A comparison between spray drying and spray freeze drying to produce an influenza subunit vaccine powder for inhalation.

The aim of this study was to investigate two different processes to produce a stable influenza subunit vaccine powder for pulmonary immunization i.e. spray drying (SD) and spray freeze drying (SFD). The formulations were analyzed by proteolytic assay, single radial immunodiffusion assay (SRID), cascade impactor analysis, and immunization studies in Balb/c mice. Proteolytic assay and SRID analysis showed that antigen integrity after SFD was best conserved when the formulation was buffered by Hepes buffer saline (HBS). Surprisingly, antigen integrity after SD was better conserved when the formulation was buffered by phosphate buffer saline (PBS) rather than by HBS. The dispersion from the dry powder inhaler, the Twincer, resulted in a fine particle fraction (aerodynamic particle size <5microm) of 37% and 23% for spray dried and spray freeze dried powders, respectively. Immunogenicity of both vaccine formulations (SFD/HBS and SD/PBS) was similar to conventional liquid formulation after i.m. immunization. In addition, compared to i.m. immunizations, the pulmonary immunization with the dry powders resulted in significantly higher IgG titers. Furthermore, both the formulations remained biochemically and physically stable for at least 3years of storage at 20 degrees C. Our results demonstrate that both optimized formulations are stable and have good inhalation characteristics.

HPLC-based quantification of haemagglutinin in the production of egg- and MDCK cell-derived influenza virus seasonal and pandemic vaccines.

The haemagglutinin (HA) content is an important specification of influenza vaccines. Recently, a reversed-phase high performance liquid chromatography (RP-HPLC) method for quantification of HA in PER.C6 cell culture-based whole virus vaccines has been reported, having a high sensitivity, precision, broad range, and high sample throughput [Kapteyn JC, Drissi Saidi M, Dijkstra R, Kars C, Tjon CMS-K, Weverling GJ et al. Haemagglutinin quantification and identification of influenza A&B strains propagated in PER.C6 cells: a novel RP-HPLC method. Vaccine 2006;24:3137-44]. This RP-HPLC assay is based on measuring the peak area of HA1, the hydrophilic subunit of HA, which turned out to be proportional to the amount of HA analyzed. Here, we present data demonstrating that this RP-HPLC method is also highly suitable for HA quantification of active and BPL- or formaldehyde-inactivated egg-based and MDCK cell-based whole virus samples, including egg allantoic harvest, and in final (monovalent) subunit vaccines, including those for pandemic H5N1 strains and for virosomal vaccines. In addition, the RP-HPLC assay was demonstrated to be a very powerful tool in the early stages of seasonal influenza vaccine production, when homologous serial radial immunodiffusion (SRID) reagents are not yet available, enabling fast and reliable viral growth studies in eggs in order to select the best growing virus strains or reassortants for the production of the seasonal trivalent influenza vaccine. Because of its high sensitivity, the RP-HPLC assay has shown its enormous value in supporting small scale MDCK-based (H5N1) influenza virus production models. Finally, the observed differences between HA1 molecules from various HA subtypes in UV absorbance, FLD response, and in the actual retention times in RP-HPLC are discussed in relation to the primary structure of the HA1 molecules studied.

Characterization of agglutinin-like sequence genes from non-albicans Candida and phylogenetic analysis of the ALS family.

The ALS (agglutinin-like sequence) gene family of Candida albicans encodes cell-surface glycoproteins implicated in adhesion of the organism to host surfaces. Southern blot analysis with ALS-specific probes suggested the presence of ALS gene families in C. dubliniensis and C. tropicalis; three partial ALS genes were isolated from each organism. Northern blot analysis demonstrated that mechanisms governing expression of ALS genes in C. albicans and C. dubliniensis are different. Western blots with an anti-Als serum showed that cross-reactive proteins are linked by beta 1,6-glucan in the cell wall of each non-albicans Candida, suggesting similar cell wall architecture and conserved processing of Als proteins in these organisms. Although an ALS family is present in each organism, phylogenetic analysis of the C. albicans, C. dubliniensis, and C. tropicalis ALS genes indicated that, within each species, sequence diversification is extensive and unique ALS sequences have arisen. Phylogenetic analysis of the ALS and SAP (secreted aspartyl proteinase) families show that the ALS family is younger than the SAP family. ALS genes in C. albicans, C. dubliniensis, and C. tropicalis tend to be located on chromosomes that also encode genes from the SAP family, yet the two families have unexpectedly different evolutionary histories. Homologous recombination between the tandem repeat sequences present in ALS genes could explain the different histories for co-localized genes in a predominantly clonal organism like C. albicans.

