H1N1 Antigens Research Articles

Detection of influenza virus by electrochemical surface plasmon resonance under potential modulation.

In this study we report the development of a novel viral pathogen immunosensor technology based on the electrochemical modulation of the optical signal from a surface plasmon wave interacting with a redox dye reporter. The device is formed by incorporating a sandwich immunoassay onto the surface of a plasmonic device mounted in a micro-electrochemical flow cell, where it is functionalized with a monoclonal antibody aimed to a specific target pathogen antigen. Once the target antigen is bound to the surface, it promotes the capturing of a secondary polyclonal antibody that has been conjugated with a redox-active methylene blue dye. The methylene blue displays a reversible change in the complex refractive index throughout a reduction-oxidation transition, which generates an optical signal that can be electrochemically modulated and detected at high sensitivity. For proof-of-principle measurements, we have targeted the hemagglutinin protein from the H5N1 avian influenza A virus to demonstrate the capabilities of our device for detection and quantification of a critical influenza antigen. Our experimental results of the EC-SPR-based immunosensor under potential modulation showed a 300pM limit of detection for the H5N1 antigen.

Prokaryotic Expression of H1N1 Influenza A Virus Haemagglutinin Protein Globular Domain (HA1)

Abstract Background: Influenza viruses are a significant cause of morbidity and mortality .The influenza virus pandemics, 1918, 1977, and especially the most recent one, A/H1N1/2009, made evident the need for generating recombinant Influenza H1N1 antigens which are essential to develop both basic and applied research programs. Among influenza virus proteins, haemagglutinin (HA) is a major surface antigen of influenza virus, thus it is highly topical in influenza research and vaccine engineering programs. Alternatively, expression of fragments of the HA (HA1 and HA2) proteins in prokaryotic systems can potentially be the most efficacious strategy for manufacture of large quantities of influenza vaccine in a short period of time. Methodology: The gene encoding the HA1 protein from the influenza A/Puerto Rico/8/34 was amplified by PCR, then cloned into pTZ57R/T cloning vector. The fidelity of HA1 open reading frame was confirmed by bidirectional sequencing, then sub-cloned into pET28a prokaryotic expression plasmid, and proteins containing HA1 N-terminally fused to His-Tag were produced in Escherichia coli BL21 through IPTG inducing. The accuracy of the expression was confirmed by running time coursed fraction samples taken before and after the IPTG induction in SDS-PAGE, Western blot analysis was also used for confirmation of the recombinant protein. Conclusion: The HA1 protein produced here could be considered and evaluated as a protective antigen which its immunogenicity potential needs to be assessed in animal models along with proper control groups. Moreover, it could be subjected for polyclonal antibody preparation, which, in turn, may be used as an essential material in western blot analyses, as well as in other immunological applications, such as ELISA, immunocytochemistry, immunohistochemistry, and other immunological and serological studies. Key words: Influenza A, H1N1 , Hemagglutinin , Prokaryotic Expression

A Universal Influenza Virus Vaccine Candidate Tested in a Pig Vaccination-Infection Model in the Presence of Maternal Antibodies.

The antigenically conserved hemagglutinin stalk region is a target for universal influenza virus vaccines since antibodies against it can provide broad protection against influenza viruses of different subtypes. We tested a universal influenza virus vaccination regimen based on sequential immunization with chimeric hemagglutinin (HA) containing viruses in a swine influenza virus pig model with maternal antibodies against pandemic H1N1. Vaccines were administered as live attenuated virus or inactivated influenza virus split vaccine (+/− Emulsigen adjuvant). As controls, we included groups that received trivalent inactivated influenza vaccine that contained pandemic H1N1 antigens, inactivated adjuvanted H1N2 vaccine (control group for vaccine associated enhanced respiratory disease in the pig model) or mock-vaccination. No induction of H1 head or stalk-specific antibody responses was observed upon vaccination, while responses against H3 and influenza B HA were elicited in the group vaccinated with the trivalent vaccine. Four weeks post vaccination, pigs were intratracheally challenged with pandemic H1N1 virus and euthanized 5 days after challenge. Despite the lack of detectable anti-stalk immunity, the chimeric hemagglutinin vaccine resulted in better clinical outcomes compared to control groups.

