Monophosphoryl lipid A adjuvant reverses a principal histologic parameter of formalin-inactivated respiratory syncytial virus vaccine-induced disease.

The mechanisms by which administration of a formalin-inactivated respiratory syncytial virus vaccine resulted in enhanced disease among children after they later became naturally infected with the virus remains largely undefined. After immunization and live virus challenge, the cotton rat demonstrated the histopathologic marker of the enhanced disease, polymorphonuclear leukocyte infiltration of lung alveolar spaces. We now report that immunization with formalin-inactivated vaccine formulated with the adjuvant, 3-deacylated monophosphoryl lipid A, dramatically reduces or eliminates the polymorphonuclear leukocyte infiltration within the alveoli of cotton rats post-challenge. We suggest, that this or similar adjuvants may be beneficial components of candidate non-replicating respiratory syncytial virus vaccines, whose development has been hampered by safety concerns.

Efficacy and safety studies of a recombinant chimeric respiratory syncytial virus FG glycoprotein vaccine in cotton rats.

Several formulations of a recombinant chimeric respiratory syncytial virus (RSV) vaccine consisting of the extramembrane domains of the F and G glycoproteins (FG) were tested in cotton rats to evaluate efficacy and safety. The FG vaccine was highly immunogenic, providing nearly complete resistance to pulmonary infection at doses as low as 25 ng in spite of inducing relatively low levels of serum neutralizing antibody at low vaccine doses. Upon RSV challenge animals primed with FG vaccine showed quite mild alveolitis and interstitial pneumonitis, which were eliminated by the addition of monophosphoryl lipid A to the formulation.

Pulmonary lesions in primary respiratory syncytial virus infection, reinfection, and vaccine-enhanced disease in the cotton rat (Sigmodon hispidus).

Infection of the cotton rat lung with a human strain of respiratory syncytial virus results in substantial virus replication and is associated with mild-to-moderate peribronchiolitis, perivasculitis, and bronchitis. Reinfection after 49 days did not result in detectable virus replication, but surprisingly, was associated with an earlier appearance and accentuation of the three types of lesions seen in cotton rats undergoing primary infection. Animals primed with formalin-inactivated virus and challenged after 49 days had pulmonary viral titers 1/10 to 1/100 of that seen in naive animals, but developed markedly accentuated lesions of the same type as in animals undergoing primary or secondary infection. In addition, the animals with the vaccine-enhanced disease developed alveolitis and interstitial pneumonitis, which seem to be specific markers for the vaccine enhancement. These latter markers may be useful in determining the safety of nonreplicating vaccines.

Adjuvants influence the quantitative and qualitative immune response in BALB/c mice immunized with respiratory syncytial virus FG subunit vaccine.

The ability of monophosphoryl lipid A (MPL), QS-21 and alum to alter the immunologic response to immunization with respiratory syncytial virus a chimeric FG construct (FG) subunit vaccine was examined in BALB/c mice. FG/MPL, FG/alum, and FG/MPL/QS-21 combinations increased non-neutralizing antibody response, while FG/QS-21 did not. FG subunit vaccine with MPL, QS-21, or both had cytokine responses more closely resembling primary infection than FG/alum, with decreased interleukin-4 mRNA levels and increased IgG2a isotype antibody. The lungs of the mice immunized with FG subunit vaccines showed a heightened inflammatory response to respiratory syncytial virus challenge as compared to live virus immunization. Adjuvants can be used to alter the humoral and cellular responses to RSV subunit immunization.

Lethal oxidative damage to human immunodeficiency virus by human recombinant myeloperoxidase.

Human recombinant myeloperoxidase was evaluated in a cell-free system for its inactivation properties on the replication of human immunodeficiency virus, HTLV-IIIB. In the presence of a hydrogen peroxide generating system (glucose and glucose oxidase) and sodium thiocyanate, the recombinant enzyme inhibited virus-induced syncytium formation and viral replication without causing any cytopathic effects on SupT1 reporter cells. In addition, U937 monocytoid cells, chronically infected with HIV1, were exposed to recombinant myeloperoxidase (10 U/ml) and monitored during 48 h for the accumulation of intracellular p24 viral antigen. Under these conditions, the recombinant enzyme significantly reduced intracellular viral replication without affecting cell viability.

Anomerization and hydrolysis of lactose by beta-galactosidase from Saccharomyces lactis.

Beta-Galactosidase from Saccharomyces lactis was found to be able to catalyze both the anomerization of alpha-lactose and the hydrolysis of beta-lactose; the rate of hydrolysis appeared to be four times higher with a 1:1 mixture of alpha and beta lactose than with a freshly prepared solution of alpha-lactose. The enzyme was also found to be unable to hydrolyze alpha-lactose. Thus, it appears that beta-galactosidase from S. lactis has its hydrolytic activity on lactose adapted only to the naturally more abundant beta-lactose.