Low external pH induces HOG1-dependent changes in the organization of the Saccharomyces cerevisiae cell wall.

Low environmental pH strongly affected the organization of the Saccharomyces cerevisiae cell wall, resulting in rapidly induced resistance to beta1,3-glucanase. At a molecular level, we found that a considerable amount of Cwp1p became anchored through a novel type of linkage for glycosylphosphatidylinositol (GPI)-dependent cell wall proteins, namely an alkali-labile linkage to beta1,3-glucan. This novel type of modification for Cwp1p did not require the presence of a GPI-derived structure connecting the protein with beta1,6-glucan. In addition, we found high levels of Cwp1p, which was double-anchored through both the novel alkali-sensitive bond to beta1,3-glucan and the alkali-resistant GPI-derived linkage to beta1,6-glucan. Further cell wall analyses demonstrated that Pir2p/Hsp150 and possibly other Pir cell wall proteins, which were already known to be linked to the beta1,3-glucan framework by an alkali-sensitive linkage, were also more efficiently retained in the cell wall at pH 3.5 than at pH 5.5. Consequently, the alkali-sensitive type of linkage of cell wall proteins to beta1,3-glucan was induced by low pH. The low pH-induced alterations in yeast cell wall architecture were demonstrated to be dependent on a functional HOG1 gene, but not on the Slt2p-mediated MAP kinase pathway. Consistent with this observation, DNA microarray studies revealed transcriptional induction of many known high-osmolarity glycerol (HOG) pathway-dependent genes, including four cell wall-related genes, namely CWP1, HOR7, SPI1 and YGP1.

The cell wall architecture of Candida albicans wild-type cells and cell wall-defective mutants.

In Candida albicans wild-type cells, the beta1, 6-glucanase-extractable glycosylphosphatidylinositol (GPI)-dependent cell wall proteins (CWPs) account for about 88% of all covalently linked CWPs. Approximately 90% of these GPI-CWPs, including Als1p and Als3p, are attached via beta1,6-glucan to beta1,3-glucan. The remaining GPI-CWPs are linked through beta1,6-glucan to chitin. The beta1,6-glucanase-resistant protein fraction is small and consists of Pir-related CWPs, which are attached to beta1,3-glucan through an alkali-labile linkage. Immunogold labelling and Western analysis, using an antiserum directed against Saccharomyces cerevisiae Pir2p/Hsp150, point to the localization of at least two differentially expressed Pir2 homologues in the cell wall of C. albicans. In mnn9Delta and pmt1Delta mutant strains, which are defective in N- and O-glycosylation of proteins respectively, we observed enhanced chitin levels together with an increased coupling of GPI-CWPs through beta1,6-glucan to chitin. In these cells, the level of Pir-CWPs was slightly upregulated. A slightly increased incorporation of Pir proteins was also observed in a beta1, 6-glucan-deficient hemizygous kre6Delta mutant. Taken together, these observations show that C. albicans follows the same basic rules as S. cerevisiae in constructing a cell wall and indicate that a cell wall salvage mechanism is activated when Candida cells are confronted with cell wall weakening.

Cell wall dynamics in yeast.

The yeast Saccharomyces cerevisiae is the first fungus for which the structure of the cell wall is known at the molecular level. It is a dynamic and highly regulated structure. This is vividly illustrated when the cell wall is damaged and a salvage pathway becomes active, resulting in compensatory changes in the wall.

The contribution of the O-glycosylated protein Pir2p/Hsp150 to the construction of the yeast cell wall in wild-type cells and beta 1,6-glucan-deficient mutants.

