Intranasal vaccination with a replication-deficient influenza virus induces heterosubtypic neutralising mucosal IgA antibodies in humans.

UNLABELLED: We investigated the cross-neutralising potential of serum and nasal wash samples from volunteers who were intranasally immunised once with a monovalent replication-deficient delNS1-H1N1 influenza virus vaccine (7.7log10TCID50/volunteer). Eight out of twelve (8/12) vaccinees responded to vaccination with a significant increase of antibody levels in serum IgG ELISA, mucosal IgA ELISA, MNA or HAI. Four responders showed delNS1-specific ELISA IgA increases and revealed excellent homosubtypic neutralising activity in serum and mucosal washings (4/4). However, 0/4 of the sera but 3/4 of the nasal washings neutralised also heterosubtypic H3N2 and H5N1 influenza viruses. Depletion experiments proved that IgA but not IgG is responsible for the cross-neutralising activity of the nasal wash sample. Our findings indicate that the induction of virus-neutralising IgA may represent a valuable correlate of cross-protection of intranasal influenza vaccines and that the delNS1 concept constitutes a promising approach to protect humans from seasonal and pandemic influenza threats. CLINICAL TRIAL REGISTRATION: NCT00724997.

Validation of the modified hemagglutination inhibition assay (mHAI), a robust and sensitive serological test for analysis of influenza virus-specific immune response.

BACKGROUND: The hemagglutination inhibition assay (HAI) is universally regarded as the gold standard in influenza virus serology. Nevertheless, difficulties in titre readouts are common and interlaboratory variations are frequently reported. OBJECTIVE: We developed and validated the modified HAI to facilitate reliable, accurate and reproducible analysis of sera derived from influenza vaccination studies. STUDY DESIGN: Clinical and preclinical serum samples, NIBSC reference sera and seasonal influenza virus type A (H1N1 and H3N2) and type B antigens were employed to validate the mHAI. Moreover, pandemic virus strains (H5N1 and H1N1pdm09) were used to prove assay robustness. RESULTS: Utilisation of a 0.08% solution of stabilised human erythrocytes, assay buffer containing bovine serum albumin and microscopical plate readout are the major differences between the modified and standard HAI assay protocols. Validation experiments revealed that the mHAI is linear, specific and up to eightfold more sensitive than the standard HAI. In 95.6% of all measurements mHAI titres were precisely measured irrespective of the assay day, run or operator. Moreover, 96.4% (H1N1) or 95.2% (H3N2 and B), respectively, of all serum samples were determined within one dilution step of the nominal values for spiked samples. Finally, the mHAI results remained unaffected by variations in virus antigens, erythrocytes, reagents, laboratory location, sample storage conditions or matrix components. CONCLUSION: The modified HAI is easy to analyse, requires only a single source of erythrocytes and allows utilisation of numerous influenza virus antigens, also including virus strains which are difficult to handle by the standard HAI (e.g. H3N2, H5N1 and H1N1pdm09).

[Preclinical studies of live intranasal H5N1 influenza vaccine with the deleted HS1 gene].

The paper gives the results of evaluating the efficiency of deINS1 pandemic H5N1 vaccine candidate VN1203delNS1 which was constructed by reverse genetics on the basis of influenza virus strain A/Vietnam/1203/04. The safety, immunogenicity and cross-protection of the vaccine strain against different H5N1 virus clades were demonstrated in mouse and macaque models. The results showed the possibility of designing a new-generation replication-deficient intranasal influenza vaccine, by applying an approach to deleting the NS1 pathogenicity factor, an antagonist of the interferon system.

Vaccine potential of influenza vectors expressing Mycobacterium tuberculosis ESAT-6 protein.