Influenza Vaccination Protects Against Pandemic H1N1 Infection in Sickle Cell Disease Mice.

Influenza infection is associated with enhanced pathology in individuals with sickle cell disease (SCD). Despite being a high priority group for annual influenza vaccination, little is known about long-term responses to influenza vaccination in this patient population. To model flu vaccination, we inoculated SCD and wild type (WT) littermate mice with the seasonal flu vaccine [containing pandemic H1N1 (pH1N1) antigen], bled the mice before and after vaccination, and intranasally challenged them with a high dose (400 PFU) of pH1N1 12 weeks later. Both WT and SCD mice were fully protected from infection, and anti-influenza immunoglobulin G titers were significantly elevated in both groups after vaccination. It appears that flu vaccination is effective in SCD mice and our data support the clinical practice of regular flu vaccination in SCD patients.

Penentuan Secara Imunopatologi Organ Target Virus Flu Burung Menggunakan Streptavidin Biotin (DETERMINATION OF TARGET ORGANS OF AVIAN INFLUENZA VIRUS USING IMMUNOPATHOLOGICAL IMMUNOHISTOCHEMISTRY STREPTAVIDIN-BIOTIN)

Avian influenza is a viral disease in poultry caused by avian influenza virus (AIV) subtype H5N1 with varying clinical signs are often similar to the clinical signs of other viral infections, such as Newcastle disease virus (NDV). The mechanism of disease pathogenesis to express clinical signs tightly correlated to the determination of the target organ seen from AIV H5N1 antigens distribution in organs, such as respiratory tract, brain and gastrointestinal tract. Immunopathological immunohistochemistry streptavidin-biotin (IHC SB) is a method for sensitive and accurate in detecting antigens of AIV on the tissues. In the present study, it was determined whether in laying hens with clinical signs of torticollis and curled toe paralysis, and pathologic anatomic lesions in the form of petechial and foci necrotic hemorrhages tested with immunopathological IHC SB is positive AIV H5N1 infection. IHC SB study results showed that the AIV H5N1 antigen were found in tissues of the lung, brain, duodenum and proventriculus. Based on these results, we can conclude that the IHC SB is a method that is highly sensitive and accurate to detect H5N1 antigens and its distribution in the host.

Perturbation of B Cell Gene Expression Persists in HIV-Infected Children Despite Effective Antiretroviral Therapy and Predicts H1N1 Response.

Despite effective antiretroviral therapy (ART), HIV-infected individuals with apparently similar clinical and immunological characteristics can vary in responsiveness to vaccinations. However, molecular mechanisms responsible for such impairment, as well as biomarkers able to predict vaccine responsiveness in HIV-infected children, remain unknown. Following the hypothesis that a B cell qualitative impairment persists in HIV-infected children (HIV) despite effective ART and phenotypic B cell immune reconstitution, the aim of the current study was to investigate B cell gene expression of HIV compared to age-matched healthy controls (HCs) and to determine whether distinct gene expression patterns could predict the ability to respond to influenza vaccine. To do so, we analyzed prevaccination transcriptional levels of a 96-gene panel in equal numbers of sort-purified B cell subsets (SPBS) isolated from peripheral blood mononuclear cells using multiplexed RT-PCR. Immune responses to H1N1 antigen were determined by hemaglutination inhibition and memory B cell ELISpot assays following trivalent-inactivated influenza vaccination (TIV) for all study participants. Although there were no differences in terms of cell frequencies of SPBS between HIV and HC, the groups were distinguishable based upon gene expression analyses. Indeed, a 28-gene signature, characterized by higher expression of genes involved in the inflammatory response and immune activation was observed in activated memory B cells (CD27+CD21−) from HIV when compared to HC despite long-term viral control (>24 months). Further analysis, taking into account H1N1 responses after TIV in HIV participants, revealed that a 25-gene signature in resting memory (RM) B cells (CD27+CD21+) was able to distinguish vaccine responders from non-responders (NR). In fact, prevaccination RM B cells of responders showed a higher expression of gene sets involved in B cell adaptive immune responses (APRIL, BTK, BLIMP1) and BCR signaling (MTOR, FYN, CD86) when compared to NR. Overall, these data suggest that a perturbation at a transcriptional level in the B cell compartment persists despite stable virus control achieved through ART in HIV-infected children. Additionally, the present study demonstrates the potential utility of transcriptional evaluation of RM B cells before vaccination for identifying predictive correlates of vaccine responses in this population.

