Safety, pharmacokinetics and pharmacodynamics of a novel anti-asthmatic drug, XC8, in healthy probands.

INTRODUCTION: XC8 (histamine glutarimide) is a novel agent which targets eosinophilic migration and mast cell degranulation and has shown anti-asthmatic effects in animal studies. OBJECTIVE: The objective of this placebo-controlled phase 1 study was to assess the safety of oral XC8 and to evaluate its pharmacokinetic and pharmacodynamic properties. METHODS: 32 healthy volunteers in three dose-escalation treatment groups (10 mg [n = 8], 50 mg [n = 8] and 200 mg [n = 16]) were randomized in a 3:1 ratio to XC8 or placebo respectively. The subjects received a single dose of the drug at Day 1 and then once-daily for 14 days (Days 8-21). RESULTS: No severe adverse events occurred. The number of adverse events was similar in the treatment arms compared to placebo and all subjects completed the study as planned. No clinically significant changes occurred in hematologic and biochemical blood tests in subjects receiving XC8. The pharmacokinetic data showed similar dose and time dependent mean plasma XC8 concentrations after single (Day 1) and multiple (Day 21) dosing. The mean maximum concentrations were 114-1993 ng/mL after single and 115-2089 ng/mL after multiple dosing. The mean times to maximum concentration were 0.68-1.01 and 0.67-0.98 h, respectively. There was no evidence for accumulation of XC8 after multiple dosing. CONCLUSION: XC8 was safe and well tolerated. A phase 2 study is being performed to further evaluate the potential role of XC8 in asthma treatment. TRIAL REGISTRATION: ClinicalTrials.gov, NCT02882217.

A Novel Oral Glutarimide Derivative XC8 Suppresses Sephadex-Induced Lung Inflammation in Rats and Ovalbumin-induced Acute and Chronic Asthma in Guinea Pigs.

BACKGROUND: Corticosteroids are the preferred option to treat asthma, however, they possess serious side effects and are inefficient in 10% of patients. Thus, new therapeutic approaches for asthma treatment are required. OBJECTIVE: To study the efficacy of a novel glutarimide derivative XC8 in a Sephadex-induced lung inflammation in rats as well as in acute and chronic ovalbumin-induced allergic asthma in guinea pigs. METHOD: Rats were treated with 0.18-18 mg/kg of XC8 intragastrically 4 times (24 h and 1 h prior to and 24 h and 45 h after endotracheal administration of Sephadex). The number of inflammatory cells in bronchoalveaolar lavages (BAL) was determined. Guinea pigs were treated with 0.045 -1.4 mg/kg (acute asthma) or with 1.4 and 7.0 mg/kg of XC8 (chronic asthma) intragastrically following the sensitization with ovalbumin and during aerosol challenge. Lung inflammation, numbers of eosinophils (BAL and lung tissue), goblet cells, degranulating mast cells and specific airway resistance (sRAW) were determined. The comparator steroid drug budesonide (0.5 mg/kg for rats and 0.16 mg/kg for guinea pigs) was administered by inhalation. RESULTS: XC8 reduced influx of eosinophils into BAL in Sephadex-induced lung inflammation model in rats (by 2.6-6.4 times). Treatment of acute asthma in guinea pigs significantly reduced eosinophils in guinea pigs in BAL (from 55% to 30%-39% of the total cell count) and goblet cells in lung tissue. In a model of acute and chronic asthma, XC8 reduced significantly the number of eosinophils and degranulating mast cells in the lung tissue. Treatment with XC8 but not with budesonide decreased the specific airway resistance in acute and chronic asthma model up to the level of naive animals. CONCLUSION: XC8 induced a profound anti-inflammatory effect by reducing eosinophils in BAL and eosinophils and degranulating mast cell numbers in the airway tissue. The anti-asthmatic effect of XC8 is comparable to that of budesonide. Moreover, in contrast to budesonide, XC8 was capable to reduce goblet cells and airway resistance.