We generated several attenuated recombinant influenza A vectors expressing the Mycobacterium tuberculosis early secretory antigenic target (ESAT-6) protein. The ESAT-6 protein was recently identified as one of the most promising protective antigens for cell-mediated immunity. The obtained vectors appeared to be capable of inducing ESAT-6 specific Th1 immune response in mice after intranasal immunization. We found that double immunization with two influenza vectors of different subtypes provided a significant level of protection in mice, when applied as prophylactic vaccine, as well as substantial therapeutic effect in mice with pre-established tuberculosis infection. Moreover, we found a strong synergistic effect when vaccination with Flu/ESAT-6 vectors was combined with isoniazid treatment, resulting in a dramatic reduction of bacterial load in the lungs of infected mice.

Effect of a single mutation in neuraminidase on the properties of Influenza B virus isolates.

Two strains of Influenza B virus were isolated in Vero cells. Subclones with improved efficiency of plaque formation were selected. The activity of the neuraminidase (NA) of the two subclones compared to their respective isolates dropped 20- and 100-fold, respectively. Both subclones had a common mutation in segment 6 leading to a change from Asp to Asn at position 457 in the NA. This mutation destroyed a salt bridge of the contact surface between the monomers, thereby causing the loss in enzymatic activity. The decreased NA activity caused improved plaque formation but had no significant impact on the replication in liquid culture.

Hyperattenuated recombinant influenza A virus nonstructural-protein-encoding vectors induce human immunodeficiency virus type 1 Nef-specific systemic and mucosal immune responses in mice.

We have generated recombinant influenza A viruses belonging to the H1N1 and H3N2 virus subtypes containing an insertion of the 137 C-terminal amino acid residues of the human immunodeficiency virus type 1 (HIV-1) Nef protein into the influenza A virus nonstructural-protein (NS1) reading frame. These viral vectors were found to be genetically stable and capable of growing efficiently in embryonated chicken eggs and tissue culture cells but did not replicate in the murine respiratory tract. Despite the hyperattenuated phenotype of influenza/NS-Nef viruses, a Nef and influenza virus (nucleoprotein)-specific CD8(+)-T-cell response was detected in spleens and the lymph nodes draining the respiratory tract after a single intranasal immunization of mice. Compared to the primary response, a marked enhancement of the CD8(+)-T-cell response was detected in the systemic and mucosal compartments, including mouse urogenital tracts, if mice were primed with the H1N1 subtype vector and subsequently boosted with the H3N2 subtype vector. In addition, Nef-specific serum IgG was detected in mice which were immunized twice with the recombinant H1N1 and then boosted with the recombinant H3N2 subtype virus. These findings may contribute to the development of alternative immunization strategies utilizing hyperattenuated live recombinant influenza virus vectors to prevent or control infectious diseases, e.g., HIV-1 infection.

Topically applied liposome encapsulated superoxide dismutase reduces postburn wound size and edema formation.

The overproduction of biochemical mediators, and activation of leukocytes and endothelial cells, generated in thermally injured tissue, gives rise to both local and distant effects. The formation of short-lived, highly reactive metabolites, such as oxygen free radicals, increases with increasing tissue ischemia, and causes further cell damage. Human recombinant Cu/Zn-superoxide dismutase (rh-Cu/Zn-SOD), an enzyme which captures these radicals, may have a beneficial effect on the postburn inflammation processes. In this study, the influence of rh-Cu/Zn-SOD application to thermally injured tissue of rabbit backskin was examined. Three different delivery strategies were compared, pure or liposomally encapsulated enzyme, or intralesionally injected rh-Cu/Zn-SOD. For control, one animal group was treated with plain gel and another group was kept untreated. At 24 h following trauma a statistically significant difference in lesion sizes between the enzyme treated and control groups was observed. After 72 h tissue swelling had diminished significantly more in the rh-Cu/Zn-SOD treated groups as compared to the control animals. The best results were achieved by spreading liposomes encapsulating the enzyme onto the wounds. Our results suggest that local treatment of burn wounds with enzymatic radical scavengers such as rh-Cu/Zn-SOD has a beneficial effect on the extent of the postburn damage.

Dental wear and growth performance in steers fed sweetpotato cannery waste.