The cell wall of yeast contains a major structural unit, consisting of a cell wall protein (CWP) attached via a glycosylphosphatidylinositol (GPI)-derived structure to beta 1,6-glucan, which is linked in turn to beta 1, 3-glucan. When isolated cells walls were digested with beta 1,6-glucanase, 16% of all CWPs remained insoluble, suggesting an alternative linkage between CWPs and structural cell wall components that does not involve beta 1,6-glucan. The beta 1,6-glucanase-resistant protein fraction contained the recently identified GPI-lacking, O-glycosylated Pir-CWPs, including Pir2p/Hsp150. Evidence is presented that Pir2p/Hsp150 is attached to beta 1,3-glucan through an alkali-sensitive linkage, without beta 1,6-glucan as an interconnecting moiety. In beta 1,6-glucan-deficient mutants, the beta 1,6-glucanase-resistant protein fraction increased from 16% to over 80%. This was accompanied by increased incorporation of Pir2p/Hsp150. It is argued that this is part of a more general compensatory mechanism in response to cell wall weakening caused by low levels of beta 1,6-glucan.

The contribution of cell wall proteins to the organization of the yeast cell wall.

Our knowledge of the yeast cell wall has increased rapidly in the past few years, allowing for the first time a description of its structure in molecular terms. Two types of cell wall proteins (CWPs) have been identified that are covalently linked to beta-glucan, namely GPI-CWPs and Pir-CWPs. Both define a characteristic supramolecular complex or structural unit. The GPI building block has the core structure GPI-CWP-->beta1,6-glucan-->beta1,3-glucan, which may become extended with one or more chitin chains. The Pir building block is less well characterized, but preliminary evidence points to the structure, Pir-CWP-->beta1,3-glucan, which probably also may become extended with one or more chitin chains. The molecular architecture of the cell wall is not fixed. The cell can make considerable adjustments to the composition and structure of its wall, for example, during the cell cycle or in response to environmental conditions such as nutrient and oxygen availability, temperature, and pH. When the cell wall is defective, dramatic changes can occur in its molecular architecture, pointing to the existence of cell wall repair mechanisms that compensate for cell damage. Finally, evidence is emerging that at least to a considerable extent the cell wall of Saccharomyces cerevisiae is representative for the cell wall of the Ascomycetes.

Loss of the plasma membrane-bound protein Gas1p in Saccharomyces cerevisiae results in the release of beta1,3-glucan into the medium and induces a compensation mechanism to ensure cell wall integrity.

Deletion of GAS1/GGP1/CWH52 results in a lower beta-glucan content of the cell wall and swollen, more spherical cells (L. Popolo, M. Vai, E. Gatti, S. Porello, P. Bonfante, R. Balestrini, and L. Alberghina, J. Bacteriol. 175:1879-1885, 1993; A. F. J. Ram, S. S. C. Brekelmans, L. J. W. M. Oehlen, and F. M. Klis, FEBS Lett. 358:165-170, 1995). We show here that gas1delta cells release beta1,3-glucan into the medium. Western analysis of the medium proteins with beta1,3-glucan- and beta1,6-glucan-specific antibodies showed further that at least some of the released beta1,3-glucan was linked to protein as part of a beta1,3-glucan-beta1,6-glucan-protein complex. These data indicate that Gas1p might play a role in the retention of beta1,3-glucan and/or beta-glucosylated proteins. Interestingly, the defective incorporation of beta1,3-glucan in the cell wall was accompanied by an increase in chitin and mannan content in the cell wall, an enhanced expression of cell wall protein 1 (Cwp1p), and an increase in beta1,3-glucan synthase activity, probably caused by the induced expression of Fks2p. It is proposed that the cell wall weakening caused by the loss of Gas1p induces a set of compensatory reactions to ensure cell integrity.

Altered extent of cross-linking of beta1,6-glucosylated mannoproteins to chitin in Saccharomyces cerevisiae mutants with reduced cell wall beta1,3-glucan content.

