Homologous Antibody Research Articles

Quantitative determination of protein molecular weight with an acoustic sensor; significance of specific versus non-specific binding.

Surface acoustic wave sensors with integrated microfluidics for multi-sample sensing have been implemented in this work towards the quantitative correlation of the acoustic signal with the molecular weight of surface bound proteins investigating different interaction/binding conditions. The results are presented for: (i) four different biotinylated molecules (30 ≤ Mw ≤ 150 kDa) specifically binding to neutravidin; (ii) the same four non-biotinylated molecules, as well as neutravidin, adsorbing onto gold; and (iii) four cardiac marker proteins (86 ≤ Mw ≤ 540 kDa) specifically binding to their homologous antibodies. Surface plasmon resonance was employed as an independent optical mass sensor. A linear relationship was found to exist between the phase change of the acoustic signal and the molecular weight of the proteins in both cases of specific binding. In contrast, non-specific binding of proteins directly onto gold exhibited no such linear relationship. In all three cases phase change was correlated with the bound mass per area. The underlying mechanism behind the different behavior between specific and non-specific binding is discussed by taking into account the geometrical restrictions imposed by the size of the specific biorecognition molecule and the corresponding bound protein. Our results emphasize the quantitative nature of the phase of the acoustic signal in determining the Mw (in the case of specific binding) with a resolution of 15% and the mass of the bound proteins (in all cases), as well as the significance of the biorecognition molecules in deriving the molecular weight from acoustic or optical detectors.

Influenza H1N1pdm-specific maternal antibodies offer limited protection against wild-type virus replication and influence influenza vaccination in ferrets.

ObjectiveThe objective was to study passively acquired influenza H1N1 pandemic (H1N1pdm) maternal antibody kinetics and its impact on subsequent influenza infection and vaccination in ferrets during an outbreak of the H1N1pdm.Design and main outcome measuresInfectivity of the H1N1pdm in the respiratory tract of ferrets was compared with the previous seasonal A/South Dakota/6/2007 (SD07, H1N1). Influenza-specific antibodies were quantitated and antibody-mediated protection against the homologous and heterologous H1N1 virus challenge infection was determined.ResultsH1N1pdm virus was approximately 10 times more infectious than SD07 in ferrets, replicated to higher viral titers in the upper respiratory tract and shed for a longer duration. Influenza-specific antibodies after natural infection persisted much longer in the circulation than passively acquired maternal antibodies. The protection conferred by the maternal antibodies was limited to the homologous virus strain and was ineffective against SD07 and H3N2 virus. Serum antibodies from maternal transmission or passive transfer interfered with homologous vaccine strain-mediated antibody responses in the ferret. A booster immunization was required to elicit a high level of antibody.ConclusionsThe findings support the rationale for a prime and boost immunization strategy in young children in whom maternal antibodies are present.

Advances in monoclonal antibody application in myocarditis

Monoclonal antibodies have become a part of daily preparation technologies in many laboratories. Attempts have been made to apply monoclonal antibodies to open a new train of thought for clinical treatments of autoimmune diseases, inflammatory diseases, cancer, and other immune-associated diseases. This paper is a prospective review to anticipate that monoclonal antibody application in the treatment of myocarditis, an inflammatory disease of the heart, could be a novel approach in the future. In order to better understand the current state of the art in monoclonal antibody techniques and advance applications in myocarditis, we, through a significant amount of literature research both domestic and abroad, developed a systematic elaboration of monoclonal antibodies, pathogenesis of myocarditis, and application of monoclonal antibodies in myocarditis. This paper presents review of the literature of some therapeutic aspects of monoclonal antibodies in myocarditis and dilated cardiomyopathy to demonstrate the advance of monoclonal antibody application in myocarditis and a strong anticipation that monoclonal antibody application may supply an effective therapeutic approach to relieve the severity of myocarditis in the future. Under conventional therapy, myocarditis is typically associated with congestive heart failure as a progressive outcome, indicating the need for alternative therapeutic strategies to improve long-term results. Reviewing some therapeutic aspects of monoclonal antibodies in myocarditis, we recently found that monoclonal antibodies with high purity and strong specificity can accurately act on target and achieve definite progress in the treatment of viral myocarditis in rat model and may meet the need above. However, several issues remain. The technology on how to make a higher homologous and weak immunogenic humanized or human source antibody and the treatment mechanism of monoclonal antibodies may provide solutions for these open issues. If we are to further stimulate progress in the area of clinical decision support, we must continue to develop and refine our understanding and use of monoclonal antibodies in myocarditis.