Attenuated Recombinant Influenza A Virus Expressing HPV16 E6 and E7 as a Novel Therapeutic Vaccine Approach.

Persistent infection with high-risk human papillomavirus (HPV) types, most often HPV16 and HPV18, causes all cervical and most anal cancers, and a subset of vulvar, vaginal, penile and oropharyngeal carcinomas. Two prophylactic virus-like particle (VLPs)-based vaccines, are available that protect against vaccine type-associated persistent infection and associated disease, yet have no therapeutic effect on existing lesions or infections. We have generated recombinant live-attenuated influenza A viruses expressing the HPV16 oncogenes E6 and E7 as experimental immunotherapeutic vaccine candidates. The influenza A virus life cycle lacks DNA intermediates as important safety feature. Different serotypes were generated to ensure efficient prime and boost immunizations. The immune response to vaccination in C57BL/6 mice was characterized by peptide ELISA and IFN-γ ELISpot, demonstrating induction of cell-mediated immunity to HPV16 E6 and E7 oncoproteins. Prophylactic and therapeutic vaccine efficacy was analyzed in the murine HPV16-positive TC-1 tumor challenge model. Subcutaneous (s.c.) prime and boost vaccinations of mice with recombinant influenza A serotypes H1N1 and H3N2, followed by challenge with TC-1 cells resulted in complete protection or significantly reduced tumor growth as compared to control animals. In a therapeutic setting, s.c. vaccination of mice with established TC-1 tumors decelerated tumor growth and significantly prolonged survival. Importantly, intralesional vaccine administration induced complete tumor regression in 25% of animals, and significantly reduced tumor growth in 50% of mice. These results suggest recombinant E6E7 influenza viruses as a promising new approach for the development of a therapeutic vaccine against HPV-induced disease.

Influenza viral vectors expressing the Brucella OMP16 or L7/L12 proteins as vaccines against B. abortus infection.

BACKGROUND: We generated novel, effective candidate vaccine against Brucella abortus based on recombinant influenza viruses expressing the Brucella ribosomal protein L7/L12 or outer membrane protein (Omp)-16 from the NS1 open reading frame. The main purpose of this work was to evaluate the safety, immunogenicity and protectiveness of vaccine candidate in laboratory animals. METHODS AND RESULTS: Four recombinant influenza A viral constructs of the subtypes Н5N1 or H1N1 expressing the Brucella proteins L7/L12 or Omp16 were obtained by a reverse genetics method: Flu-NS1-124-L7/L12-H5N1, Flu-NS1-124-Omp16-H5N1, Flu-NS1-124-L7/L12-H1N1 and Flu-NS1-124-Omp16-H1N1. Despite of substantial modification of NS1 gene, all constructs replicated well and were retain their Brucella inserts over five passages in embryonated chicken eggs (CE). Administration of the mono- or bivalent vaccine formulation via prime-boost intranasal (i.n.), conjunctival (c.) or subcutaneous (s.c.) immunization was safe in mice; no deaths, body weight loss or pathomorphological changes were observed over 56 days. Moreover, guinea pigs vaccinated i.n. with vaccine vectors did not shed the vaccine viruses through their upper respiratory tract after the prime and booster vaccination. These findings confirmed the replication-deficient phenotype of viral vectors. The highest antibody response to Brucella antigen was obtained with constructs expressing L7/L12 (ELISA, GMT 242.5-735.0); whereas the highest T-cell immune response- with construct expressing Omp16 (ELISPOT, 337 ± 52-651 ± 45 spots/4×105cells), which was comparable (P > 0.05) to the response induced by the commercial vaccine B. abortus 19. Interestingly, c. immunization appeared to be optimal for eliciting T-cell immune response. In guinea pigs, the highest protective efficacy after challenge with B. abortus 544 was achieved with Omp16 expressing constructs in both monovalent or bivalent vaccine formulations; protective efficacy was comparable to those induced by a commercial live B. abortus 19 vaccine. CONCLUSION: Thus, influenza vectors expressing Brucella protective antigens can be developed as novel influenza vectored vaccine against B. abortus infection.

