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.

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.

Cell surface antigens of human renal cancer defined by autologous typing.

Sera from 28 patients with renal cancer were tested for reactivity with surface antigens of cultured autologous renal cancer cells. Four serological assays were used to survey sera for autologous antibody. Immune adherence, protein A, and C3-mixed hemadsorption assays detected reactivity in a high percentage of patients (80-100%), whereas mixed hemadsorption assays were negative with sera from all but one patient. Reactive sera from six patients were analyzed by absorption tests with autologous, allogeneic, and restricted to autologous renal cancer cells; class 2 antigens, present on certain allogeneic renal and nonrenal cancer cells; and class 3 antigens, found on a wide variety of normal and malignant cell types. The sera of one patient detected class 1, 2, and 3 antigens, the sera of three patients detected class 2 antigens, and the sera of two patients detected class 3 antigens. This analysis of renal cancer, with the recognition of three classes of surface antigens recognized by autologous sera, resembles the results of autologous typing of three other human malignancies: malignant melanoma, acute leukemia, and astrocytoma. Evidence provided by autologous typing of these cancers indicates that class 1 and class 2 antigens are tumor-restricted and that under certain circumstances these antigens are immunogenic for the autologous host.

Production of hemadsorption-negative areas by serums containing Australia antigen.

Exposure of human Wi-38 cells to human serums containing Australia antigen, and presumably serum hepatitis virus, renders the cells refractory to infection by Newcastle disease virus as detected by the hemadsorption-negative plaque test for intrinsic interference. Induction of the Newcastle disease virus refractory state could be passed in cell culture with up to a 1 : 100,000 dilution of material obtained from cells "infected" with serums containing Australia antigen after filtration (0.45-microm pores) and heating to 60 degrees C for 1 hour. Human antiserums to the Australia antigen prevented induction of the Newcastle disease virus refractory state.

Cell rigidity: Effect on concanavalin A-mediated agglutinability of fibroblasts after fixation.

A quantitative hemadsorption assay distinguishes the effects of membrane fixation on concanavalin A-mediated agglutinability of fixed cells with unfixed cells. We observed undiminished adherence of unfixed erythrocytes to glutaraldehyde-fixed normal and virus-transformed hamster fibroblasts coated with concanavalin A. Fixation of the erythrocytes abolished agglutination with fixed fibroblasts. The agglutinability of fixed cells is more likely related to increased cell rigidity than to decreased membrane fluidity.

Rubella virus: growth and cytopathic effect in primary cultures of cells of rabbit embryos.

Primary cultures of rabbit (New Zealand white) embryo cells support growth of rubella virus. Distinct cytopathic changes are discernible within 6 to 8 days after inoculation. This cell system has been successful for the recovery of rubella virus from clinical materials and the demonstration of neutralizing antibody in patient serum.

Temperature-dependent rosette formation by mouse lymphoma cells as a result of viral hemadsorption.

Cells from several mouse lymphomas formed rosettes with nonsensitized foreign erythrocytes through C-type virus particles clustered on the cell surface in serum-free medium held at 4 degrees C. This type of rosetting was found most typically in a lymphoma induced by Rauscher leukemia virus in tissue culture (RD-12), but it also occurred in 23 of 61 spontaneous thymic lymphomas in AKR mice. Chemically or X-ray-induced leukemias and spontaneous reticulum cell sarcomas did not form rosettes. The nature of the rosette formation may be interpreted as viral hemadsorption, with a possible relationship to hemagglutination by murine leukemia viruses. The receptor on virus particles was trypsin sensitive and showed high affinity to serum inhibitors (RIF). Serum rosette-inhibiting activity was assessed by a quantitative rosette inhibition test; rosette inhibition proved widely distributed among species. Physicochemical properties of serum RIF and their function both in vivo and in vitro were described. Rosette formation with similar temperature requirements, previously reported in a mouse lymphoma carrying membrane-bound heterophile cold hemagglutinin, was readily distinguished from viral hemadsorption by its insensitivity to mouse serum RIF.

Enhanced expression of melanoma-associated antigens and beta 2-microglobulin on cultured human melanoma cells by interferon.