OBJECTIVE: To determine whether feeding sweetpotato cannery waste (SPCW) to cattle had adverse effects on dental wear, growth performance, or ruminal tissues. DESIGN: Clinical trial. ANIMALS: 36 Holstein steers. PROCEDURE: Steers were assigned to 1 of 3 groups. All steers received ryegrass hay ad libitum. In addition, steers in group 1 were fed 3.2 kg of corn and soybean meal/steer/d, steers in group 2 were fed 0.45 kg of soybean meal/steer/d and SPCW ad libitum, and steers in group 3 were fed a mixture of SPCW and broiler litter ad libitum. Samples of rumen fluid were collected on day 56. Steers were slaughtered on day 84, and samples of rumen were submitted for histologic examination. Teeth from control steers were removed, and calcium ion loss in response to etching with 2.28% lactic acid solutions buffered to pH of 3.75, 4.0, 4.25, 4.5, and 4.75 was determined. RESULTS: Average daily gain was lower for steers fed SPCW than for steers in the other 2 groups. Steers fed the SPCW-broiler litter mixture had only mild increases in tooth wear and tooth color scores, compared with control steers, whereas steers fed unbuffered SPCW had substantial increases in tooth wear and tooth color scores. Histologic abnormalities were detected in rumens from steers fed diets containing SPCW. Calcium ion loss decreased as pH of the etching solution increased. CLINICAL IMPLICATIONS: Results indicate that feeding cattle unbuffered SPCW can cause dental erosion, ruminal epithelial changes, and poor growth; however, SPCW buffered with broiler litter can be used as a cattle feed.

Chimeric influenza virus replicating predominantly in the murine upper respiratory tract induces local immune responses against human immunodeficiency virus type 1 in the genital tract.

Previously, a mucosal model of immunization against human immunodeficiency virus type 1 (HIV-1) was established by using influenza virus as a vector for the neutralizing gp41 epitope ELDKWA. Whether replication of this chimeric influenza virus in the upper respiratory tract of mice is sufficient for inducing mucosal immune responses in the genital tract was investigated. An immunization strategy was established that permits the virus to replicate in the murine upper respiratory tracts but not in the lungs. Intranasal application of the chimeric virus induced HIV-1-specific antibodies in sera and genital tract. In addition, chimeric virus-specific antibody-secreting cells were detected in lymphocyte populations obtained from lungs, spleens, and urogenital tracts. These results indicate that replication of the chimeric influenza/ELDKWA virus in the upper respiratory tract is sufficient to induce systemic immune responses as well as local immune responses in the genital tract.

Transfectant influenza A viruses with long deletions in the NS1 protein grow efficiently in Vero cells.

We established a reverse genetics system for the nonstructural (NS) gene segment of influenza A virus. This system is based on the use of the temperature-sensitive (ts) reassortant virus 25A-1. The 25A-1 virus contains the NS gene from influenza A/Leningrad/134/57 virus and the remaining gene segments from A/Puerto Rico (PR)/8/34 virus. This particular gene constellation was found to be responsible for the ts phenotype. For reverse genetics of the NS gene, a plasmid-derived NS gene from influenza A/PR/8/34 virus was ribonucleoprotein transfected into cells that were previously infected with the 25A-1 virus. Two subsequent passages of the transfection supernatant at 40 degreesC selected viruses containing the transfected NS gene derived from A/PR/8/34 virus. The high efficiency of the selection process permitted the rescue of transfectant viruses with large deletions of the C-terminal part of the NS1 protein. Viable transfectant viruses containing the N-terminal 124, 80, or 38 amino acids of the NS1 protein were obtained. Whereas all deletion mutants grew to high titers in Vero cells, growth on Madin-Darby canine kidney (MDCK) cells and replication in mice decreased with increasing length of the deletions. In Vero cells expression levels of viral proteins of the deletion mutants were similar to those of the wild type. In contrast, in MDCK cells the level of the M1 protein was significantly reduced for the deletion mutants.

A host restriction-based selection system for influenza haemagglutinin transfectant viruses.