Conserved peptides enhance immune efficiency of inactive vaccines against emerging avian influenza viruses in chicken

Avian influenza viruses (AIVs) such as H5N1 and H7N9 are a great threat to poultry economics and public health. Vaccination can effectively inhibit the spread of AIV in poultry, which is also a viable strategy for controlling virus transmission from poultry to human. Adjuvants that are commonly used in current inactivated vaccines to provide stronger anti-AIV immune responses are often limited in their capacity to quantitatively induce both humoral and cellular immune responses. Herein, we assessed the levels of immune responses generated by a vaccine formulation comprising inactivated H5N1 antigen and synthetic peptides covering conserved CD4+, CD8+ T cell, and B cell epitopes. We found that the synthetic peptides enhanced the antibody responses against conserved influenza virus antigen M2e. Notably, the hemagglutination inhibition test results indicated that the peptides significantly augmented the antibody responses of inactivated H5N1 antigen even in the 1/10 or 1/5 dose group, in the identical antibody level as antigen alone used at the full dose. This indicates that the peptide can significantly reduce the use of inactivated virus, lowering the cost of the vaccine. Moreover, the peptides increased the transcript levels of interleukin-4 and interferon-γ cytokines in chicken peripheral blood mononuclear cells, which may facilitate both humoral and cellular immune responses. Our data suggest that this peptide combined with inactivated H5N1 antigen enhances both the humoral and cellular immune responses, which may benefit the prediction and design of synthetic peptide-based adjuvants for vaccines in chicken.

Heterologous post-infection immunity against Egyptian avian influenza virus (AIV) H9N2 modulates the course of subsequent infection by highly pathogenic AIV H5N1, but vaccination immunity does not.

In Egypt, zoonotic A/goose/Guangdong/1/96 (gs/GD-like) highly pathogenic avian influenza virus (HPAIV) H5N1 of clade 2.2.1.2 is entrenched in poultry populations and has co-circulated with low-pathogenic avian influenza virus H9N2 of the G1 lineage since 2010. Here, the impact of H9N2 infection or vaccination on the course of consecutive infection with a lethal Egyptian HPAIV H5N1 is studied. Three-week-old chickens were infected with H9N2 or vaccinated with inactivated H9N2 or H5N1 antigens and challenged three weeks later by an HPAIV H5N1. Interestingly, pre-infection of chickens with H9N2 decreased the oral excretion of H5N1 to levels that were comparable to those of H5N1-immunized chickens, but vaccination with inactivated H9N2 did not. H9N2 pre-infection modulated but did not conceal clinical disease by HPAIV H5N1. By contrast, homologous H5 vaccination abolished clinical syndromic surveillance, although vaccinated clinical healthy birds were capable of spreading the virus.

Evaluation of Haemagglutinin Content by RP-HPLC to Generate Pandemic Influenza Vaccine.

The potency of influenza vaccine is determined based on its hemagglutinin (HA) content. In general, single radial immunodiffusion (SRID) assay has been utilized as the standard method to measure HA content. However, preparation of reagents for SRID such as antigen and antibody takes approximately 2~3 months, which causes delays in the development of influenza vaccine. Therefore, quantification of HA content by other alternative methods is required. In this study, we measured HA contents of H1N1 antigen and H1N1 influenza vaccine by reverse phase-high performance liquid chromatography (RP-HPLC) methods. The presence of HA1 and HA2 was investigated by silver staining and Western blot assay. In addition, accuracy and repeatability of HA measurement by RP-HPLC were evaluated. Comparison of HA concentration by SRID and RP-HPLC revealed a precise correlation between the two methods. Our results suggest that RP-HPLC assay can replace SRID in the event of a pandemic flu outbreak for rapid vaccine development.