The effect of human leukocyte interferon (IFN) on the in vitro growth and expression of melanoma-associated antigens (MAA). beta 2-microglobulin (beta 2m) and HLA-DR antigen on cultured human melanoma cells was studied. Exposure of melanoma cells to IFN for 64 hours resulted in a dose-dependent inhibition of growth with 46% reduction in cell number at 10(3) U IFN/ml and 74% reduction at 10(5) U/ml. Quantitative absorption experiments in the mixed hemadsorption assay determined that the expression of MAA and beta 2m on treated cells was enhanced at 10(2)-10(5) U IFN/ml, twofold to fivefold for MAA and fivefold to twelvefold for beta 2m. No change was seen in HLA-DR antigen expression. The IFN-induced enhancement of MAA and beta 2m could be detected as early as after 16 hours and a maximum expression was reached at 96 hours after IFN exposure. The IFN-induced enhancement of MAA and beta 2m on melanoma cells was reversible. Studies with melanoma cells grown in stationary phase and serum-deprived conditions indicated that IFN-induced augmentation of MAA and beta 2m did not require cell proliferation. The data suggest that the effect of IFN on antigen expression is independent of its effect on cell growth. Further studies are needed to fully elucidate the mechanism.

Use of the blocking procedure in mixed hemadsorption tests for the analysis of surface antigens in liver tissue culture cells.

For detection of surface species-specific and specific tumor antigens in the various tissue culture cell lines, including interspecies hybrid cell line, the blocking procedure was included in a microhemadsorption test (MHT). Use of the appropriate high-titered blocking antisera unadequate to the indicatory system of MHT permits the detection of different species-specific and tumor antigens on the surface of the liver tissue culture cells with the use of heterologous unabsorbed immune sera.

Decreased antigenicity of mouse blastocysts after activation for implantation from experimental delay.

The antigenicity of mouse blastocysts in experimentally delayed implantation and after activation from delay by estradiol administration were determined by two different methods. CBA blastocysts were incubated in C57BL/6J anti-CBA serum, and the amount of antibodies bound to the blastocysts was traced by -125-I-conjugated sheep antimouse gamma-globulin (isotope antiglobulin technique) and sensitized sheep red cells (mixed haemadsorption technique). With both techniques it was possible to demonstrate that mouse blastocysts in experimental delay of implantation possess alloantigens, and that this antigen expression has decreased markedly 14 hr after activation for implantation. It is suggested that this phenomenon may be of significance for noncognition of the allogeneic conceptus during pregnancy.

Early detection of influenza virus in cell culture by means of immunofluorescence.

An indirect fluorescent antibody technique using human serum for the rapid detection of influenza A virus in monkey kidney cell cultures is described. Influenza A2 virus was isolated from 38 of 88 nose/throat swabs received from patients with suspected influenza during the winter of 1971-72.Thirty-two strains isolated in monkey kidney cell cultures were identified by immunofluorescence on the day haemadsorption was observed, 22 of them within a week of receipt of the specimen. Human antiserum was found to be very satisfactory for this purpose.

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.

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.

Multiple sclerosis and parainfluenza 1 virus. History of the isolation of the virus and expression of phenotypic differences between the isolated virus and Sendai virus.

54 cultures were established from brain tissue obtained 2-3 hrs after death from 1 case of multiple sclerosis and 30 cultures from another case. Following fusion with indicator cells in the presence of lysolecithin, a parainfluenza type 1 virus (6/94 virus) was isolated from cultures representing one plaque area in the first case and one plaque area in the second case. A cell line chronically infected with the 6/94 virus has been maintained for more than 100 passages in vitro. A close relationship to the Sendai Hemagglutinating Virus of Japan (HVJ) is indicated from RNA-RNA hybridization and the patterns of electrophoretic mobilities of viral polypeptides. Conversely, differences in optima for growth-requirement temperatures, hemolytic activity and the capability to fuse mammalian cells, distinguishes 6/94 virus and HVJ as distinct phenotypic entities of a closely related genotype.

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.

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.

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.

Persistent SV5 virus infection in continuous cell cultures.

A continuous line of guinea pig kidney cells (CGPK/H) and a continuous line of mouse fibroblasts (L/H) spontaneously infected with parainfluenza virus SV5 were found. These cultures showed no enhanced cell degeneration or symplast formation, nor was haemagglutinin accumulation or infectious virus demonstrated in them. However, regular reproduction of ribonucleoprotein (RNP) characteristic of parainfluenza viruses, morphologically complete virions and antigens producing antibody to SV5 virus were found in the cells. Focal haemadsorption neutralized by antiserum to SV5 virus was also demonstrated. The infection persisted in the cell populations for over 2 years (the observation period) under standard conditions of cell dispersion and subcultivation.