Development of monoclonal antibodies to highly pathogenic avian influenza H5N1 virus and their application to diagnostics, prophylaxis, and therapy

A panel of 17 monoclonal antibodies (MAbs) against highly pathogenic avian influenza virus (HPAIV) A/Duck/Novosibirsk/56/05 A/H5N1 (subclade 2.2) isolated in Russian Federation was developed. Immunoblot analysis showed that 12 MAbs were specific for the hemagglutinin (HA) and 5 MAbs for nucleoprotein (NP). All anti-HA MAbs were reactive in ELISA and immunofluorescence (IF) test and 10 of them were reactive in hemagglutination-inhibition (HI) and neutralization tests. Quantitative competitive ELISA revealed that anti-HA MAbs recognized at least 4 non-overlapping antigenic determinants and anti-NP MAbs recognized at least 3 non-overlapping antigenic determinants. Four sandwich ELISA procedures were developed using the obtained MAbs. These procedures are useful for 1) identification of avian, human, and swine influenza A viruses, 2) differentiation of avian influenza virus (AIV) from human and swine influenza viruses, 3) differentiation of AIV H5 from other AIV subtypes, and 4) differentiation between 2.2 and 2.3.2 subclades of H5N1 influenza viruses. Prophylactic and therapeutic efficacy of anti-HA MAbs with high neutralization activity was tested in BALB/c mice. A complete protection was achieved by single injection of MAbs (20 mg/kg) 24 hrs before challenge with 10 LD50 of HPAIV H5N1. Therapeutic efficacy was 90% that was similar to those of Rimantadine and Tamiflu.     

The Role of the Serotoninergic System in the Regulation of Thyroid Function in Old Rats

The effect of serotoninotropic substances with positive and negative effects on serum concentrations of thyrotropic hormone, total triiodothyronine, and total thyroxin was studied in old male rats. Chronic treatment with L-tryptophan and citalopram reduced serum level of total thyroxin in old intact animals and serum concentration of thyrotropic hormone in old thyrectomied rats. DL-p-chlorophenylalanine increased the serum concentrations of total thyroxin in old intact rats and in thyroidectomied animals treated with triiodothyronine. Chronic methysergide therapy was associated with reduction of serum concentration of thyrotropic hormone in old intact and old thyroidectomied rats and of total triiodothyronine in old intact animals.     

Detection of hepatitis C markers--nucleocapsid protein, RNA and virus-specific antibodies in blood donor plasma

Comparative study of hepatitis C virus (HCV) markers (core protein, RNA, and virus-specific antibodies) was carried out in plasma samples from 80 donors. A method based on sandwich ELISA with monoclonal antibodies to recombinant protein was developed for measuring core protein. Nucleocapsid protein was detected after various treatments of precipitates obtained after concentration of virus-containing material from plasma samples. These treatments allowed differentiation of core protein in virions, free nucleocapsids, and immune complexes circulating in peripheral blood. The minimal detectable concentration was 5 pg/ml, maximal 850 pg/ml. The detection of core protein virtually coincided with the detection of HCV RNA: 94.4% RNA-positive samples contained the virus protein. Other parameters (activities of antibodies to HCV in ELISA and level of SGPT) did not allow differentiation of plasma samples by the presence of actively replicating virus. Assay of nucleocapsid protein in the plasma of subjects infected with HCV in various populations of virus particles is important from practical (for blood service) and theoretical viewpoints (for studies of virus pathogenesis mechanisms).     

SOFFCO, Montpellier 31 mai–2 juin 2012

Index

SOFFCO, Montpellier 31 mai–2 juin 2012 Index des auteurs     

Escape mutants of pandemic influenza A/H1N1 2009 virus: variations in antigenic specificity and receptor affinity of the hemagglutinin

The emergence of porcine circovirus type 2 (PCV2) associated diseases and particularly postweaning multisystemic wasting syndrome (PMWS) was a shock for the swine industry and formulated a considerable challenge for researchers in the area of viral immunology in swine. The unique features of PMWS of which emaciation and lymphoid depletion were the most evident indicated a deep involvement of the immune system of the pig in the pathogenesis of this condition and indicated that PCV2 was a singular pathogen. Also, the multifactorial nature of the disease complicated the understanding of PMWS pathogenesis. Nowadays, it is known that PCV2 deeply affects the functionality of the immune system of the pig but also the industry has been able to produce efficacious vaccines. In the present paper some of the most relevant immunological features of PMWS and of PCV2 infection in general will be reviewed.     

Cation-osmotic Haemolysis in Patients with Retinal Vein Occlusion

The purpose of this work was to use a new technique to assess erythrocyte deformability in patients with retinal vein occlusion. Erythrocyte microrheology changes were measured by cation-osmotic haemolysis (COH) in healthy donors and in patients with both central (n= 6) and branch (n= 16) retinal vein occlusion up to 12 months after the occlusion. The patient group consisted of five patients with ischaemic and 17 with oedematous vein occlusion. The control group consisted of the same number of age-, sex- and risk factor (hypertension)-matched donors. In patients with retinal vein occlusion, COH was significantly decreased compared to the control group. The decrease was most marked at low and high concentrations of incubating media where the differences reached high statistical significance (p<0.01–0.001). In our previous experiments we showed that COH and erythrocyte deformability (ED) are closely related and that COH reflects basic information about erythrocyte deformability. Thus, decreased COH in patients with retinal vein occlusion points to reduced ED in comparison with the control group. We, therefore, assume that impairment in ED together with other microrheological abnormalities contributes to the pathophysiology of changes in patients with retinal vein occlusion.     

Sequence Similarities and Evolutionary Relationships of Influenza Virus A Hemagglutinins

This study brings the analysis of amino acid sequences of hemagglutinin (HA) from the influenza virus A that can infect a wide variety of birds and mammals. 191 sequences belonging to all known 15 HA subtypes were compared. The emphasis was given on functional sites (receptor-binding cavity with its right and left edges) and degree of their conservation in each subtype. Three evolutionary trees of 15 avian HA representatives were constructed: one tree based on the alignment of the entire HA sequences and two trees based on the alignment of HA1 and HA2 chains, respectively. The results have shown that, despite low degree of sequence similarity among the 191 sequences of HA1 subunit, the active site is well conserved, and that there are only marginal differences in the clustering of the individual HA subtypes between the two subunit trees. In this respect, the subtype H9 seems to be the most fluctuating example. The proposals of the probable avian HAs that could be the closest relatives to human (mammalian) HAs were also provided for several HA subtypes.     

Effect of a Deficiency of Ceruloplasmin Copper in Blood Plasma on Copper Metabolism in the Brain

Copper deficiency in adult rats was induced by addition of silver chloride to the feed. The concentrations of silver, copper, iron, and zinc and relative activity of genes for copper transporting proteins and copper enzymes were measured in the cortex, cerebellum, hippocampus, amygdala, pituitary gland, and hypothalamus. Silver was accumulated only in the hypothalamic—pituitary system. These changes were accompanied by a decrease in the concentration of copper and increase in the contents of iron and zinc. Activity of genes for copper transport enzymes (high-affinity copper transporter; and two copper transport ATPases, ATP7A and ATP7B) and copper enzymes that were formed in the intracellular secretory pathway did not decrease in the brain of rats with copper deficiency. Relative activity of genes for intracellular copper enzymes (Cu²⁺/Zn²⁺ superoxide dismutase and subunit IV of cytochrome c oxidase), concentration of immunoreactive polypeptides of superoxide dismutase, and enzymatic activity of superoxide dismutase remained unchanged under these conditions.