Evaluation of influenza vaccine-immunogenicity in cell-mediated immunity

The immunological effect of influenza vaccines cannot be evaluated accurately using an antibody titer. Therefore, we used a new method that measures cell-mediated immunity to investigate changes in the amount of interferon-gamma (IFN-γ) produced after vaccination in response to the vaccine antigen. The study was conducted during the 2014–2015 influenza season in 23 adults, using a vaccine that contained three types of antigen. The IFN-γ level increased by at least 1.5 times in 65% (15/23) of cases in response to the H1N1 antigen, in 57% (13/23) of cases in response to the H3N2 antigen, and in 57% (13/23) of cases in response to the B antigen. During the study period, 4 subjects developed type A influenza. Our data showed that the IFN-γ level did not increase by 1.5 times in these subjects. We propose that the efficacy of influenza vaccines may be evaluated by measuring changes in the level of IFN-γ produced in response to influenza vaccine.

Pharmacokinetics and biodistribution of squalene-containing emulsion adjuvant following intramuscular injection of H5N1 influenza vaccine in mice

Squalene is a component of oil-in-water emulsion adjuvants developed for potential use in some influenza vaccines. The biodistribution of the squalene-containing emulsion adjuvant (AddaVax™) alone and as part of complete H5N1 vaccine was quantified in mechanistically and toxicologically relevant target tissues up to 336 h (14 days) following injection into quadriceps muscle. At 1 h, about 55% of the intramuscularly injected dose of squalene was detected in the local quadriceps muscles and this decreased to 26% at 48 h. Twenty-four hours after the injection, approximately 5%, 1%, and 0.6% of the injected dose was detected in inguinal fat, draining lymph nodes, and sciatic nerve, respectively. The peak concentration for kidney, brain, spinal cord, bone marrow, and spleen was each less than 1% of the injected dose, and H5N1 antigen did not significantly alter the biodistribution of squalene to these tissues. The area-under-blood-concentration curve (AUC) and peak blood concentration (Cmax) of squalene were slightly higher (20–25%) in the presence of H5N1 antigen. A population pharmacokinetic model-based statistical analysis identified body weight and H5N1 antigen as covariates influencing the clearance of squalene. The results contribute to the body of knowledge informing benefit-risk analyses of squalene-containing emulsion vaccine adjuvants.

Recognition of influenza H3N2 variant virus by human neutralizing antibodies.

Since 2011, over 300 human cases of infection, especially in exposed children, with the influenza A H3N2 variant (H3N2v) virus that circulates in swine in the US have been reported. The structural and genetic basis for the lack of protection against H3N2v induced by vaccines containing seasonal H3N2 antigens is poorly understood. We isolated 17 human monoclonal antibodies (mAbs) that neutralized H3N2v virus from subjects experimentally immunized with an H3N2v candidate vaccine. Six mAbs exhibited very potent neutralizing activity (IC50 < 200 ng/ml) against the H3N2v virus but not against current human H3N2 circulating strains. Fine epitope mapping and structural characterization of antigen-antibody complexes revealed that H3N2v specificity was attributable to amino acid polymorphisms in the 150-loop and the 190-helix antigenic sites on the hemagglutinin protein. H3N2v-specific antibodies also neutralized human H3N2 influenza strains naturally circulating between 1995 and 2005. These results reveal a high level of antigenic relatedness between the swine H3N2v virus and previously circulating human strains, consistent with the fact that early human H3 seasonal strains entered the porcine population in the 1990s and reentered the human population, where they had not been circulating, as H3N2v about a decade later. The data also explain the increased susceptibility to H3N2v viruses in young children, who lack prior exposure to human seasonal strains from the 1990s.