Development of a High Density Hemagglutinin Protein Microarray to Determine the Breadth of Influenza Antibody Responses

We have developed an influenza hemagglutinin protein microarray to assess humoral recognition of diverse influenza strains induced by vaccination and infection. Each array consists of controls and 127 hemagglutinin antigens from 60 viruses, spotted in replicates to generate a single array of 1296 spots. Six arrays are configured on a single slide, which in the following analysis was probed simultaneously with 2 isotype-specific fluorescent secondary antibodies yielding over 15,000 data points per slide. Here we report the use of this system to evaluate mouse, ferret, and human sera. The array allows simultaneous examination of the magnitude of antibody responses, the isotype of such antibodies, and the breadth of influenza strain recognition. We are advancing this technology as a platform for rapid, simple, high-throughput assessment of homologous and heterologous antibody responses to influenza disease and vaccination.

Ab initio structure prediction of the antibody hypervariable H3 loop

Antibodies have the capability of binding a wide range of antigens due to the diversity of the six loops constituting the complementarity determining region (CDR). Among the six loops, the H3 loop is the most diverse in structure, length, and sequence identity. Prediction of the three-dimensional structures of antibodies, especially the CDR loops, is an important step in the computational design and engineering of novel antibodies for improved affinity and specificity. Although it has been demonstrated that the conformation of the five non-H3 loops can be accurately predicted by comparing their sequences against databases of canonical loop conformations, no such connection has been established for H3 loops. In this work, we present the results for ab initio structure prediction of the H3 loop using conformational sampling and energy calculations with the program Prime on a dataset of 53 loops ranging in length from 4 to 22 residues. When the prediction is performed in the crystal environment and including symmetry mates, the median backbone root mean square deviation (RMSD) is 0.5 Å to the crystal structure, with 91% of cases having an RMSD of less than 2.0 Å. When the prediction is performed in a noncrystallographic environment, where the scaffold is constructed by swapping the H3 loops between homologous antibodies, 70% of cases have an RMSD below 2.0 Å. These results show promise for ab initio loop predictions applied to modeling of antibodies.

Characterization of cross protection of Swine-Origin Influenza Virus (S-OIV) H1N1 and reassortant H5N1 influenza vaccine in BALB/c mice given a single-dose vaccination

BackgroundInfluenza virus has antigen drift and antigen shift effect, vaccination with some influenza vaccine might not induce sufficient immunity for host to the threat of other influenza virus strains. S-OIV H1N1 and H5N1 influenza vaccines in single-dose immunization were evaluated in mice for cross protection to the challenge of A/California/7/2009 H1N1 or NIBRG-14 H5N1 virus.ResultsBoth H1N1 and H5N1 induced significant homologous IgG, HAI, and microneutralization antibody responses in the mice, while only vaccines plus adjuvant produced significant heterogeneous IgG and HAI antibody responses. Both alum and MPLA adjuvants significantly reduced the S-OIV H1N1 vaccine dose required to elicit protective HAI antibody titers from 0.05 μg to 0.001 μg. Vaccines alone did not protect mice from challenge with heterogeneous influenza virus, while H5N1 vaccine plus alum and MPLA adjuvants did. Mouse body weight loss was also less significant in the presence of adjuvant than in the vaccine without adjuvant. Furthermore, both H1N1 and H5N1 lung viral titers of immunized mice were significantly reduced post challenge with homologous viruses.ConclusionOnly in the presence of MPLA adjuvant could the H5N1 vaccine significantly reduce mouse lung viral titers post H1N1 virus challenge, and not vice versa. MPLA adjuvant induced cross protection with a single dose vaccination to the challenge of heterogeneous influenza virus in mice. Lung viral titer seemed to be a better indicator compared to IgG, neutralization antibody, and HAI titer to predict survival of mice infected with influenza virus.