Design and characterization of a peptide mimotope of the HIV-1 gp120 bridging sheet.

The Bridging Sheet domain of HIV-1 gp120 is highly conserved among the HIV-1 strains and allows HIV-1 binding to host cells via the HIV-1 coreceptors. Further, the bridging sheet domain is a major target to neutralize HIV-1 infection. We rationally designed four linear peptide epitopes that mimic the three-dimensional structure of bridging sheet by using molecular modeling. Chemically synthesized peptides BS3 and BS4 showed a fair degree of antigenicity when tested in ELISA with IgG purified from HIV(+) broadly neutralizing sera while the production of synthetic peptides BS1 and BS2 failed due to their high degree of hydrophobicity. To overcome this limitation, we linked all four BS peptides to the COOH-terminus of GST protein to test both their antigenicity and immunogenicity. Only the BS1 peptide showed good antigenicity; however, no envelope specific antibodies were elicited upon mice immunization. Therefore we performed further analyses by linking BS1 peptide to the NH2-terminus of the E2 scaffold from the Geobacillus Stearothermophylus PDH complex. The E2-BS1 fusion peptide showed good antigenic results, however only one immunized rabbit elicited good antibody titers towards both the monomeric and oligomeric viral envelope glycoprotein (Env). In addition, moderate neutralizing antibodies response was elicited against two HIV-1 clade B and one clade C primary isolates. These preliminary data validate the peptide mimotope approach as a promising tool to obtain an effective HIV-1 vaccine.

Sublingual immunization with a live attenuated influenza a virus lacking the nonstructural protein 1 induces broad protective immunity in mice.

The nonstructural protein 1 (NS1) of influenza A virus (IAV) enables the virus to disarm the host cell type 1 IFN defense system. Mutation or deletion of the NS1 gene leads to attenuation of the virus and enhances host antiviral response making such live-attenuated influenza viruses attractive vaccine candidates. Sublingual (SL) immunization with live influenza virus has been found to be safe and effective for inducing protective immune responses in mucosal and systemic compartments. Here we demonstrate that SL immunization with NS1 deleted IAV (DeltaNS1 H1N1 or DeltaNS1 H5N1) induced protection against challenge with homologous as well as heterosubtypic influenza viruses. Protection was comparable with that induced by intranasal (IN) immunization and was associated with high levels of virus-specific antibodies (Abs). SL immunization with DeltaNS1 virus induced broad Ab responses in mucosal and systemic compartments and stimulated immune cells in mucosa-associated and systemic lymphoid organs. Thus, SL immunization with DeltaNS1 offers a novel potential vaccination strategy for the control of influenza outbreaks including pandemics.

Single HA2 mutation increases the infectivity and immunogenicity of a live attenuated H5N1 intranasal influenza vaccine candidate lacking NS1.

BACKGROUND: H5N1 influenza vaccines, including live intranasal, appear to be relatively less immunogenic compared to seasonal analogs. The main influenza virus surface glycoprotein hemagglutinin (HA) of highly pathogenic avian influenza viruses (HPAIV) was shown to be more susceptible to acidic pH treatment than that of human or low pathogenic avian influenza viruses. The acidification machinery of the human nasal passageway in response to different irritation factors starts to release protons acidifying the mucosal surface (down to pH of 5.2). We hypothesized that the sensitivity of H5 HA to the acidic environment might be the reason for the low infectivity and immunogenicity of intranasal H5N1 vaccines for mammals. METHODOLOGY/PRINCIPAL FINDINGS: We demonstrate that original human influenza viruses infect primary human nasal epithelial cells at acidic pH (down to 5.4), whereas H5N1 HPAIVs lose infectivity at pH ≤ 5.6. The HA of A/Vietnam/1203/04 was modified by introducing the single substitution HA2 58K→I, decreasing the pH of the HA conformational change. The H5N1 reassortants containing the indicated mutation displayed an increased resistance to acidic pH and high temperature treatment compared to those lacking modification. The mutation ensured a higher viral uptake as shown by immunohistochemistry in the respiratory tract of mice and 25 times lower mouse infectious dose50. Moreover, the reassortants keeping 58K→I mutation designed as a live attenuated vaccine candidate lacking an NS1 gene induced superior systemic and local antibody response after the intranasal immunization of mice. CONCLUSION/SIGNIFICANCE: Our finding suggests that an efficient intranasal vaccination with a live attenuated H5N1 virus may require a certain level of pH and temperature stability of HA in order to achieve an optimal virus uptake by the nasal epithelial cells and induce a sufficient immune response. The pH of the activation of the H5 HA protein may play a substantial role in the infectivity of HPAIVs for mammals.

