IFNγ and B-cell subsets did not change. Subjects with baseline HAI ≥1:40 had significantly greater increases in IgG ASC and AI after immunization compared with those with HAI less than 1:40. Neutralizing titers and AI after vaccination increased with older age. High pH1N1 HAI responses were associated with increased IgG ASC, IFNγ, IL-2, microneutralizion titers, and AI. Microneutralization titers after vaccination increased with high IgG ASC and IL-2 responses. IgG ASC also increased with high IFNγ responses. CD4% and viral load did not predict the immune responses post-vaccination, but the B-cell distribution did. Notably, vaccine immunogenicity increased with high CD19+CD21+CD27+% resting memory, high CD19+CD10+CD27+% immature activated, low CD19+CD21-CD27-CD20-% tissue-like, low CD19+CD21-CD27-CD20-% transitional and low CD19+CD38+HLADR+% activated B-cell subsets.HIV-infected children on HAART mount a broad B-cell memory response to pH1N1 vaccine, which was higher for subjects with baseline HAI≥1:40 and increased with age, presumably due to prior exposure to pH1N1 or to other influenza vaccination/infection. The response to the vaccine was dependent on B-cell subset distribution, but not on CD4 counts or viral load.ClinicalTrials.gov NCT00992836.

This study describes the development of aptamers as a therapy against influenza virus infection. Aptamers are oligonucleotides (like ssDNA or RNA) that are capable of binding to a variety of molecular targets with high affinity and specificity. We have studied the ssDNA aptamer BV02, which was designed to inhibit influenza infection by targeting the hemagglutinin viral protein, a protein that facilitates the first stage of the virus' infection. While testing other aptamers and during lead optimization, we realized that the dominant characteristics that determine the aptamer's binding to the influenza virus may not necessarily be sequence-specific, as with other known aptamers, but rather depend on general 2D structural motifs. We adopted QSAR (quantitative structure activity relationship) tool and developed computational algorithm that correlate six calculated structural and physicochemical properties to the aptamers' binding affinity to the virus. The QSAR study provided us with a predictive tool of the binding potential of an aptamer to the influenza virus. The correlation between the calculated and actual binding was R2 = 0.702 for the training set, and R2 = 0.66 for the independent test set. Moreover, in the test set the model's sensitivity was 89%, and the specificity was 87%, in selecting aptamers with enhanced viral binding. The most important properties that positively correlated with the aptamer's binding were the aptamer length, 2D-loops and repeating sequences of C nucleotides. Based on the structure-activity study, we have managed to produce aptamers having viral affinity that was more than 20 times higher than that of the original BV02 aptamer. Further testing of influenza infection in cell culture and animal models yielded aptamers with 10 to 15 times greater anti-viral activity than the BV02 aptamer. Our insights concerning the mechanism of action and the structural and physicochemical properties that govern the interaction with the influenza virus are discussed.

Existing methods to measure influenza vaccine immunogenicity prohibit detailed analysis of epitope determinants recognized by immunoglobulins. The development of highly multiplex proteomics platforms capable of capturing a high level of antibody binding information will enable researchers and clinicians to generate rapid and meaningful readouts of influenza-specific antibody reactivity.We developed influenza hemagglutinin (HA) whole-protein and peptide microarrays and validated that the arrays allow detection of specific antibody reactivity across a broad dynamic range using commercially available antibodies targeted to linear and conformational HA epitopes. We derived serum from blood draws taken from 76 young and elderly subjects immediately before and 28±7 days post-vaccination with the 2008/2009 trivalent influenza vaccine and determined the antibody reactivity of these sera to influenza array antigens.Using linear regression and correcting for multiple hypothesis testing by the Benjamini and Hochberg method of permutations over 1000 resamplings, we identified antibody reactivity to influenza whole-protein and peptide array features that correlated significantly with age, H1N1, and B-strain post-vaccine titer as assessed through a standard microneutralization assay (pless than 0.05, q less than 0.2). Notably, we identified several peptide epitopes that were inversely correlated with regard to age and seasonal H1N1 and B-strain neutralization titer (pless than 0.05, q less than 0.2), implicating reactivity to these epitopes in age-related defects in response to H1N1 influenza. We also employed multivariate linear regression with cross-validation to build models based on age and pre-vaccine peptide reactivity that predicted vaccine-induced neutralization of seasonal H1N1 and H3N2 influenza strains with a high level of accuracy (84.7% and 74.0%, respectively).Our methods provide powerful tools for rapid and accurate measurement of broad antibody-based immune responses to influenza, and may be useful in measuring response to other vaccines and infectious agents.

This chapter describes some commonly used methods of influenza virus titration, antigenic characterization, and serological methods by antibody detection. These methods are essential not only for virus characterization but also for identifying new antigenic variants, vaccine strain selection, and sero-epidemiologic studies of influenza virus transmission and prevalence. Virus titration methods such as the hemagglutination assay, 50% egg or tissue culture infectious dose, and plaque assay are employed to determine the amount of virus particles in a sample. The hemagglutination inhibition assay is a reliable, relatively simple and inexpensive technique to antigenically characterize isolates of influenza viruses. Serological methods such as virus neutralization and hemagglutination inhibition are the fundamental tools used in sero-epidemiologic studies of influenza virus transmission and prevalence and in the evaluation of vaccine immunogenicity. While serological methods rarely yield an early diagnosis of acute influenza virus infection, well-timed, paired acute, and convalescent serum samples may establish the diagnosis of a recent influenza infection even when attempts to detect the virus are negative.