The Mitochondrion-Like Organelle of Trimastix pyriformis Contains the Complete Glycine Cleavage System

All eukaryotic organisms contain mitochondria or organelles that evolved from the same endosymbiotic event like classical mitochondria. Organisms inhabiting low oxygen environments often contain mitochondrial derivates known as hydrogenosomes, mitosomes or neutrally as mitochondrion-like organelles. The detailed investigation has shown unexpected evolutionary plasticity in the biochemistry and protein composition of these organelles in various protists. We investigated the mitochondrion-like organelle in Trimastix pyriformis, a free-living member of one of the three lineages of anaerobic group Metamonada. Using 454 sequencing we have obtained 7 037 contigs from its transcriptome and on the basis of sequence homology and presence of N-terminal extensions we have selected contigs coding for proteins that putatively function in the organelle. Together with the results of a previous transcriptome survey, the list now consists of 23 proteins – mostly enzymes involved in amino acid metabolism, transporters and maturases of proteins and transporters of metabolites. We have no evidence of the production of ATP in the mitochondrion-like organelle of Trimastix but we have obtained experimental evidence for the presence of enzymes of the glycine cleavage system (GCS), which is part of amino acid metabolism. Using homologous antibody we have shown that H-protein of GCS localizes into vesicles in the cell of Trimastix. When overexpressed in yeast, H- and P-protein of GCS and cpn60 were transported into mitochondrion. In case of H-protein we have demonstrated that the first 16 amino acids are necessary for this transport. Glycine cleavage system is at the moment the only experimentally localized pathway in the mitochondrial derivate of Trimastix pyriformis.

Assessment of Antigen-Specific and Cross-Reactive Antibody Responses to an MF59-Adjuvanted A/H5N1 Prepandemic Influenza Vaccine in Adult and Elderly Subjects

Preparedness against an A/H5N1 influenza pandemic requires well-tolerated, effective vaccines which provide both vaccine strain-specific and heterologous, cross-clade protection. This study was conducted to assess the immunogenicity and safety profile of an MF59-adjuvanted, prepandemic influenza vaccine containing A/turkey/Turkey/01/2005 (H5N1) strain viral antigen. A total of 343 participants, 194 adults (18 to 60 years) and 149 elderly individuals (≥61 years), received two doses of the investigational vaccine given 3 weeks apart. Homologous and heterologous antibody responses were analyzed by hemagglutination inhibition (HI), single radial hemolysis (SRH), and microneutralization (MN) assays 3 weeks after administration of the first vaccine dose and 3 weeks and 6 months after the second dose. Immunogenicity was assessed according to European licensure criteria for pandemic influenza vaccines. After two vaccine doses, all three European licensure criteria were met for adult and elderly subjects against the homologous vaccine strain, A/turkey/Turkey/1/2005, when analyzed by HI and SRH assays. Cross-reactive antibody responses were observed by HI and SRH analyses against the heterologous H5N1 strains, A/Indonesia/5/2005 and A/Vietnam/1194/2004, in adult and elderly subjects. Solicited local and systemic reactions were mostly mild to moderate in severity and occurred less frequently in the elderly than in adult vaccinees. In both adult and elderly subjects, MF59-adjuvanted vaccine containing 7.5 μg of A/Turkey strain influenza virus antigen was highly immunogenic, well tolerated, and able to elicit cross-clade, heterologous antibody responses against A/Indonesia and A/Vietnam strains 6 weeks after the first vaccination.