Lactoferrin: its role as a regulator of human granulopoiesis?

Lactoferrin has been proposed recently as a physiological regulator of the granulocyte-monocyte progenitor (CFU-GM). This glycoprotein, when saturated with iron, has been said to limit the CFU-GM growth by decreasing production and release of colony stimulating activity by monocytes and macrophages. Human milk lactoferrin saturated with iron, at concentrations ranging from 10(-8) M, was added either to endogenously stimulated bone marrow cells or to mononucleated cells used as feeder layers for adherent cell-depleted marrow. Irrespective of the concentration of lactoferrin within the culture system used, no significant inhibition of the CFU-GM growth was observed. Moreover, the CFU-GM stimulating activity of medium conditioned by a 4 day incubation of 1 X 10(6) mononucleated blood cells in the presence or in the absence of lactoferrin was the same. Various possible explanations for not confirming the reported inhibiting activity of iron-saturated lactoferrin were explored: (a) masking inhibition of the system by prostaglandin E2 (PGE2), (b) masking inhibition of the system by bovine lactoferrin present in the fetal calf serum, (c) preinhibition of the system by leukemic-associated inhibitory activity possibly present in the culture system, (d) the iron and calcium content of the culture medium used, (e) the fixation of lactoferrin to plastic compounds, (f) the source of the human lactoferrin used, and (g) the marrow cell separation methods used. None of these factors was shown to play a role in vitro in the activity of lactoferrin and thus no evidence was found for a significant role of lactoferrin in the regulation of human granulopoiesis.

Lactoferrin catabolism in the rat liver.

The hepatic uptake and degradation of human diferric 125I-lactoferrin by the liver of the intact rat were studied. After intravenous injection, the tracer was rapidly cleared by the liver, probably by adsorptive pinocytosis, as inferred from observations with a 3,470-fold dose range. Endocytosed lactoferrin was transferred, with a delay, from a light-density subcellular particle to an organelle that had a density similar to lysosomes. The loss of protein bound 125I from the liver was very slow (half-life 2.7 h), and its rate matched closely that of human asialotransferrin type 3. Lactoferrin was found to be a poor substrate for lysosomal hydrolases in vitro. Fucoidin effected the release of a portion of lactoferrin from the liver back into the plasma. By using this agent, indirect evidence was obtained suggesting that a fraction of lactoferrin is being repeatedly endo- and exocytosed (diacytosed) by the liver over prolonged periods of time. Fucosylation failed to impart lactoferrinlike properties on human asialotransferrin type 1, although the derivatized protein exhibited a less than or equal to 10-fold increase in affinity for the liver relative to the parent molecule.

Galactose-specific adsorptive endocytosis: an ultrastructural qualitative and quantitative study in cultured rat hepatocytes.

Using galactosylated bovine serum albumin coupled to colloidal gold (galBSA-CG) as a probe, the receptor-mediated endocytosis pathway has been analyzed qualitatively and quantitatively at the ultrastructural level in cultured rat hepatocytes. The results showed that galBSA-CG was specifically recognized by the asialoglycoprotein receptor of the hepatocytes, thus confirming biochemical findings. The probe was preferentially bound in coated pits of the cell surface. When bound elsewhere on the plasma membrane, it apparently moved towards coated regions. In both cases, it was then internalized via coated vesicles. The galBSA-CG passed through pleiomorphic tubular structures and in endosomes, a pool of smooth-surfaced vesicles of various size (50-350 nm), before transiently accumulating in multivesicular bodies. The latter then fused with lysosomes where the glycoproteinic moiety of the probe was degraded, as judged by the flocculated aspect of the accumulated gold particles. About 10% of the internalized ligand was recycled back to the cell surface via secreting vesicles containing lipoprotein-like particles without having apparently passed through lysosomes, which suggests the existence of a pre-lysosomal sorting mechanism of the endocytosed material. Functional recovery of the morphologically restored biliary polarity of hepatocytes in culture was indicated by the fact that galBSA-CG finally appeared in the reconstituted bile canaliculi.

Lactoferrin: no evidence for its role in regulation of CSA production by human lymphocytes and monocytes.