Mutations affecting the stability of the haemagglutinin molecule impair the immunogenicity of live attenuated H3N2 intranasal influenza vaccine candidates lacking NS1.

The isolation and cultivation of human influenza viruses in embryonated hen eggs or cell lines often leads to amino acid substitutions in the haemagglutinin (HA) molecule. We found that the propagation of influenza A H3N2 viruses on Vero cells may trigger the appearance of HA destabilising mutations, affecting viral resistance to low pH or high temperature treatment. Two ΔNS1 reassortants, containing the HA sequences identical to the original human H3N2 influenza virus isolates were constructed. Passages of these viruses on Vero cells led to the appearance of single mutations in the HA(1) L194P or HA(2) G75R subunits that impaired virus stability. The original HA sequences and the stable phenotypes of the primary isolates were preserved if reassortants were passaged by infection at pH 5.6 and cultivation in medium at pH 6.5. Corresponding ΔNS1 reassortants were compared for their immunogenicity in ferrets upon intranasal immunisation. Vaccine candidates containing HA mutations demonstrated significantly lower immunogenicity compared to those without mutations. Thus, the retaining of the original HA sequences of human viruses during vaccine production might be crucial for the efficacy of live attenuated influenza vaccines.

Influenza virus-like particles as an antigen-carrier platform for the ESAT-6 epitope of Mycobacterium tuberculosis.

Various virus-like particles (VLPs) have been shown to induce cytotoxic T-cell immune response as well as B-cell immune response. This makes VLPs promising candidates for antigen-carrier platforms for various epitopes. Influenza A VLPs were produced displaying a 20 amino acid sequence from Mycobacterium tuberculosis early secretory antigenic target 6 protein (ESAT-6). As this sequence is known to comprise a potent T-cell epitope it was chosen as a model for a foreign epitope to be presented on an influenza VLP scaffold. The ESAT-6 epitope was engineered into the antigenic region B of the influenza hemagglutinin (HA) from strain A/New Caledonia/20/99. VLPs were expressed in insect cells and subjected to immunization studies in mice. High serum antibody titers detected against recombinant ESAT-6 demonstrated the feasibility of influenza A VLPs serving as an efficient platform for epitope presentation.

A novel type of influenza vaccine: safety and immunogenicity of replication-deficient influenza virus created by deletion of the interferon antagonist NS1.

BACKGROUND. The nonstructural protein NS1 of influenza virus counteracts the interferon-mediated immune response of the host. By deleting the open reading frame of NS1, we have generated a novel type of influenza vaccine. We studied the safety and immunogenicity of an influenza strain lacking the NS1 gene (DeltaNS1-H1N1) in healthy volunteers. METHODS. Healthy seronegative adult volunteers were randomized to receive either a single intranasal dose of the DeltaNS1-H1N1 A/New Caledonia vaccine at 1 of 5 dose levels (6.4, 6.7, 7.0, 7.4, and 7.7 log(10) median tissue culture infective dose) (n = 36 recipients) or placebo (n = 12 recipients). RESULTS. Intranasal vaccination with the replication-deficient DeltaNS1-H1N1 vaccine was well tolerated. Rhinitis-like symptoms and headache were the most common adverse events identified during the 28-day observation period. Adverse events were similarly distributed between the treatment and placebo groups. Vaccine-specific local and serum antibodies were induced in a dose-dependent manner. In the highest dose group, vaccine-specific antibodies were detected in 10 of 12 volunteers. Importantly, the vaccine also induced neutralizing antibodies against heterologous drift variants. CONCLUSIONS. We show that vaccination with an influenza virus strain lacking the viral interferon antagonist NS1 induces statistically significant levels of strain-specific and cross-neutralizing antibodies despite the highly attenuated replication-deficient phenotype. Further studies are warranted to determine whether these results translate into protection from influenza virus infection. TRIAL REGISTRATION. ClinicalTrials.gov identifier: NCT00724997 .