β‐hexosaminidase from Xenopus laevis eggs and oocytes: From gene to immunochemical characterization

Glycosidases are present both in sperm and eggs in vertebrates and have been associated with different fertilization steps as gamete binding, egg coat penetration, and polyspermy prevention. In this manuscript, we have analyzed the activity of different glycosidases of Xenopus laevis eggs. The main activity corresponded to N-acetyl-β-D-glucosaminidase (Hex), which was reported to participate both in gamete binding and polyspermy prevention among phylogenetically distant animals. We have raised homologous antibodies against a recombinant N-terminal fragment of a X. laevis Hex, and characterized egg's Hex both by Western blot and immunohistochemical assays. Noteworthy, Hex was mainly localized to the cortex of animal hemisphere of full-grown oocytes and oviposited eggs, and remained unaltered after fertilization. Hex is constituted by different pair arrangements of two subunits (α and β), giving rise to three possible Hex isoforms: A (αβ), B (ββ), and S (αα). However, no information was available regarding molecular identity of Hex in amphibians. We present for the first time the primary sequences of two isoforms of X. laevis Hex. Interestingly, our results suggest that α- and β-like subunits that constitute Hex isoforms could be synthesized from a same gene in Xenopus, by alternative exon use. This finding denotes an evolutionary divergence with mammals, where α and β Hex subunits are synthesized from different genes on different chromosomes.

Detection of anti-Borrelia burgdorferi antibodies in buffaloes (Bubalus bubalis) in the state of Pará, Brazil

This study aimed to investigate the frequency of homologous antibodies of IgG class against Borrelia burgdorferi in buffaloes in the state of Pará, Brazil. Blood serum samples from 491 buffaloes were analyzed by means of the indirect ELISA test, using crude antigen produced from a cultivar of the North American strain G39/40 of B. burgdorferi. There were 412 positive samples (83.91%), and there was no statistically significant difference in the proportions of positive animals between the 81.69% (232/284) originating from Marajó Island and the 86.96% (180/207) from the continental area of the state of Pará. In all the municipalities studied, the frequency of positive findings of antibodies against B. burgdorferi among the animals ranged from 63.6% to 92.9%. The high numbers of seropositive animals can be explained by the frequent presence of the tick Rhipicephalus (Boophilus)microplus, and by the possible existence of spirochetes of the genus Borrelia infecting buffaloes in the region studied, although specific studies are needed to confirm this relationship. These factors suggest that a cross-reaction exists between the North American strain G39/40 of B. burgdorferi, which is used as an antigenic substrate, and the species of Borrelia spp. that possibly infects buffaloes in the state of Pará.

Homologous and heterologous antibody responses to a one-year booster dose of an MF59®: Adjuvanted A/H5N1 pre-pandemic influenza vaccine in pediatric subjects

Background: Primary immunization with two doses of MF59®-adjuvanted A/H5N1 influenza vaccine has been shown to be highly immunogenic and well tolerated in children and adolescents. Assessment of long-term antibody persistence after priming, and the effects of a one-year booster dose in children and adolescents was needed.Objectives This study assessed homologous and heterologous antibody responses to a one-year booster dose of MF59-adjuvanted A/H5N1 influenza vaccine in previously primed children.Subjects and methods: Twelve months after primary vaccination, toddlers, children and adolescents received a single booster dose of the same A/H5N1 vaccine. Paired sera were collected before and three weeks after booster vaccination. Homologous antibody responses against the A/Vietnam/1194/2004 vaccine strain were measured by hemagglutination inhibition (HI), single radial hemolysis (SRH) and microneutralization (MN) assays. Heterologous antibody responses against A/Indonesia/5/2005 and A/Anhui/1/2005 strains were assessed by MN assay only.Results: Twelve months after primary vaccination, persistent, homologous, seroprotective HI antibody titers (≥ 40) were observed in 46%, 26% and 30% of toddlers, children and adolescents; following booster vaccination, seroprotection rates increased to 99%, 98% and 91%, respectively. All toddlers and children, and 99% of adolescents achieved MN antibody titers ≥ 40. Cross-reactive A/H5N1 antibodies were detected in 94–98% of subjects after booster vaccination.Conclusions Two priming doses of MF59-adjuvanted A/H5N1 vaccine resulted in homologous and heterologous antibody responses which persisted for up to one year after immunization. A one-year booster dose was highly immunogenic, generating high homologous and cross-reactive A/H5N1 antibody titers.