The yeast cell wall contains beta1,3-glucanase-extractable and beta1,3-glucanase-resistant mannoproteins. The beta1,3-glucanase-extractable proteins are retained in the cell wall by attachment to a beta1,6-glucan moiety, which in its turn is linked to beta1,3-glucan (J. C. Kapteyn, R. C. Montijn, E. Vink, J. De La Cruz, A. Llobell, J. E. Douwes, H. Shimoi, P. N. Lipke, and F. M. Klis, Glycobiology 6:337-345, 1996). The beta1,3-glucanase-resistant protein fraction could be largely released by exochitinase treatment and contained the same set of beta1,6-glucosylated proteins, including Cwp1p, as the B1,3-glucanase-extractable fraction. Chitin was linked to the proteins in the beta1,3-glucanase-resistant fraction through a beta1,6-glucan moiety. In wild-type cell walls, the beta1,3-glucanase-resistant protein fraction represented only 1 to 2% of the covalently linked cell wall proteins, whereas in cell walls of fks1 and gas1 deletion strains, which contain much less beta1,3-glucan but more chitin, beta1,3-glucanase-resistant proteins represented about 40% of the total. We propose that the increased cross-linking of cell wall proteins via beta1,6-glucan to chitin represents a cell wall repair mechanism in yeast, which is activated in response to cell wall weakening.

Large scale identification of genes involved in cell surface biosynthesis and architecture in Saccharomyces cerevisiae.

The sequenced yeast genome offers a unique resource for the analysis of eukaryotic cell function and enables genome-wide screens for genes involved in cellular processes. We have identified genes involved in cell surface assembly by screening transposon-mutagenized cells for altered sensitivity to calcofluor white, followed by supplementary screens to further characterize mutant phenotypes. The mutated genes were directly retrieved from genomic DNA and then matched uniquely to a gene in the yeast genome database. Eighty-two genes with apparent perturbation of the cell surface were identified, with mutations in 65 of them displaying at least one further cell surface phenotype in addition to their modified sensitivity to calcofluor. Fifty of these genes were previously known, 17 encoded proteins whose function could be anticipated through sequence homology or previously recognized phenotypes and 15 genes had no previously known phenotype.

Architecture of the yeast cell wall. Beta(1-->6)-glucan interconnects mannoprotein, beta(1-->)3-glucan, and chitin.

In a previous study (Kollár, R., Petráková, E., Ashwell, G., Robbins, P. W., and Cabib, E. (1995) J. Biol. Chem. 270, 1170-1178), the linkage region between chitin and beta(1-->3)-glucan was solubilized and isolated in the form of oligosaccharides, after digestion of yeast cell walls with beta(1-->3)-glucanase, reduction with borotritide, and subsequent incubation with chitinase. In addition to the oligosaccharides, the solubilized fraction contained tritium-labeled high molecular weight material. We have now investigated the nature of this material and found that it represents areas in which all four structural components of the cell wall, beta(1-->3)-glucan, beta(1-->6)-glucan, chitin, and mannoprotein are linked together. Mannoprotein, with a protein moiety about 100 kDa in apparent size, is attached to beta(1-->6)-glucan through a remnant of a glycosylphosphatidylinositol anchor containing five alpha-linked mannosyl residues. The beta(1-->6)-glucan has some beta(1-->3)-linked branches, and it is to these branches that the reducing terminus of chitin chains appears to be attached in a beta(1-->4) or beta(1-->2) linkage. Finally, the reducing end of beta(1-->6)-glucan is connected to the nonreducing terminal glucose of beta(1-->3)-glucan through a linkage that remains to be established. A fraction of the isolated material has three of the main components but lacks mannoprotein. From these results and previous findings on the linkage between mannoproteins and beta(1-->6)-glucan, it is concluded that the latter polysaccharide has a central role in the organization of the yeast cell wall. The possible mechanism of synthesis and physiological significance of the cross-links is discussed.

Retention of Saccharomyces cerevisiae cell wall proteins through a phosphodiester-linked beta-1,3-/beta-1,6-glucan heteropolymer.

