Towards a novel influenza vaccine: engineering of hemagglutinin on a platform of adenovirus dodecahedron.

BACKGROUND: The production process for the current influenza vaccine takes about 6 months and its antigenic composition must be modified annually. In the attempt towards developing influenza vaccine production that would be faster, safer and cheaper we engineered an influenza vaccine in which multiple copies of hemagglutinin (HA) would be delivered by a vector, adenovirus dodecahedron (Ad Dd). Dd is a virus-like particle, formed by assembly of twelve copies of pentameric penton base (Pb) proteins responsible for virus penetration. In order to attach HA to the vector, an adaptor containing WW domains was used. The WW domain is a linear peptide fragment identified as a partner of proline-proline-x-tyrosine (PPxY) motif present at the N-terminal extremity of the Pb protein, which is a building block of Dd. That tandem of three WW domains in fusion with the protein of interest enables interaction with Dd and efficient translocation to the cytoplasm of cells in culture. RESULTS: Since HA is an oligomeric protein with complicated processing, we prepared six different constructs of HA (A/swan/Poland/467/2006(H5N1)) in fusion with the WW adaptor. Herein we report baculovirus expression and functional analysis of six HA-WW variants. The best behaving variant was successfully delivered into human cells in vitro. CONCLUSIONS: Engineering of a soluble complex of HA with Dd, a virus-like particle that serves as a vector, an adjuvant and as a multivalent presentation platform, is an important step toward a novel influenza vaccine.

Characterization of the antigenic and functional domains of a Mycoplasma synoviae variant vlhA gene.

The Mycoplasma synoviae haemagglutinin gene, vlhA, encodes two major immunodominant and surface-exposed membrane proteins, MSPB and MSPA. Both products are antigenically variable but only MSPA mediates binding to erythrocytes. Previously we have shown that M. synoviae type strain WVU 1853 could express a variant vlhA gene, referred to as MS2/28.1, with a considerably shorter and divergent MSPA region. A finding that prompted detailed characterization of its antigenic and functional properties. Here we mutagenized each of the six opal codons of the variant MS2/28.1 vlhA member into tryptophan, thus allowing its expression in Escherichia coli as well as its cleavage products, MSPB and MSPA. In addition, we expressed 5 contiguous regions of MS2/28.1 extending from the last part of MSPB to the C-terminus of MSPA. Colony immunostaining with region-specific antisera mapped antigenic variation to the N-terminal half of MS2/28.1 MSPA. No haemagglutinating activity was observed for MSPB, but consistent haemadsorption inhibition was mapped to the region extending from amino acid 325 to 344. Inhibition of both haemagglutination and haemadsorption activities were obtained with sera directed against the C-terminal region of MSPA, with the highest titers (1/320 and 1/160, respectively) being recorded for its last 60 residues. Importantly, antibodies to this region also yielded the highest metabolic inhibition titer of 1/1280. Overall, aside from mapping the functional domains of a M. synoviae highly divergent haemagglutinin gene, this study shows that the C-terminal half of its MSPA region induced the highest titers of antibodies inhibiting haemagglutination, haemadsorption, and metabolism.

Presence of a full-length highly polymorphic region (HPR) in the ISAV haemagglutinin-esterase does not affect the primary functions of receptor binding and esterase activity.

The putatively avirulent infectious salmon anaemia virus (ISAV) HPR0 variant has key phenotypic differences to isolates from disease outbreaks in Atlantic salmon farms. It appears to not cause disease, potentially displays a different tissue tropism and has yet to be isolated in conventional ISAV-permissive cell lines. This study focussed on identifying the biological basis for the observed differences by examining the properties of the haemagglutinin-esterase (HE) proteins derived from NWM10 (HPR0), Nevis 390/98 (HPR7 pathogenic strain) and mutant combinations of the two. Using a transfection-based system and haemadsorption analysis in salmon cell lines, this study demonstrated for the first time that an HPR0 HE was fully functional in terms of receptor-binding and -destroying activity and also suggested that the presence of a full-length HPR alone did not appear to affect these functions.

Cross-reactivity to olive tree pollen and orchard grass pollen in patients with pollinosis.