Prokaryotic Expression of H1N1 Influenza A Virus Haemagglutinin Protein Globular Domain (HA1)

Background: Influenza viruses are a significant cause of morbidity and mortality. The influenza virus pandemics, 1918, 1977, and especially the most recent one, A/H1N1/2009, made evident the need for generating recombinant Influenza H1N1 antigens which are essential to develop both basic and applied research programs. Among influenza virus proteins, haemagglutinin (HA) is a major surface antigen of influenza virus, thus it is highly topical in influenza research and vaccine engineering programs. Alternatively, expression of fragments of the HA (HA1 and HA2) proteins in prokaryotic systems can potentially be the most efficacious strategy for manufacture of large quantities of influenza vaccine in a short period of time. Materials and Methods: The gene encoding the HA1 protein of the influenza A/Puerto Rico/8/34 was amplified by PCR, then cloned into pTZ57R/T cloning vector. The fidelity of the HA1 open reading frame was confirmed by bidirectional sequencing, then sub-cloned into pET28a prokaryotic expression plasmid, and proteins containing HA1 N-terminally fused to His-Tag were produced in Escherichia coli BL21 through IPTG inducing. The accuracy of the expression was confirmed by running time coursed fraction samples taken before and after the IPTG induction in SDS-PAGE, Western blot analysis were also used for confirmation of the recombinant protein. Results and Conclusion: The HA1 protein produced here could be considered and evaluated as a protective antigen, which its immunogenicity potential needs to be assessed in animal models along with proper control groups. Moreover, it could be subjected for polyclonal antibody preparation, which, in turn, may be used as an essential material in western blot analyses, as well as in other immunological applications, such as ELISA, immunocytochemistry, immunohistochemistry, and other immunological and serological studies. K e y w o r ds: Influenza A, H1N1, Hemagglutinin, Prokaryotic Expression

Randomized Double-Blind Study of the Safety and Immunogenicity of Standard-Dose Trivalent Inactivated Influenza Vaccine versus High-Dose Trivalent Inactivated Influenza Vaccine in Adult Hematopoietic Stem Cell Transplantation Patients

Hematopoietic stem cell transplantation (HCT) survivors are less likely than matched healthy controls to mount a strong immune response to trivalent inactivated influenza vaccine (TIV). High-dose (HD) or standard-dose (SD) TIV were given to adult HCT subjects 18 years or older at least 6 months after transplantation. Subjects were randomized 2:1 to receive either the HD (60 μg hemagglutinin [HA]/strain/dose) or the SD (15 μg HA/strain/dose) TIV. Injection-site and systemic reactions were documented after each vaccination and immune responses were measured before and after each vaccination. A total of 44 subjects were enrolled (25 in year 1 and 19 in year 2), with 15 in the SD group and 29 in the HD group. The median time to vaccination after transplantation was 7.9 months (range, 6 to 106 months), the median age was 50 years (range, 19.6 to 73 years), and 61% were male. No differences in demographic or lab data were noted between groups; however, the HD group had higher median baseline total IgG level (676 versus 469 mg/dL, P = .025). No differences in individual injection-site or systemic reactions were noted between groups; however, more events of any injection-site symptom combined were reported in the HD group. No serious adverse events were attributed to vaccination. After vaccination, the HD group had a higher percentage of individuals with titers ≥1:40 and a higher geometric mean titer (GMT) against the H3N2 strain compared with that of the SD group. HD and SD TIV were found to be safe and well tolerated in adult HCT recipients. However, the HD group had higher frequency of injection-site reactions but the majority of the reactions were mild and resolved. The HD group had a higher percentage of individuals with post-vaccination titer ≥ 1:40 and GMT for H3N2 antigen, indicating better immunogenicity. These data support the need for a phase II immunogenicity trial in HCT recipients.

Effects of swine flu (H1N1) vaccine on albino rats

The pandemic flu (2009) is a global outbreak of a new strain of H1N1 influenza virus that referred as “swine flu”. The 2009 flu pandemic vaccines (H1N1 vaccines) are the biological preparation of influenza vaccines that have been developed to protect and improve immunity against the pandemic H1N1/09 virus. Squalene has been added to vaccine as immunologic adjuvant to stimulate the immune system, increase the response to the vaccine and reduce amount of usage vaccine. Thimerosal has been added to some vaccines as a preservative because it is effective in preventing bacterial contamination, particularly in multidose containers. To investigate the possible immunological effects of swine flu vaccine, Albino rats were injected with H1N1 antigen contained thimerosal, adjuvant and H1N1 vaccine. Results obtained in the present study showed that squalene and thimerosal contributed to the regiment of adverse reactions and symptoms, which act as detergents instead of its helper role of vaccine that undergone toxicity effectors in the biological pathways including; immunopathological and histopathological effects; the damage was done by vaccine, particularly when potentiated by powerful “immunoenhancers” caused a strong immunostimulation that emphasized by results of this research as well as its biological hazards which are more pronounced when act together as whole vaccine than they act individually so the adverse events that resulted from treatment with squalene-adjuvanted vaccine are more potent than those of either antigen or squalene- treated groups