Development of a serotyping enzyme-linked immunosorbent assay system based on recombinant truncated hantavirus nucleocapsid proteins for New World hantavirus infection

New World hantaviruses were divided into five groups based on the amino acid sequence variability of the internal variable region (around 230-302 amino acids) of hantavirus nucleocapsid protein (NP). Sin Nombre virus (SNV), Andes virus, Black Creek Canal virus (BCCV), Carrizal virus (CARV) and Cano Delgadito virus belong to groups 1, 2, 3, 4 and 5, respectively. Patient and rodent sera were serotyped successfully by an enzyme-linked immunosorbent assay (ELISA) with recombinant truncated NP lacking 99 N-terminal amino acids (trNP100) of SNV, CARV and BCCV. The trNP100 of BCCV showed lower reactivity to heterologous sera. In contrast, whole recombinant NP antigens detected both homologous and heterologous antibodies equally. The results together with results of a previous study suggest that trNP100 can distinguish infections among viruses in groups 1, 2, 3 and 4 of New World hantaviruses. The serotyping ELISA with trNP100 is useful for epidemiological surveillance in humans and rodents.

Performance evaluation of a new fourth-generation HIV combination antigen–antibody assay

Education and diagnostic tests capable of early detection represent our most effective means of preventing transmission of human immunodeficiency virus (HIV). The importance of early detection is underlined by studies demonstrating increased life expectancy following early initiation of antiviral treatment. The Elecsys® HIV combi PT assay is a fourth-generation antigen–antibody combination assay developed to allow earlier detection of seroconversion, and to have increased sensitivity and improved specificity. We aimed to determine how early the assay could detect infection compared with existing assays; whether all HIV variants could be detected; and the assay’s specificity using samples from blood donors, routine specimens, and patients with potential cross-reacting factors. Samples were identified as positive by the Elecsys® assay 4.9 days after a positive polymerase chain reaction result (as determined by the panel supplier), which was earlier than the 5.3–7.1 days observed with comparators. The analytical sensitivity of the Elecsys® HIV combi PT assay for the HIV-1 p24 antigen was 1.05 IU/mL, which compares favorably with the comparator assays. In addition, the Elecsys® assay identified all screened HIV subtypes and displayed greater sensitivity to HIV-2 homologous antigen and antibodies to HIV-1 E and O and HIV-2 than the other assays. Overall, the specificity of the Elecsys® assay was 99.88 % using samples from blood donors and 99.81 % when analyzing unselected samples. Potential cross-reacting factors did not interfere with assay performance. The Elecsys® HIV combi PT assay is a sensitive and specific assay that has been granted the CE mark according to Directive 2009/886/EC.

Immunogenicity and specificity of Norovirus Consensus GII.4 virus-like particles in monovalent and bivalent vaccine formulations (113.27)

Abstract Noroviruses, a major cause of acute gastroenteritis worldwide, present antigenic diversity that must be considered for the development of an effective vaccine. In this study, we explored approaches to increase the broad reactivity of virus-like particle (VLP) norovirus vaccine candidates. The immunogenicity of a GII.4 “Consensus” VLP that was engineered from sequences of three distinct naturally-occurring GII.4 strains was examined for its ability to induce cross-reactive immune responses against different noroviruses. Rabbits immunized with GII.4 Consensus VLPs developed high serum antibody titers against VLPs derived from a number of distinct wild-type GII.4 viruses Because the sera exhibited low cross-reactivity with antigenically-distinct GI norovirus strains, we investigated the serum antibody response to a bivalent vaccine formulation containing GI.1 and GII.4 Consensus VLPs that was administered to animals under varying conditions. In these studies, the highest homologous and heterologous antibody titers to the bivalent vaccine were elicited following immunization of animals by the IM route using Alhydrogel as adjuvant. Our data indicate that the use of both genetically-engineered norovirus VLPs that incorporate relevant epitopes from multiple strains and multivalent vaccine formulations increase the breadth of the immune response to diverse variants within a genotype and, thus, prove helpful in the rational design of VLP-based vaccines against human noroviruses.