We studied 92 patients with allergic rhinitis in Syodoshima, Japan, during the pollen season between April and June to evaluate the cross-reactivity to different antigens, including pollen from the olive tree (Olea europaea) and orchard grass (Dactylis glomerata). Olive tree pollen was present in the atmosphere for 23 days, from May 19 to June 12, 1994. Specific IgE antibodies for olive tree pollen antigen were present in 21 (26.9%) of the 78 patients with allergic rhinitis. Nine (24.3%) of the 37 patients with allergic rhinitis exhibited positive skin reactivity to an extract of olive tree pollen. Fifteen (88.2%) of the 17 patients who had IgE reactivity in their sera to olive tree pollen antigen demonstrated allergic reactions to an extract of olive tree pollen. Specific IgE antibodies for orchard grass pollen antigen were present in 43 (48.3%) of the 89 patients with allergic rhinitis and 20 (95.2%) of the 21 patients who had IgE reactivity in their sera to olive tree pollen antigen. The inhibition test using the CAP System revealed that the reactivity of the IgE antibody specific for olive tree pollen antigen was inhibited dose-dependently by an extract of orchard grass pollen. These findings show that there is a reaction in some patients with grass (Gramineae) pollinosis that might be induced by olive tree pollen.

The antiviral spectrum of Listerine antiseptic.

The mechanism of activity and the antiviral spectrum of Listerine antiseptic have not been examined thoroughly. We therefore tested its effect on laboratory strains of herpes simplex type 1 and type 2 (enveloped DNA viruses), influenza A virus (enveloped RNA virus), rotavirus (nonenveloped RNA virus), and adenovirus type 5 (nonenveloped DNA virus). Each virus was mixed with an equal volume of Listerine for 30 seconds to 5 minutes, and the residual infectivity of the virus was assessed. An antiviral effect was defined as greater than 95% reduction of infectivity. Exposure to Listerine for 30 seconds had an antiviral effect against herpes simplex type-1 and type-2 (96.3% and 100% reduction in infectious virus, respectively) and influenza A (100% reduction). In contrast, rotavirus-induced plaque formation was reduced by 12.2% after 30 seconds of exposure to Listerine, whereas 5 minutes of exposure to Listerine resulted in a 21.5% increase in plaque formation. Exposure of adenovirus to Listerine had a minimal effect on the cytopathocity of the virus, with a 33.4% reduction in virus levels after 5 minutes. The antiviral activity of Listerine is thus not related to the viral genome but is probably directed to the viral envelope.

IgM antibody capture haemadherence test (MACHAT) for the detection of measles specific IgM.

An antibody capture haemadherence test (MACHAT) for detecting measles-specific IgM is described. The assay is based on the antibody capture principle with rhesus monkey erythrocytes as detector system in place of labelled antisera. MACHAT was compared with a commercial indirect enzyme immunoassay (EIA) for measles-specific IgM using 382 sera from patients notified as measles. There was good agreement between the two tests; 106 sera were found to contain measles IgM by both tests, 7 further sera were positive only in the commercial EIA and 9 only in MACHAT. One sera gave an equivocal result in MACHAT and another in the commercial EIA. Twelve of the 18 sera with discrepant results were also tested by MACRIA; in 7 MACRIA gave the same results as MACHAT, in 3 the MACRIA results agreed with the commercial test and in 2 the MACRIA results were equivocal. Specificity was established by a lack of MACHAT reactivity in sera collected from blood donors (n = 83) and from cases of recent rubella, dengue and parvovirus B19 infection (n = 51). The MACHAT is a simple, cheap test that can be read by eye and is suitable for measles surveillance programmes in the developing world.

[Observation on application of solid phase indirect hemadsorption assay for detection IgM antibodies to tuberculosis in non-serum specimens].

We used solid phase indirect hemadsorption assay (SPIHA) to detect specific IgM antibodies in specimens from cerebrospinal fluid, pleural fluid and ascitic fluid in patients with tuberculosis and compared with the enzyme linked immunosorbent assay (ELISA) and indirect hemagglutination assay (IHA). The result showed that detection of tuberculous specific IgM antibodies in cerebrospinal fluid by SPIHA is important for diagnosing the tuberculous meningitis.

Molecular analysis of virus-producing and non-producing clones derived from a defective SSPE virus Yamagata-1 strain.