During the 1996 influenza epidemic in Vienna we obtained influenza A virus specimens (Vienna/47/96, Vienna/81/96) which grow efficiently in African green monkey kidney (Vero) cells but not in embryonated chicken eggs. Amplification of the specimens in Vero cells resulted in progeny that agglutinated human but not chicken erythrocytes. Reassortment analysis suggested that the haemagglutinin (HA) might be responsible for the host restriction. Vero cells were infected with the Vienna/47/96 virus and then transfected with reconstituted ribonucleoprotein complexes containing HA genes from egg-adapted strains. Subsequent selective passages in embryonated chicken eggs resulted in selection of transfectant viruses, growing in eggs and containing the transfected HAs. The results demonstrate that host restriction of the Vero-adapted Vienna/47/96 virus is due to its HA. Moreover, the experiments showed that the Vienna/47/96 strain can be used as helper virus for reverse genetics experiments.

In vitro effects of an acidic by-product feed on bovine teeth.

OBJECTIVE: To evaluate in vitro erosive effects of sweet potato cannery waste (SPCW) on bovine incisor enamel. SAMPLE POPULATION: 20 bovine mandibles. PROCEDURE: Mandibles were collected and incisors were classified into 3 categories: lacking observable wear, advanced normal wear, or abnormal wear associated with feeding SPCW. Intact mandibles were radiographed. Contralateral normal teeth from the same jaw were used to compare Ca2+ loss (etching) with SPCW, lactic acid (pH 3.2), or SPCW neutralized with NaOH to pH 5.0 or 5.5. Scanning electron microscopy was performed to compare etched and unetched specimens. Two abnormally worn teeth were evaluated histologically. Knoop hardness testing was conducted on unexposed areas of surface enamel and enamel exposed to SPCW. RESULTS: Radiography revealed large periapical abscesses in the mandibles exposed to SPCW. Nearly identical amounts of Ca2+ were removed by SPCW and lactic acid solution at the same pH. Scanning electron microscopy did not indicate consistent differences between etch patterns resulting from exposure to SPCW or lactic acid. Mean rate of calcium removal was 56% higher in deciduous than permanent teeth. Knoop hardness data suggested that softening occurred in enamel exposed to SPCW. Neutralizing SPCW to pH 5.5 eliminated calcium removal. Histologic examination of sections indicated that SPCW degraded and removed some dentin matrix proteins. CONCLUSIONS: Exposure to SPCW results in enamel erosion in vitro; low pH is the most likely cause of erosion. Neutralizing SPCW to pH 5.5 eliminated erosive effects. CLINICAL RELEVANCE: Confirmation of SPCW's erosive effects on enamel in vitro supported the field diagnosis.

Immunogenic presentation of a conserved gp41 epitope of human immunodeficiency virus type 1 on recombinant surface antigen of hepatitis B virus.

In view of the high antigenic variability of human immunodeficiency virus type 1 (HIV-1), a vaccine against AIDS must induce an immune response to epitopes as invariable as possible among the various virus strains and clones. Previously the highly conserved six amino acid sequence Glu-Leu-Asp-Lys-Trp-Ala (ELDKWA) from gp41, defining the epitope of the human MAb 2F5, was shown to elicit HIV-1-neutralizing antibodies when presented on haemagglutinin of influenza virus. We investigated the immunogenic potential of the MAb 2F5 epitope and two of its major escape epitopes as internal fusions to the hepatitis B virus (HBV) surface antigen (HBsAg). Recombinant HBsAg-HIV proteins produced in the methylotrophic yeast Pichia pastoris self-assembled into 22 nm lipoprotein particles. Mice immunized with these particles developed an anti-HBsAg immune response in a range that is considered to be protective against HBV infection in humans. More importantly, antisera had extremely high titres of antibodies reactive with a structurally flexible form of the HIV-1 epitope, whereby strong cross-reactivity with the escape variants of the epitope was observed. Although HIV-1 gp 160 and the ectodomain of gp41 containing the epitope were significantly recognized, the antisera failed to neutralize HIV-1 in vitro. These data, together with those on the haemagglutinin-ELDKWAS fusion suggest that the ability of the MAb 2F5 epitope to induce neutralizing antibodies depends on the molecular context in which it is presented. Therefore, further characterization of secondary and tertiary structure requirements of the epitope is indispensable for the full exploitation of its potential as a vaccine component.