A multi-virus detectable microfluidic electrochemical immunosensor for simultaneous detection of H1N1, H5N1, and H7N9 virus using ZnO nanorods for sensitivity enhancement

This paper describes a multi-detectable and nano-flow immunosensor based on ZnO nanorods (NRs) grown on the inner surface of PDMS sensor region for sensing H1N1, H5N1, and H7N9 influenza viruses simultaneously using electrochemical method. Nanostructured ZnO NRs with a high isoelectric point (IEP ∼9.5) tend to interact electrostatically with proteins with lower IEP such as H1N1, H5N1, and H7N9 antibodies. ZnO NRs were hydrothermally grown on the upper inner surface of the nano-flow PDMS sensor region. The forementioned three influenza viruses were successfully detected from three separate sensing regions by measuring the oxidation current of 3,3′,5,5′-tetramethylbenzidine (TMB) by horseradish peroxidase (HRP) conjugated on capture antibody of those influenza viruses when proper potential was applied. The proposed immunosensors were evaluated using 1pg/ml, 10pg/ml, 100pg/ml, 1ng/ml, and 10ng/ml of H1N1, H5N1, and H7N9 antigens by amperometry. These immunosensors showed high selectivity toward H1N1, H5N1, and H7N9, which was successfully confirmed by distinguishing the target virus individually from a mixture of three virus antigens. A low limit of detection was demonstrated by detecting as low as 1pg/ml of each virus and it is believed that this was possible by enhancing the sensitivity with the ZnO NRs grown on the PDMS surface in the sensing region. The steady-state oxidation current output linearly increased with respect to the logarithm of the H1N1, H5N1, and H7N9 virus concentrations in the range of 1–10ng/ml.

Effect of Immunosuppression on T-Helper 2 and B-Cell Responses to Influenza Vaccination.

Influenza vaccine immunogenicity is suboptimal in immunocompromised patients. However, there are limited data on the interplay of T- and B- cell responses to vaccination with simultaneous immunosuppression. We collected peripheral blood mononuclear cells from transplant recipients before and 1 month after seasonal influenza vaccination. Before and after vaccination, H1N1-specific T- and B-cell activation were quantified with flow cytometry. We also developed a mathematical model using T- and B-cell markers and mycophenolate mofetil (MMF) dosage. In the 47 patients analyzed, seroconversion to H1N1 antigen was demonstrated in 34%. H1N1-specific interleukin 4 (IL-4)-producing CD4(+) T-cell frequencies increased significantly after vaccination in 53% of patients. Prevaccine expression of H1N1-induced HLA-DR and CD86 on B cells was high in patients who seroconverted. Seroconversion against H1N1 was strongly associated with HLA-DR expression on B cells, which was dependent on the increase between prevaccine and postvaccine H1N1-specific IL-4(+)CD4(+) T cells (R(2) = 0.35). High doses of MMF (≥ 2 g/d) led to lower seroconversion rates, smaller increase in H1N1-specific IL-4(+)CD4(+) T cells, and reduced HLA-DR expression on B cells. The mathematical model incorporating a MMF-inhibited positive feedback loop between H1N1-specific IL-4(+)CD4(+) T cells and HLA-DR expression on B cells captured seroconversion with high specificity. Seroconversion is associated with influenza-specific T-helper 2 and B-cell activation and seems to be modulated by MMF.

Antigenic differences between AS03 adjuvanted influenza A (H1N1) pandemic vaccines: implications for pandemrix-associated narcolepsy risk.