Yeast cell wall proteins, including Cwp1p and alpha-agglutinin, could be released by treating the cell wall with either beta-1,3-or beta-1,6-glucanases, indicating that both polymers are involved in anchoring cell wall proteins. It was shown immunologically that both beta-1,3- and beta-1,6-glucan were linked to yeast cell wall proteins, including Cwp1p and alpha-agglutinin. It was further shown that beta-1,3-glucan was linked to the wall protein through a beta-1,6-glucan moiety. The beta-1,6-glucan moiety could be removed from Cwp1p and other cell wall proteins by cleaving phosphodiester bridges either enzymatically using phosphodiesterases or chemically using ice-cold aqueous hydrofluoric acid. These observations are consistent with the notion that cell wall proteins in Saccharomyces cerevisiae are linked to a beta-1,3-/beta-1,6-glucan heteropolymer through a phosphodiester linkage and that this polymer is responsible for anchoring cell wall proteins. It is proposed that this polymer is identical to the alkali-soluble beta-1,3-/beta-1,6-glucan heteropolymer characterized by Fleet and Manners (1976, 1977).

Covalent association of beta-1,3-glucan with beta-1,6-glucosylated mannoproteins in cell walls of Candida albicans.

Yeast and hyphal walls of Candida albicans were extracted with sodium dodecyl sulfate (SDS). Some of the extracted proteins reacted with a specific beta-1,6-glucan antiserum but not with a beta-1,3-glucan antiserum. They lost their beta-1,6-glucan epitope after treatment with ice-cold aqueous hydrofluoric acid, suggesting that beta-1,6-glucan was linked to the protein through a phosphodiester bridge. When yeast and hyphal walls extracted with SDS were subsequently extracted with a pure beta-1,3-glucanase, several mannoproteins that were recognized by both the beta-1,6-glucan antiserum and the beta-1,3-glucan antiserum were released. Both epitopes were sensitive to aqueous hydrofluoric acid treatment, suggesting that beta-1,3-glucan and beta-1,6-glucan are linked to proteins by phosphodiester linkages. The possible role of beta-glucans in the retention of cell wall proteins is discussed.

Glucosylation of cell wall proteins in regenerating spheroplasts of Candida albicans.

The cell wall of Candida albicans contains mannoproteins that are covalently associated with beta-1,6-glucan. When spheroplasts were allowed to regenerate a new cell wall, initially non-glucosylated cell wall proteins accumulated in the medium. While the spheroplasts became osmotically stable, beta-1,6-glucosylated proteins could be identified in their cell wall by SDS-extraction or beta-1,3-glucanase digestion. At later stages of regeneration, beta-1,3-glucosylated proteins were also found. Hence, incorporation of proteins into the cell wall is accompanied by extracellular coupling to beta-1,6-/beta-1,3-glucan. The SDS-extractable glucosylated proteins probably represent degradation products of wall proteins rather than their precursors. Tunicamycin delayed, but did not prevent the formation of beta-1,6-glucosylated proteins, demonstrating that beta-1,6-glucan is not attached to N-glycosidic side-chains of wall proteins.

Identification of beta-1,6-glucosylated cell wall proteins in yeast and hyphal forms of Candida albicans.

Several cell wall proteins released from yeast and hyphal cells of Candida albicans by laminarinase reacted with an affinity-purified antiserum raised against beta-1,6-glucan. Binding of the antiserum was competitively inhibited by beta-1,6-glucan, but not by beta-1,3-glucan or isolated N-chains. Immunodetection was completely abolished when the proteins were treated with periodate. These results demonstrate that the laminarinase-released wall proteins of C. albicans possess an epitope consisting of beta-1,6-linked glucose residues. The yeast form of C. albicans contained four beta-1,6-glucosylated wall proteins, an Endo H-resistant protein of 125 kDa and three glycoproteins which became only detectable after Endo H digestion and had a molecular mass of 320, 170 and 44 kDa, respectively. As for the hyphal form, a different set of beta-1,6-glucosylated wall proteins was found consisting of two Endo H-resistant glycoproteins of 125 and 80 kDa, respectively, and two glycoproteins that after Endo H digestion had a molecular mass of 320 and 38 kDa, respectively. Sodium dodecyl sulfate-extractable wall proteins and medium proteins did not react with the beta-1,6-glucan antiserum. The beta-1,6-glucan epitope could be removed by aqueous hydrofluoric acid indicating that the epitope is phosphodiester-linked to the cell wall proteins. It is speculated that the epitope forms part of a GPI-anchor and might be involved in the anchoring of mannoproteins into the cell wall.