Mucosal model of immunization against human immunodeficiency virus type 1 with a chimeric influenza virus.

Previously, we constructed a chimeric influenza virus that expresses the highly conserved amino acid sequence ELDKWA of gp41 of human immunodeficiency virus type 1 (HIV-1). Antisera elicited in mice by infection with this chimeric virus showed neutralizing activity against distantly related HIV-1 isolates (T. Muster, R. Guinea, A. Trkola, M. Purtscher, A. Klima, F. Steindl, P. Palese, and H. Katinger, J. Virol. 68:4031-4034, 1994). In the present study, we demonstrated that intranasal immunizations with this chimeric virus are also able to induce a humoral immune response at the mucosal level. The immunized mice had ELDKWA-specific immunoglobulins A in respiratory, intestinal, and vaginal secretions. Sustained levels of these secretory immunoglobulins A were detectable for more than 1 year after immunization. The results show that influenza virus can be used to efficiently induce secretory antibodies against antigens from foreign pathogens. Since long-lasting mucosal immunity in the genital and intestinal tracts might be essential for protective immunity against HIV-1, influenza virus appears to be a promising vector for HIV-1-derived immunogens.

Parameters governing steam sterilization of deadlegs.

Use of saturated steam for sterilization-in-place (SIP) is limited by factors effecting displacement of air from deadlegs. Effects of tube diameter, length, orientation and position within a deadleg were quantitatively studied by examining temperature profiles and rates of kill of Bacillus stearothermophilus spores. Tube diameter had the greatest effect on sterilization. For small diameter tubes, 0.4 cm inside diameter (ID), air displacement was minimal and due mainly to diffusion. 8.8 cm long tubes with 0.4 cm IDs could not be sterilized at 121 degrees C. As tube diameter was increased and buoyant driven convective flow became dominant over viscous forces, sterilization was achieved and tube orientation became critical. Sterilization time, as defined by a twelve log reduction in spore population, was 75 minutes in a 19.0 cm long vertical tube with 1.7 cm ID, whereas 167 minutes were required for an 8.8 cm long tube with 1.0 cm ID. For 8.8 cm long tubes, only the 1.7 cm ID tube could be sterilized when orientated 5 degrees above horizontal. Data show that length to diameter ratios, L/Ds, do not provide a general guideline which can be used to predict sterilization. In the absence of steam bleeders, equipment should be designed to assure strong buoyancy driven convective flow to assure adequate air removal. This requires elimination of small diameter deadlegs (0.4 cm ID and less) and vertical positioning of deadlegs.

Temperature profiles and sterilization within a dead-ended tube.

Use of steam-in-place (SIP) sterilization has increased as the complexity of biotechnology processing equipment has increased. Extensive biological testing is required prior to use of this equipment as no quantitative guidelines exist for the design of SIP sterilizable equipment. Dead-ended geometries present the most difficult challenge to SIP sterilization, but data are not available as to the effects of tube orientation, length and diameter on time required for sterilization. This study examines the effects on sterilization of location within a dead-ended tube and orientation of the tube with respect to the gravitational vector. Temperature profiles and biological kill of Bacillus stearothermophilus were determined for four tube orientations. Kill kinetics were characterized by time to start of kill and cycle log reduction (CLR) times. Both values increased with increasing distance up the tube and orientation of the tube in a more horizontal position. CLR values were as much as ten times greater than those resulting from saturated steam. Projected sterilization times were determined and found to be very dependent on tube orientation. Recommendations are given for sterilization and validation testing of dead-ended geometries.