Lactoferrin (LF) has been recently proposed as a physiologic regulator of the granulocyte monocyte progenitor (CFU-GM). This glycoprotein, when saturated with iron, has been said to limit CFU-GM growth by decreasing production and release of colony stimulating activity (CSA) by monocytes and macrophages. Human milk LF saturated with iron, at concentrations ranging from 10(-18) to 10(-8) M was added either to endogenously stimulated bone marrow cells or to mononucleated cells used as feeder layers for adherent cell-depleted marrow. Irrespective of the concentration of LF within the culture system used, no significant inhibition of CFU-GM growth was observed. Moreover, the CFU-GM stimulating activity of medium conditioned by a 4-day incubation of 1 X 10(6) mononucleated blood cells in the presence or in the absence of LF was the same. Various possible explanations for not confirming the reported inhibiting activity of iron saturated LF were explored: 1) masking inhibition of the system by prostaglandin E2 (PGE2), 2) masking inhibition of the system by bovine LF still detectable in the fetal calf serum after heating, 3) preinhibition of the system by leukemic-associated inhibitory activity (LIA) possibly present in the culture system, 4) the iron and calcium content of the culture medium used, 5) the fixation of LF to plastic compounds, 6) the source of the human LF used, 7) the marrow cell separation methods used. None of these factors was shown to play a role in vitro in the activity of LF and thus no evidence was found for a significant role of LF in the regulation of CSA production by monocytes. Peripheral blood human monocytes isolated by elutriation and incubated in albumin free medium in the presence of either 125I-LF or colloidal gold-labeled LF showed no LF binding.

Cell adhesion mediated by a purified fucosyltransferase.

Human embryonic skin fibroblasts attach and spread on surfaces on which a fucosyltransferase purified from human milk has been immobilized. The adhesion-enhancing effect of the transferase involves specific interactions of the enzyme surface with the cell surface carbohydrate acceptors, as suggested by the following findings. About 80% of human embryonic skin fibroblasts attach and spread in 1 hr on fucosyltransferase surfaces; in contrast, bovine serum albumin, fetuin, asialofetuin, and asialotransferrin surfaces fail to enhance adhesion. The adhesion-mediating activity of the transferase is destroyed by alkylation of the sulfhydryl groups or by heating. The adhesion on fucosyltransferase surfaces is inhibited by glycoprotein, glycolipid, and oligosaccharide acceptors containing the sugar sequence galactosyl-(beta 1 leads to 4)-N-acetylglucosamine, in agreement with the substrate specificity of the enzyme. The results suggest that glycosyltransferases are able to stimulate cell adhesion in a manner similar to that proposed for lectins.

Disulfide content of reduced hen egg white and human milk lysozymes during the folding process.

In order to obtain a better understanding of the possible influence of the primary sequence of a protein on its folding pathway, renaturation of reduced human milk lysozyme was compared to that of reduced hen egg white lysozyme. Following disulfide bond formation, under identical conditions, similar products were found during the folding of both lysozymes, but the kinetics of appearance and disappearance of these intermediates as well as the appearance of the native conformation were different.

Enzymic properties of an N-acetylglucosaminide 3-alpha-L-fucosyltransferase of a wheat-germ agglutinin-resistant melanoma clone.

A fucosyltransferase was solubilized by extraction with Triton CF-54 from a wheat-germ agglutinin-resistant variant of mouse B16 melanoma. Through affinity chromatography on GDP hexanolamine--Sepharose a 44-fold enrichment of its specific activity was obtained. Analysis of its specificity indicated that the enzyme is an N-acetylglucosaminide 3-alpha-L-fucosyltransferase, which is able to transfer fucose to oligosaccharides containing Gal(beta 1-4)GlcNAc and Gal(beta 1-4)Glc structures. The enzyme is activated by divalent cations and has a maximum of activity at pH 5. It is unable to transfer fucose to sialylated glycoproteins, 6-alpha-sialyllactose or 3-alpha-sialyllactose. As suggested by its precipitation in the presence of antibodies raised in rabbit against a soluble human milk N-acetylglucosaminide 3-alpha-L-fucosyltransferase, these two enzymes seem to be structurally related.

Comparison between the folding of reduced hen egg white lysozyme and that of reduced human milk lysozyme.

In vitro, renaturation of reduced and unfolded lysozyme is catalyzed by a mixture of reduced and oxidized glutathione. After initiation of disulfide bond formation associated with the folding process of reduced human lysozyme, molecules have been trapped in a stable form with iodoacetic acid (preserving disulfide bonds) at various times of reoxidation. Each population of molecules trapped in this way was then analyzed by acrylamide gel electrophoresis which separates intermediates on the basis of the number of disulfide bonds they contain and the mean volume of the polypeptide chain. Moreover, the rate of reoxidation of the regeneration mixture was monitored by changes in enzymatic activity, fluorescence quantum yield, and global sulfhydryl group titer. Enzymatic activity was observed to appear after an induction period, and no intermediate, except the fully regenerated species, is active. The first two disulfide bonds reoxidize rapidly, and very few intermediates containing one or two disulfide bonds could be trapped. On the other hand, the intermediates containing three and four disulfide bonds are more predominant, and their formation proceeds more slowly. A folding pathway is suggested, based on the kinetic studies of appearance and disappearance of the various observed intermediates. When these results are compared with those obtained for hen egg white lysozyme and with those found in literature, it can be concluded that the reduced human protein recovers its native conformation more progressively and with more difficulty than the hen egg white protein. This difference might be explained by a greater organization and a greater hydrophobicity in the human molecule.