Swine-origin pandemic H1N1 influenza virus-like particles produced in insect cells induce hemagglutination inhibiting antibodies in BALB/c mice.

Recent outbreaks of influenza A highlight the importance of rapid and sufficient supply for pandemic and inter-pandemic vaccines. Classical manufacturing methods for influenza vaccines fail to satisfy this demand. Alternatively, cell culture-based production systems and virus-like particle (VLP)-based technologies have been established. We developed swine-origin pandemic H1N1 influenza VLPs consisting of hemagglutinin (A/California/04/2009) and matrix protein. Hemagglutinin and matrix protein were co-expressed in insect cells by the baculovirus expression system. VLPs were harvested from infection supernatants, purified and used for intraperitoneal immunization of BALB/c mice. Immunization induced high serum antibody titers against A/California/04/2009 as well as hemagglutination inhibiting antibodies. Additionally, we compared VLP production in two different insect cell lines, Sf9 and BTI-TN5B1-4 (High Five). Taken together VLPs represent a potential strategy for the fight against new pandemic influenza viruses.

Preclinical evaluation of a replication-deficient intranasal DeltaNS1 H5N1 influenza vaccine.

BACKGROUND: We developed a novel intranasal influenza vaccine approach that is based on the construction of replication-deficient vaccine viruses that lack the entire NS1 gene (DeltaNS1 virus). We previously showed that these viruses undergo abortive replication in the respiratory tract of animals. The local release of type I interferons and other cytokines and chemokines in the upper respiratory tract may have a "self-adjuvant effect", in turn increasing vaccine immunogenicity. As a result, DeltaNS1 viruses elicit strong B- and T- cell mediated immune responses. METHODOLOGY/PRINCIPAL FINDINGS: We applied this technology to the development of a pandemic H5N1 vaccine candidate. The vaccine virus was constructed by reverse genetics in Vero cells, as a 5:3 reassortant, encoding four proteins HA, NA, M1, and M2 of the A/Vietnam/1203/04 virus while the remaining genes were derived from IVR-116. The HA cleavage site was modified in a trypsin dependent manner, serving as the second attenuation factor in addition to the deleted NS1 gene. The vaccine candidate was able to grow in the Vero cells that were cultivated in a serum free medium to titers exceeding 8 log(10) TCID(50)/ml. The vaccine virus was replication deficient in interferon competent cells and did not lead to viral shedding in the vaccinated animals. The studies performed in three animal models confirmed the safety and immunogenicity of the vaccine. Intranasal immunization protected ferrets and mice from being infected with influenza H5 viruses of different clades. In a primate model (Macaca mulatta), one dose of vaccine delivered intranasally was sufficient for the induction of antibodies against homologous A/Vietnam/1203/04 and heterologous A/Indonesia/5/05 H5N1 strains. CONCLUSION/SIGNIFICANCE: Our findings show that intranasal immunization with the replication deficient H5N1 DeltaNS1 vaccine candidate is sufficient to induce a protective immune response against H5N1 viruses. This approach might be attractive as an alternative to conventional influenza vaccines. Clinical evaluation of DeltaNS1 pandemic and seasonal influenza vaccine candidates are currently in progress.

Azidothymidine inhibits melanoma cell growth in vitro and in vivo.