BackgroundNarcolepsy results from immune-mediated destruction of hypocretin secreting neurons in hypothalamus, however the triggers and disease mechanisms are poorly understood. Vaccine-attributable risk of narcolepsy reported so far with the AS03 adjuvanted H1N1 vaccination Pandemrix has been manifold compared to the AS03 adjuvanted Arepanrix, which contained differently produced H1N1 viral antigen preparation. Hence, antigenic differences and antibody response to these vaccines were investigated.Methods and FindingsIncreased circulating IgG-antibody levels to Pandemrix H1N1 antigen were found in 47 children with Pandemrix-associated narcolepsy when compared to 57 healthy children vaccinated with Pandemrix. H1N1 antigen of Arepanrix inhibited poorly these antibodies indicating antigenic difference between Arepanrix and Pandemrix. High-resolution gel electrophoresis quantitation and mass spectrometry identification analyses revealed higher amounts of structurally altered viral nucleoprotein (NP) in Pandemrix. Increased antibody levels to hemagglutinin (HA) and NP, particularly to detergent treated NP, was seen in narcolepsy. Higher levels of antibodies to NP were found in children with DQB1*06∶02 risk allele and in DQB1*06∶02 transgenic mice immunized with Pandemrix when compared to controls.ConclusionsThis work identified 1) higher amounts of structurally altered viral NP in Pandemrix than in Arepanrix, 2) detergent-induced antigenic changes of viral NP, that are recognized by antibodies from children with narcolepsy, and 3) increased antibody response to NP in association of DQB1*06∶02 risk allele of narcolepsy. These findings provide a link between Pandemrix and narcolepsy. Although detailed mechanisms of Pandemrix in narcolepsy remain elusive, our results move the focus from adjuvant(s) onto the H1N1 viral proteins.

Serological evidence for non-lethal exposures of Mongolian wild birds to highly pathogenic avian influenza H5N1 virus.

Surveillance for highly pathogenic avian influenza viruses (HPAIV) in wild birds is logistically demanding due to the very low rates of virus detection. Serological approaches may be more cost effective as they require smaller sample sizes to identify exposed populations. We hypothesized that antigenic differences between classical Eurasian H5 subtype viruses (which have low pathogenicity in chickens) and H5N1 viruses of the Goose/Guangdong/96 H5 lineage (which are HPAIV) may be used to differentiate populations where HPAIVs have been circulating, from those where they have not. To test this we performed hemagglutination inhibition assays to compare the reactivity of serum samples from wild birds in Mongolia (where HPAIV has been circulating, n = 1,832) and Europe (where HPAIV has been rare or absent, n = 497) to a panel of reference viruses including classical Eurasian H5 (of low pathogenicity), and five HPAIV H5N1 antigens of the Asian lineage A/Goose/Guangdong/1/96. Antibody titres were detected against at least one of the test antigens for 182 Mongolian serum samples (total seroprevalence of 0.10, n = 1,832, 95% adjusted Wald confidence limits of 0.09–0.11) and 25 of the European sera tested (total seroprevalence of 0.05, n = 497, 95% adjusted Wald confidence limits of 0.03–0.07). A bias in antibody titres to HPAIV antigens was found in the Mongolian sample set (22/182) that was absent in the European sera (0/25). Although the interpretation of serological data from wild birds is complicated by the possibility of exposure to multiple strains, and variability in the timing of exposure, these findings suggest that a proportion of the Mongolian population had survived exposure to HPAIV, and that serological assays may enhance the targeting of traditional HPAIV surveillance toward populations where isolation of HPAIV is more likely.

SAW sensor for Influenza A virus detection enabled with efficient surface functionalization

Surface acoustic wave (SAW) sensors using Love wave were designed and fabricated with SiO2-coated piezoelectric LiNbO3 wafers for Influenza A viral antigen detection, in which the targeted analyte was selectively captured at the surface through the antigen and antibody interaction enabled with appropriate surface functionalization. It was found that triethoxysilylbutylaldehyde (ALTES) and triethoxysilylundecanal ethylene glycol acetal (ACTES) are effective chemical reagents for immobilizing Influenza A hemagglutinin (HA) antibodies on the SiO2/LiNbO3 SAW surface. The detection limit of Influenza A HA H1N1 antigen at an analyte concentration as low as 1ngmL−1 was demonstrated without any compensation design at room temperature with our obtained SAW sensors with the HA antibodies immobilized at ALTES-functionalized surface.