Lactoferrin binding to lysozyme-treated Micrococcus luteus.

When the cell lysis of Micrococcus luteus by hen egg white or human lysozyme is performed in the presence of bovine or human lactoferrin, a temporary increase of the turbidity of the solution as followed at 450 nm is observed. Examination of the suspension under light microscopy has proven that the protoplasts produced upon lysozyme action are agglutinated by lactoferrin. The rate of agglutination depends on pH, lactoferrin, lysozyme and cells concentrations. Agglutination is maximal at pH 5.5. Around 1.4 X 10(6) binding sites for lactoferrin per cell have been determined through a Scatchard plot analysis. The binding to the cells is not mediated by the glycosidic moiety of lactoferrin but rather by a charge-to charge interaction as succinylation of about four out of the 39 lysines of lactoferrin completely abolishes its ability to agglutinate the cells. Binding does not depend on ionic iron nor on the iron content of lactoferrin itself.

The influence of membrane mutations on metastasis.

In an effort to assess the effect of surface carbohydrates upon the metastasizing properties of tumor cells, lectin-resistant mouse melanoma cells were selected. Wheat-germ-agglutinin-resistant lines displayed mainly decreased metastasis properties as well as well-defined alterations in surface carbohydrates: in a glycopeptide with four side chains, two of them were missing their terminal sialic acid residues while two fucoses were newly attached to the oligosaccharide. The enzymatic defect could be pinpointed to an over-60-fold increase in fucosyltransferase, while the sialyltransferase did not decrease significantly. Revertants were again selected with lectins and their fucosyltransferase activities returned to normal values again. The metastasizing potential of the revertants was not yet assessed carefully but a return of some of the metastasizing potential was noted.

Enzymatic basis for a lectin-resistant phenotype: increase in a fucosyltransferase in mouse melanoma cells.

In the search for the biochemical basis of the control of glycosylation of cell surface carbohydrates, revertant clones were isolated from previously characterized wheat germ agglutinin-resistant clones of B16 mouse melanoma cells by selection for resistance to Lotus tetragonolobus lectin or to ricin. Comparison of the wheat germ agglutinin-resistant clones with the parent and revertant clones indicated that this phenotype was correlated with an increased sensitivity to the Lotus lectin, a 60- to 70-fold increase in alpha 1 leads to 3 fucosyltransferase activity and a decreased sialic acid content of the N-glycosidic chains of glycoproteins. The results suggest a novel type of control mechanism for lectin resistance, an increase in a glycosyltransferase activity. The presence of alpha 1 leads to 3 bound fucose on N-acetylglucosamine residues would interfere with the addition of sialic acid by alpha 2 leads to 3 linkages to galactose residues in the carbohydrate units, and this change could explain the resistance to wheat germ agglutinin and the increased sensitivity to the Lotus lectin. A change in a regulatory gene for the fucosyltransferase as a possible primary cause for the changed phenotype is discussed.

Co-purification of the Lewis blood group N-acetylglucosaminide alpha 1 goes to 4 fucosyltransferase and an N-acetylglucosaminide alpha 1 goes to 3 fucosyltransferase from human milk.

The Lewis blood group-specified N-acetylglucosaminide alpha 1 goes to 4 fucosyltransferase and an N-acetylglucosaminide alpha 1 goes to 3 fucosyltransferase have been copurified over 500,000-fold from human milk by affinity chromatography on GDP-hexanolamine agarose. The purified enzyme preparation migrates as two major bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with apparent Mr = 53,000 and 51,000. Analysis of the acceptor substrate specificity of the transferase(s) and structural characterization of the reaction products indicate that the enzyme(s) forms the Fuc alpha 1 goes to 4GlcNAc, Fuc alpha 1 goes to 3GlcNAc, and Fuc alpha 1 goes to 3Glc linkages with oligosaccharide acceptors containing the nonreducing terminal sequences Gal beta 1 goes to 3GlcNAc, Gal beta 1 goes to 4GlcNAc, and Gal beta 1 goes to 4Glc, respectively. The two fucosyltransferase activities are activated to the same extent by a variety of divalent metal ions, inactivated at identical rates by thermal denaturation or reaction with N-ethylmaleimide, and inhibited to the same extent by rabbit antiserum prepared against the purified fucosyltransferase(s). In addition, kinetic analysis of the initial rate data obtained using acceptors for one of the fucosyltransferase activities as an inhibitor of the second suggests that acceptors for both fucosyltransferase activities bind at a single active site.