Serotype specificity of recombinant fusion protein containing domain III and capsid protein of dengue virus 2

Recombinant fusion protein containing domain III of the dengue envelope protein fused to capsid protein from dengue 2 virus was immunogenic and conferred protection in mice against lethal challenge in previously report. Here, the antigenic specificity of this recombinant protein using anti-dengue antibodies from mice and humans and the cross-reactive humoral and cellular response induced in immunized mice were evaluated. The homologous anti-dengue antibodies showed a higher reactivity to the recombinant protein compared to the wide cross-reactivity observed for viral antigen as determined by ELISA. The IgG anti-dengue and functional antibodies, induced by the recombinant proteins in mice, were highly serotype specific by ELISA, hemaglutination inhibition and plaque reduction neutralizing tests. Accordingly, the cellular immune response determined by the IFNγ and TNFα secretion, was serotype specific. The specificity of serotype associated to this recombinant protein in addition to its high antigenicity, immunogenicity and protecting capacity suggest its advantage as a possible functional and safe vaccine candidate against dengue in a future tetravalent formulation.

Immunogenicity and specificity of norovirus Consensus GII.4 virus-like particles in monovalent and bivalent vaccine formulations

Noroviruses, a major cause of acute gastroenteritis worldwide, present antigenic diversity that must be considered for the development of an effective vaccine. In this study, we explored approaches to increase the broad reactivity of virus-like particle (VLP) norovirus vaccine candidates. The immunogenicity of a GII.4 “Consensus” VLP that was engineered from sequences of three genetically distinct naturally occurring GII.4 strains was examined for its ability to induce cross-reactive immune responses against different clusters of GII.4 noroviruses. Rabbits immunized with GII.4 Consensus VLPs developed high serum antibody titers against VLPs derived from a number of distinct wild-type GII.4 viruses, including some that had been circulating over 30 years ago. Because the sera exhibited low cross-reactivity with antigenically distinct GI norovirus strains, we investigated the serum antibody response to a bivalent vaccine formulation containing GI.1 (Norwalk virus) and GII.4 Consensus VLPs that was administered to animals under varying conditions. In these studies, the highest homologous and heterologous antibody titers to the bivalent vaccine were elicited following immunization of animals by the intramuscular route using Alhydrogel (Al(OH)3) as adjuvant. Our data indicate that the use of both genetically engineered norovirus VLPs that incorporate relevant epitopes from multiple strains and multivalent vaccine formulations increase the breadth of the immune response to diverse variants within a genotype and, thus, prove helpful in the rational design of VLP-based vaccines against human noroviruses.

Elicitation of Anti-1918 Influenza Virus Immunity Early in Life Prevents Morbidity and Lower Levels of Lung Infection by 2009 Pandemic H1N1 Influenza Virus in Aged Mice

The Spanish influenza virus pandemic of 1918 was responsible for 40 million to 50 million deaths and is antigenically similar to the swine lineage 2009 pandemic influenza virus. Emergence of the 2009 pandemic from swine into humans has raised the possibility that low levels of cross-protective immunity to past shared epitopes could confer protection. In this study, influenza viruslike particles (VLPs) were engineered to express the hemagglutinin (HA) and genes from the 1918 influenza virus to evaluate the duration of cross-protection to the H1N1 pandemic strain by vaccinating young mice (8 to 12 weeks) and then allowing the animals to age to 20 months. This immunity was long lasting, with homologous receptor-blocking antibodies detected throughout the lifespan of vaccinated mice. Furthermore, the 1918 VLPs fully protected aged mice from 2009 pandemic H1N1 virus challenge 16 months after vaccination. Histopathological assessment showed that aged vaccinated mice had significant protection from alveolar infection but less protection of the bronchial tissue than adult vaccinated mice. Additionally, passive transfer of immune serum from aged vaccinated mice resulted in protection from death but not morbidity. This is the first report describing the lifelong duration of cross-reactive immune responses elicited by a 1918 VLP vaccine in a murine model. Importantly, these lifelong immune responses did not result in decreased total viral replication but did prevent infection of the lower respiratory tract. These findings show that immunity acquired early in life can restrict the anatomical location of influenza viral replication, rather than preventing infection, in the aged.