Azidothymidine (AZT), currently used for HIV treatment, was also shown to induce cell growth inhibition and apoptosis in different human tumors. The objective of this study was to investigate the ability of AZT to inhibit the growth of human melanoma cells in vitro and in vivo. In cytotoxicity assays, treatment of cells with varying concentrations of AZT-induced inhibition of cell growth and apoptosis in three human melanoma cell lines without affecting the growth of nontumorigenic cells. AZT-dependent inhibition of proliferation was accompanied by a significant S-phase arrest of the cell cycle. Coexposure of cells to AZT during cisplatin treatment showed a synergistic effect on cytotoxicity. Moreover, AZT monotreatment of melanoma in a severe combined immunodeficiency-mouse xenotransplantation model resulted in significant tumor reduction. These results demonstrate for the first time the antimelanoma activity of AZT, suggesting its clinical utilization either as a sole agent or in combination with other chemotherapeutic agents.

Expression, purification, and in vivo administration of a promising anti-idiotypic HIV-1 vaccine.

To date only a few neutralizing antibodies against HIV-1 exist. Since these neutralizing antibodies are only rarely found in sera of HIV-1 infected individuals an active vaccine is required. We recently developed murine anti-idiotypic antibody Ab2/3H6 against monoclonal antibody (mAb) 2F5, which is one of the most prominent neutralizing antibodies. Anti-idiotypic antibody Ab2/3H6 has been partially humanized and expressed as whole immunoglobulin G in Chinese hamster ovary cells in order to minimize the human anti-mouse antibody response. Here we describe the expression, purification, and immunohistochemical characterization of the chimeric Ab2/3H6 Fab fragment, which was finally used beside the whole IgG1 as an antigen for immunization of guinea pigs. The crude sera were screened for specific antibodies against the epitope of mAb 2F5 ELDKWA as well as for reactivity against HIV-1 gp41.

Live attenuated influenza virus expressing human interleukin-2 reveals increased immunogenic potential in young and aged hosts.

Despite the reported efficacy of commercially available influenza virus vaccines, a considerable proportion of the human population does not respond well to vaccination. In an attempt to improve the immunogenicity of live influenza vaccines, an attenuated, cold-adapted (ca) influenza A virus expressing human interleukin-2 (IL-2) from the NS gene was generated. Intranasal immunization of young adult and aged mice with the IL-2-expressing virus resulted in markedly enhanced mucosal and cellular immune responses compared to those of mice immunized with the nonrecombinant ca parent strain. Interestingly, the mucosal immunoglobulin A (IgA) and CD8(+) T-cell responses in the respiratory compartment could be restored in aged mice primed with the IL-2-expressing virus to magnitudes similar to those in young adult mice. The immunomodulating effect of locally expressed IL-2 also gave rise to a systemic CD8(+) T-cell and distant urogenital IgA response in young adult mice, but this effect was less distinct in aged mice. Importantly, only mice immunized with the recombinant IL-2 virus were completely protected from a pathogenic wild-type virus challenge and revealed a stronger onset of virus-specific CD8(+) T-cell recall response. Our findings emphasize the potential of reverse genetics to improve the efficacy of live influenza vaccines, thus rendering them more suitable for high-risk age groups.

GMP production of liposomes--a new industrial approach.

A new scalable liposome production system is presented, which is based on the ethanol injection technique. The system permits liposome manufacture regardless of production scale, as scale is determined only by free disposable vessel volumes. Once the parameters are defined, an easy scale up can be performed by just changing the process vessels. These vessels are fully sterilizeable and all raw materials are transferred into the sanitized and sterilized system via 0.2 microm filters to guarantee an aseptic production. Liposome size can be controlled by the local lipid concentration at the injection point depending on process parameters like injection pressure, lipid concentration and injection rate. These defined process parameters are furthermore responsible for highly reproducible results with respect to vesicle diameters and encapsulation rates Compared to other technologies like the film method which is normally followed by size reduction through high pressure homogenization, ultrasonication or extrusion, no mechanical forces are needed to generate homogeneous and narrow distributed liposomes. Another important advantage of this method is the suitability for the entrapment of many different drug substances such as large hydrophilic proteins by passive encapsulation, small amphiphilic drugs by a one step remote loading technique or membrane association of antigens for vaccination approaches.