Two virus clones were isolated from a defective SSPE virus, the Yamagata-1 strain, and designated as the YA and YF clones. The YA clone-infected cells produced neither cell-free virus nor cell-associated virus, whereas the YF clone-infected cells produced both cell-associated and cell-free virus. No difference of epitopes on structural proteins was observed between these two clones. Both clones had hemadsorption activity. Quantitation of structural protein by Western dot blots showed relatively a lower amount of M protein in the YA-infected cells than that in the YF-infected cells. The ratio, P plus M dicistronic/M monocistronic mRNA, in the YA-infected cells was about twice that in the YF-infected cells. Sequence analysis of cDNA corresponding to P plus M dicistronic mRNA revealed that the deduced M protein of the YF virus was smaller than that of the YA virus by five amino acids from the carboxy terminal. These results suggest that abundant production of P plus M dicistronic mRNA is responsible for the reduced amount of M protein in the non-productive YA clone.

Evaluation of a new bladder tumor marker.

Two monoclonal antibodies have been developed that bind to a shed bladder tumor-associated antigen. Preliminary data have demonstrated that antigen-positive tumors shed detectable amounts of antigen in the urine while antigen-negative tumors do not. This antigen may be differentially metabolized by normal and malignant urothelial cells. Further characterization of this antigen and its evaluation as a urinary marker for antigen positive bladder cancers is continuing.

Hemadsorption by colonies of Ureaplasma urealyticum.

Hemadsorption by colonies of Ureaplasma urealyticum and Mycoplasma pneumoniae differed quantitatively and qualitatively. Using standard methodology, few strains of U. urealyticum hemadsorbed; with a modified method, most strains hemadsorbed, indicating a second type of association. Scanning electron microscopy of tannin-osmium-stained preparations showed guinea pig erythrocytes embedded in ureaplasma colonies and craters left when erythrocytes were dislodged.

Genetic markers in human bone: II. Studies on ABO (and IGH) grouping.

A combination absorption-elution, two-dimensional absorption-inhibition procedure was used to determine the ABH antigen composition of a series of human bone specimens of known ABO type that had been aged up to nine months under dry and humid conditions at ambient temperature, 37 degrees C, and 56 degrees C; at ambient temperature in dry and wet soil; and buried in soil outdoors. Grouping data for the separate elution and inhibition testing, as well as for the combination procedure, are given. The combination method was found to be a highly reliable procedure for bone tissue ABH typing. Some data on microbial contaminants of human bone specimens aging in soil, and their effects on ABH typing results, are presented. No direct correlation between the properties of microbial contaminants and specific changes in the ABH antigenic composition of aging bone tissue specimens could be ascertained. Data on IGH antigen determination and on the quantitation of immunoglobulin G (IgG) in human bone tissue extracts indicated that immunoglobulin levels were typically too low to expect routinely successful Gm antigen testing results. However, these factors can sometimes be determined in fresh bone tissue extracts, particularly if the extracts are concentrated.

Solid-phase hemadsorption assay for IgM antibody to Treponema pallidum in serum and umbilical cord blood.

A solid-phase hemadsorption assay (SPHA) for Treponema pallidum was evaluated using serum and umbilical cord blood. A total of 133 sera reactive using immunofluorescent antibody assay were tested by SPHA for IgM antibody against T. pallidum. They were categorized into clinical stages of syphilis, including eight sera with primary, 32 sera with secondary, 68 sera with latent, 21 sera with unclassified, and four sera with congenital syphilis. Sera were reactive for 75%, 90.6%, and 33.8% using SPHA in the primary, secondary, and latent phases, respectively. The method gave 57.1% reactivity for the unclassified phase of syphilitic sera and 100% reactive for the congenital syphilitic sera. All 80 immunofluorescent nonreactive sera were nonreactive by SPHA. Of 19 reactive cord blood samples tested, seven samples were reactive by SPHA. However, all 20 nonreactive umbilical cord blood samples were nonreactive by SPHA. The SPHA assay for the determination of IgM antibody to T. pallidum in serum appears to be sensitive for primary, secondary, and congenital syphilis. The SPHA assay in the cord blood is sensitive and specific.

[ELISA diagnosis for Mycoplasma pneumoniae infection with human normal immunoglobulin products as control sera].