In this study, the effect of innate serum inhibitors on influenza virus infection was addressed. Seasonal influenza A(H1N1) and A(H3N2), 2009 pandemic A(H1N1) (H1N1pdm) and highly pathogenic avian influenza (HPAI) A(H5N1) viruses were tested with guinea pig sera negative for antibodies against all of these viruses as evaluated by hemagglutination-inhibition and microneutralization assays. In the presence of serum inhibitors, the infection by each virus was inhibited differently as measured by the amount of viral nucleoprotein produced in Madin-Darby canine kidney cells. The serum inhibitors inhibited seasonal influenza A(H3N2) virus the most, while the effect was less in seasonal influenza A(H1N1) and H1N1pdm viruses. The suppression by serum inhibitors could be reduced by heat inactivation or treatment with receptor destroying enzyme. In contrast, all H5N1 strains tested were resistant to serum inhibitors. To determine which structure (hemagglutinin (HA) and/or neuraminidase (NA)) on the virus particles that provided the resistance, reverse genetics (rg) was applied to construct chimeric recombinant viruses from A/Puerto Rico/8/1934(H1N1) (PR8) plasmid vectors. rgPR8-H5 HA and rgPR8-H5 HANA were resistant to serum inhibitors while rgPR8-H5 NA and PR8 A(H1N1) parental viruses were sensitive, suggesting that HA of HPAI H5N1 viruses bestowed viral resistance to serum inhibition. These results suggested that the ability to resist serum inhibition might enable the viremic H5N1 viruses to disseminate to distal end organs. The present study also analyzed for correlation between susceptibility to serum inhibitors and number of glycosylation sites present on the globular heads of HA and NA. H3N2 viruses, the subtype with highest susceptibility to serum inhibitors, harbored the highest number of glycosylation sites on the HA globular head. However, this positive correlation cannot be drawn for the other influenza subtypes.

Studies of influenza virus evolution under controlled experimental conditions can provide a better understanding of the consequences of evolutionary processes with and without immunological pressure. Characterization of evolved strains assists in the development of predictive algorithms for both the selection of subtypes represented in the seasonal influenza vaccine and the design of novel immune refocused vaccines. To obtain data on the evolution of influenza in a controlled setting, naïve and immunized Guinea pigs were infected with influenza A/Wyoming/2003 (H3N2). Virus progeny from nasal wash samples were assessed for variation in the dominant and other epitopes by sequencing the hemagglutinin (HA) gene to quantify evolutionary changes. Viral RNA from the nasal washes from infection of naïve and immune animals contained 6% and 24.5% HA variant sequences, respectively. Analysis of mutations relative to antigenic epitopes indicated that adaptive immunity played a key role in virus evolution. HA mutations in immunized animals were associated with loss of glycosylation and changes in charge and hydrophobicity in and near residues within known epitopes. Four regions of HA-1 (75-85, 125-135, 165-170, 225-230) contained residues of highest variability. These sites are adjacent to or within known epitopes and appear to play an important role in antigenic variation. Recognition of the role of these sites during evolution will lead to a better understanding of the nature of evolution which help in the prediction of future strains for selection of seasonal vaccines and the design of novel vaccines intended to stimulated broadened cross-reactive protection to conserved sites outside of dominant epitopes.

Influenza virus invades the olfactory bulb (OB) and enhances cytokine mRNAs therein at the time of illness onset. Here we show that viral antigen immunoreactivity co-localized with glial markers in the OB but could not be detected in other brain areas. Interleukin 1beta- and tumor necrosis factor alpha-immunoreactivity co-localized with neuronal markers in olfactory and central autonomic systems, and the number of cytokine-immunoreactive neurons increased at the time of illness onset [15 h post-inoculation (PI)] but not before (10 h PI). These results suggest that the OB virus influences the brain cytokines and therefore the onset of illness.

An epitope-blocking enzyme-linked immunosorbent assay (bELISA) was developed for the detection of antibodies to influenza A virus in taxonomically diverse domestic and wild vertebrate species. In contrast to the bELISAs published previously that require reagent production, manipulation by the end-user, or have not been evaluated for use with both mammalian and avian species, this assay is performed using commercially available recombinant nucleoprotein antigen and corresponding nucleoprotein-specific monoclonal antibody and has been shown to work with multiple avian and mammalian species. The efficacy of the bELISA as a serum screening assay was compared to the agar gel immunodiffusion (AGID) assay using 251 serum samples obtained from experimentally infected mallards (Anas platyrhynchos) and raccoons (Procyon lotor). The concordance between the AGID assay and bELISA was 94.1% (95% CI=89.9, 98.3) for raccoons, and 71.2% (95% CI=63.5, 78.9) for mallards and 82.8% (95% CI=78.2, 87.3) overall. The bELISA was more sensitive than the AGID assay as demonstrated by the detection of antibodies to influenza A virus at earlier time points in experimental infection studies and at higher serial dilutions. The efficacy of the bELISA to monitor natural influenza A virus exposure was also compared to the AGID assay using an additional 745 serum samples from six avian species and six mammalian species. This bELISA provides a rapid, reliable, and inexpensive technique for large-scale surveillance of influenza A virus exposure in taxonomically diverse vertebrate species.

Mouse monoclonal antibodies, directed against antigenic sites on influenza A and B viruses and found to be type-specific in an immunoassay, were assessed for use as diagnostic reagents in an indirect immunofluorescence assay on nasopharyngeal secretions. The influenza A antibodies were directed against nucleoprotein or matrix protein antigens and the influenza B antibodies against nucleoprotein and haemagglutinin antigens. The influenza A anti-matrix monoclonal antibody was found to give a strong intranuclear particulate fluorescence in normal baboon kidney cells and cells from nasopharyngeal secretions negative for influenza A virus, including those from a patient infected with respiratory syncytial virus. Pools of the remaining monoclonal antibodies gave satisfactory results on 25 specimens from patients with influenza A H1N1 and H3N2 subtypes and 12 from patients with influenza B.