Influenza virus NS vectors expressing the mycobacterium tuberculosis ESAT-6 protein induce CD4+ Th1 immune response and protect animals against tuberculosis challenge.

Infection with Mycobacterium tuberculosis remains a major cause of morbidity and mortality all over the world. Since the effectiveness of the only available tuberculosis vaccine, Mycobacterium bovis bacillus Calmette-Guérin (BCG), is suboptimal, there is a strong demand to develop new tuberculosis vaccines. As tuberculosis is an airborne disease, the intranasal route of vaccination might be preferable. Live influenza virus vaccines might be considered as potential vectors for mucosal immunization against various viral or bacterial pathogens, including M. tuberculosis. We generated several subtypes of attenuated recombinant influenza A viruses expressing the 6-kDa early secretory antigenic target protein (ESAT-6) of M. tuberculosis from the NS1 reading frame. We were able to demonstrate the potency of influenza virus NS vectors to induce an M. tuberculosis-specific Th1 immune response in mice. Moreover, intranasal immunization of mice and guinea pigs with such vectors induced protection against mycobacterial challenge, similar to that induced by BCG vaccination.

Virus-coated layer-by-layer colloids as a multiplex suspension array for the detection and quantification of virus-specific antibodies.

BACKGROUND: Suspension array technology has surpassed ELISA for automated, simultaneous detection and quantification of soluble biomarkers such as virus-specific antibodies. We describe assays in which antigens are attached to a lipid bilayer surrounding color-coded particles. METHODS: We used layer-by-layer technology to establish a multiplex suspension array with distinguishable microbeads coated with authentic viral surfaces to catch and quantify virus-specific antibodies in a flow cytometric analysis. Antigenic surfaces were generated by chimeric and wild-type baculoviruses plus 2 different influenza A virus subtypes fused to a lipid bilayer surrounding distinctly colored particles. Specificity of binding of chosen antibodies and sera was detected by immunofluorescence. Results of multiplex analysis were compared with results of ELISA. RESULTS: Titrations of virus-specific antibodies in the multiplex suspension array demonstrated specific binding to the viral surface proteins. The multiplex suspension array gave positive results for up to log 5-diluted primary antibodies with an approximately 5- to 10-fold reduced dynamic range compared with the respective ELISA. CONCLUSIONS: The bead-based multiplex suspension array is customizable and easy to establish. By displaying native influenza A virus surfaces and recombinant HIV-1 epitopes, the new assay provides a tool for the detection of major viral infections in humans.

Immunoglobulin G specifically binding plant N-glycans with high affinity could be generated in rabbits but not in mice.

Xylosylated and core alpha1,3-fucosylated N-glycans from plants are immunogenic, and they play a still obscure role in allergy and in the field of plant-made protein pharmaceuticals. We immunized mice to generate monoclonal antibodies (mAbs) binding plant N-glycans specifically via the epitope containing either the xylose or the core alpha1,3-fucose residue. Splenocytes expressing N-glycan-specific antibodies derived from C57BL/6 mice previously immunized with plant glycoproteins were preselected by cell sorting to generate hybridoma lines producing specific antibodies. However, we obtained only mAbs unable to distinguish fucosylated from xylosylated N-glycans and reactive even with the pentasaccharide core Man3GlcNAc2. In contrast, immunization of rabbits yielded polyclonal sera selectively reactive with either fucosylated or xylosylated N-glycans. Purification of these sera using glyco-modified neoglycoproteins coupled to a chromatography matrix provided polyclonal sera suitable for affinity determination. Surface plasmon resonance measurements using sensor chips with immobilized glyco-modified transferrins revealed dissociation constants of around 10(-9) M. This unexpectedly high affinity of IgG antibodies toward carbohydrate epitopes has repercussions on our conception of the binding strength and significance of antiglycan IgE antibodies in allergy.