It was found that the human normal immunoglobulin G product, prepared from pooled plasma of at least 1,000 normal human donors, contained some specific IgG antibodies to Mycoplasma pneumoniae by Western blotting. M. pneumoniae-ELISA OD values increased linearly from 25 to 800 micrograms/ml in the concentration of the product, there were no differences in the M. pneumoniae-ELISA OD value of the products among lots. Then we used this product as control serum for measurement of IgG antibodies to M. pneumoniae in human sera by ELISA and compared the results with those obtained by complement fixation (CF) and indirect hemadosorption (IHA) tests. ELISA titers determined by this method had a mutual relation with CF and IHA titers.

Replication and plaque formation of swine hemagglutinating encephalomyelitis virus (67N) in swine cell line, SK-K culture.

Swine hemagglutinating encephalomyelitis virus (HEV), 67N strain, adapted to suckling mouse brain, grew readily in a porcine cell line, SK-K cell culture with cytopathic effect (CPE) consisting of syncytium formation and detachment of fused cells and round cells from glass surface. After further passages in SK-K cell monolayers with undiluted culture fluid, CPE developed earlier and became complete within 48 h postinoculation (p.i.). Viral specific antigen was detected in the cytoplasm of the infected SK-K cells by indirect immunofluorescence using rabbit antiserum against the mouse-passaged virus. The SK-K-passaged virus as well as the original mouse-passaged virus formed clear plaques on SK-K cell monolayers under simple overlay medium. The plaque assay system for HEV 67N was established by studying various factors influencing the plaque formation in the SK-K cell cultures. By this system more than 10(6) PFU/0.2 ml of the virus yield was detected in the fluid phase of the infected cultures at 48 h p.i. The SK-K-passaged virus caused fatal infection in 4-week-old mice by intracerebral inoculation, but was inhibited by rabbit antiserum against the mouse-passaged virus. Plaque formation and hemagglutinating activity of the virus were specifically inhibited by antisera against the mouse-passaged and SK-K-passaged 67N virus.

[Study of the multiplication in cell cultures of two strains of para-influenza virus type 3. III. Multiplication in BHK 21 cells]. / Etude de la multiplication sur cultures de cellules de deux souches de virus paragrippal du type 3. Note III. Multiplication sur cellules BHK 21.

Study was conducted on the multiplication of two strains (C243 and D) of parainfluenza virus type 3 in BHK 21 cells. Multiplication curve of the virus was established and immunohistochemical aspects of the process were investigated. Chronological study of successive steps of the formation and development of viral components allowed to see that the virus multiplication rate is low in this cell system. The parainfluenza antigen became detectable by immunofluorescence in the infected cell perinuclear region after a relatively long eclipse period (18 h) and synthetized virus has few RNA and induced no inclusion information in the cytoplasm or the nucleus. However, an important nuclear participation was noted: 72 h after inoculation, nuclear fluorescence was observed, as well as a nuclear DNA rising and frequent aberrant mitoses. Comparison between the two investigated strains led to the observation that the autochthonous D strain induced more frequent aberrant mitoses and more important cell destruction than the C243 one. Differences were also noted as regards the infecting and hemagglutinating titers.

[Assay of thermolabile enterotoxins of Escherichia coli and Vibrio cholerae by inhibition of erythroadsorption on the GM1 ganglioside]. / Dosage de l'entérotoxine thermolabile de Escherichia coli et de Vibro cholerae par inhibition de l'érythro-adsorption sur le ganglioside GM1.

A GM1 erythroassay (GERYDO) for heat-labile Escherichia coli enterotoxin (LT) and cholera toxin (CT) is described. This assay was developed for use in poorly equipped laboratories in developing countries. It uses GM1-coated polystyrene plates and is based on the competition between the toxin to be assayed and CT covalently bound to sheep red blood cells. GERYDO can detect 0.9 or 0.5 ng of CT per ml depending on the method of sensitization of erythrocytes. Good quantitative and qualitative correlation with the enzyme-linked immunosorbent assay and the Vero cell test was observed.

Serologic test combinations for safe detection of rubella infections.

Today a great number of serologic tests, including commercial kits, are available for rubella antibody testing. The performance and quality of various commercial tests (ELISA Enzygnost [Behring], Rubazyme [Abbott], Rubenz M [Northumbria/Biosigma], single radial hemolysis [e.g., Koch & Merk]) with a great number of selected sera were compared with those of conventional methods (hemagglutination-inhibition test, sucrose density gradient ultracentrifugation), and certain test combinations were selected. These proved useful for safe and rapid diagnosis of the stage of natural, vaccinal, and congenital rubella infection and for the determination of the immune status in pregnant women.