Genome organization, serology and phylogeny of Grapevine leafroll-associated viruses 4 and 6: Taxonomic implications

Complete nucleotide sequences of the type isolate of Grapevine leafroll-associated virus 4 (GLRaV-4) and of an isolate of GLRaV-6 from cv ‘Estellat’ (GLRaV-6Est) were generated and compared mutually and with related viruses. The genome organization of both viruses resembled that of members of Subgroup I in the genus Ampelovirus (fam. Closteroviridae). The availability of these sequences, along with previously existing data on related GLRaVs, allowed critical review of the taxonomy and nomenclature of these viruses. In phylogenetic analyses, GLRaV-4 and -6Est consistently grouped with GLRaV-5, -9, and -Pr forming a poorly resolved sub-cluster (“GLRaV-4 group”) within the genus Ampelovirus. In-depth study showed that genetic distances between these viruses do not exceed the intra-species diversity observed in other closteroviruses. In Western blots, partially purified preparations of GLRaVs -4, -5, -6 and -9 reacted only with homologous monoclonal antibodies, but were all recognized by polyclonal antisera to GLRaV-5 and GLRaV-9. Serological relatedness among these viruses was further confirmed in DAS-ELISA. In immuno-electron microscopy, GLRaV-6 particles appeared uniformly decorated with homologous monoclonal antibodies, whereas GLRaV-2, used as a control, showed “bipolar” morphology of the virion. Results of this study challenge taxonomy and nomenclature of several GLRaVs suggesting that they are divergent isolates of Grapevine leafroll-associated virus 4 and not, as has been assumed, distinct species (definitive and/or putative) in the genus Ampelovirus.

Evaluation of a virosomal H5N1 vaccine formulated with Matrix M™ adjuvant in a phase I clinical trial

The avian influenza H5 virus epizootic continues to cause zoonosis with human fatalities, highlighting the continued need for pandemic preparedness against this subtype. This study evaluated the tolerability and immunogenicity of a Matrix M™ adjuvanted virosomal H5N1 vaccine in a phase I clinical trial. Sixty healthy adults were vaccinated intramuscularly with two doses of influenza H5N1 (NIBRG-14) virosomal vaccine alone (30 μg haemagglutinin (HA)) or 1.5, 7.5 or 30 μg HA formulated with 50 μg Matrix M™ adjuvant. The antibody response was analysed by haemagglutination inhibition (HI), microneutralisation (MN) and single radial haemolysis (SRH) assays. The vaccine was well tolerated in all groups but injection site pain was more frequently observed in the Matrix M™ adjuvanted groups. The vaccine elicited homologous and heterologous H5N1-specific antibody responses and the Matrix M™ adjuvanted formulations met all the EU regulatory criteria. In conclusion, Matrix M™ adjuvant was well tolerated and augmented the antibody response allowing considerable dose sparing down to 1.5 μg HA.

Development of a portable, high throughput biosensor system for rapid plant virus detection

Biosensors based on living cells are characterized by high sensitivity, selectivity and rapid response times. A novel portable cell biosensor system for the detection of plant viruses, based on immobilized ‘Vero’ cells carrying on their membrane virus specific antibodies was developed and was designated as High Throughput Bioelectric Recognition Assay (BERA-HTP). BERA-HTP was tested for the detection of purified Potato virus Y (PVY), Cucumber mosaic virus (CMV) and Tobacco rattle virus (TRV) and of CMV and TRV in single, as well as in mixed infections in two different plant host species. The sensor was based on live, mammalian cells, the membrane of which has been artificially saturated with antibodies specific to different plant viruses. The attachment of PVY, CMV or TRV viral particles to the homologous electroinserted antibodies caused a virus-specific change of the cell membrane electric potential that was not observed with virus-free samples or with heterologous viruses. Fluorescence microscopy observations showed that attachment of virus particles to the cell membrane bearing the homologous antibody, was associated with a decrease of [Ca2+]cyt. The perspective for the development of BERA-HTP as a portable, reliable and rapid (duration of assay for 96 samples: ∼70min) detection method of plant viruses in the field is discussed.