Promising Results from Alzheimer's Disease Passive Immunotherapy Support the Development of a Preventive Vaccine.

The apparently near-term effects of the monoclonal antibody BAN2401 in slowing the progression of prodromal Alzheimer's disease (AD) has created cautious optimism about the therapeutic use of antibodies that neutralize cytotoxic soluble amyloid-ß aggregates, rather than removing plaque. Plaque being protective, as it immobilizes cytotoxic amyloid-ß, rather than AD's causative agent. The presence of natural antibodies against cytotoxic amyloid-ß implies the existence of a protective anti-AD immunity. Hence, for vaccines to induce a similar immunoresponse that prevents and/or delays the onset of AD, they must have adjuvants that stimulate a sole anti-inflammatory Th2 immunity, plus immunogens that induce a protective immunoresponse against diverse cytotoxic amyloid-ß conformers. Indeed, amyloid-ß pleomorphism may explain the lack of long-term protection by monoclonal antibodies that neutralize single conformers, like aducanumab. A situation that would allow new cytotoxic conformers to escape neutralization by previously effective monoclonal antibodies. Stimulation of a vaccine's effective immunoresponse would require the concurrent delivery of immunogen to dendritic cells and their priming, to induce a polarized Th2 immunity. An immunoresponse that would produce besides neutralizing antibodies against neurotoxic amyloid-ß oligomers, anti-inflammatory cytokines; preventing inflammation that aggravates AD. Because of age-linked immune decline, vaccines would be significantly more effective in preventing, rather than treating AD. Considering the amyloid-ß's role in tau's pathological hyperphosphorylation and their synergism in AD, the development of preventive vaccines against both amyloid-ß and tau should be considered. Due to convenience and cost, vaccines may be the only option available to many countries to forestall the impending AD epidemic.

Altered immunomodulating and toxicological properties of degraded Quillaja saponaria Molina saponins.

Quillaja saponins are readily hydrolyzed under physiological conditions, yielding deacylated forms that are significantly less toxic than their precursors. Yet, deacylated saponins are unable to stimulate a strong primary immune response. Although deacylated saponins elicit a strong total IgG response, their capacity to stimulate a Thl type IgG isotype profile (i.e. high levels of IgG2a and IgG2b) has been significantly diminished. Instead, an IgG profile closer to that of a Th2 immune response is stimulated (i.e. high IgG1 levels). Deacylated saponins have also lost their capacity to elicit an effective T cell immunity, as shown by their stimulation of a marginal lymphoproliferative response and their inability to elicit the production of cytotoxic lymphocytes (CTL). Modification of the immune-modulating properties brought by the degradation of quillaja saponins during vaccine storage may change the intended immune response from a Th1 to a Th2 type. This alteration would have negligible effects on vaccines depending on Th2 immunity mediated by neutralizing antibodies. However, the performance of vaccines directed against intracellular pathogens as well as therapeutic cancer vaccines may be seriously affected by the loss of their capacity to stimulate both a Th1 immune response and the production of CTL.

Development of semisynthetic triterpenoid saponin derivatives with immune stimulating activity.

Aldehyde-containing triterpene saponins have adjuvant properties, but only those from Quillaja saponaria Molina stimulate the production of cytotoxic T lymphocytes (CTL) against exogenous antigens. Quillaja saponins have two normonoterpene ester moieties, linked linearly to their fucosyl residue, that play a critical role in the stimulation of CTL. These ester moieties are also responsible for these saponins' instability and toxicity. Based on the structure-activity relationships for the different groups of Q. saponaria saponins, new semi-synthetic analogs were developed that have the adjuvanticity of quillaja saponins, yet with less toxicity and greater stability in aqueous solutions. The quillaja saponin analogs were prepared by replacing their hydrolytically unstable ester groups with another lipophilic chain linked by a stable amide bond on these saponins' glucuronic acid residue. One of these analogs, GPI-0100, is a dodecylamide saponin derivative that stimulates an antibody isotype profile that corresponds to a Th1 type immune response, as well as CTL production against exogenous antigens.

DS-1, a modified Quillaja saponin, enhances ocular and nasal absorption of insulin.

The purpose of this study was to test DS-1, a modified Quillaja saponin, for its efficacy as an absorption enhancer. Anesthetized rats receiving eyedrops or nosedrops formulated with regular pork insulin in saline showed no hypoglycemic response, indicating no systemic absorption of insulin. However, rats receiving eyedrops or nosedrops formulated with insulin plus 0.025-0.10% DS-1 showed rapid absorption of insulin and a concomitant decrease in serum D-glucose levels. No response was observed following sublingual or buccal delivery of insulin. In conclusion, the modified saponin DS-1 was efficacious at enhancing nasal or ocular insulin delivery at extremely low concentrations. The mechanism of DS-1 action is not yet known.

Characterization of six murine monoclonal antibodies specific for toxin B of Clostridium difficile.

Six murine hybridoma cell lines producing monoclonal antibodies (MAbs) specific for Toxin B of Clostridium difficile have been generated from toxin-immunized female RBF/DnJ mice. All six antibodies were reactive in Western blots with a > 200-kD protein in the supernatants of the toxigenic strain 10463 and were unreactive with similarly prepared material from the nontoxigenic strain 2037. Polyclonal antisera from rabbits immunized with Toxin B reacted on Western blots primarily with Toxin B, a 40-kD and a 55-kD band with a minor set of triplet bands at approximately 100 kD. None of the MAbs reacted in a direct EIA with purified Toxin A from C. difficile but two MAbs reacted weakly with a trypsin-sensitive band (> 200 kD) in Western blots of C. sordellii. Polyclonal antisera developed against Toxin B reacted strongly with supernatants from C. sordellii, C. bifermentans, and the nontoxigenic strain 2037. Toxin B-specific antisera was unreactive with supernatants from C. perfringens or purified Toxin A from C. difficile in direct EIA. Toxin B-specific MAbs linked to an affinity column were able to deplete bacterial supernatant of cytotoxigenic activity.

Genetically-engineered subunit vaccine against feline leukaemia virus: protective immune response in cats.

A recombinant retroviral subunit vaccine has been developed that successfully protects cats from infectious feline leukaemia virus (FeLV) challenge. The antigen used is a non-glycosylated protein derived from the envelope glycoprotein of FeLV subgroup A, expressed in Escherichia coli. This recombinant protein, rgp70D, includes the entire exterior envelope protein gp70, plus the first 34 amino acids from the transmembrane protein p15E. The vaccine consists of purified rgp70D absorbed on to aluminium hydroxide and used in conjunction with a novel saponin adjuvant. Cats immunized with this formulation developed a strong humoral immune response, including neutralizing and feline oncornavirus-associated cell membrane antigen antibodies. Vaccinated animals showed an anamnestic response upon intraperitoneal challenge with FeLV-A, and were protected from viral infection. In contrast, the control animals developed viraemia shortly after the challenge, which in most cases became chronic. Formulation of the same antigen with other widely used adjuvants elicited poor protective immune responses in cats.

Fluorometric enzyme immunoassay for measurement of infectious feline leukemia virus and its neutralization.

A fluorometric enzyme immunoassay using an alkaline phosphatase-conjugated monoclonal antibody was developed to quantitate feline leukemia virus (FeLV) infection. Monoclonal antibodies, directed against the FeLV structural protein p27, were conjugated with alkaline phosphatase using a modified maleimide method. The enzyme immunoassay requires only 4 days to reproducibly measure FeLV production instead of the 12 days required for the commonly used transformation assay using C81 cells. A linear correlation was found between the virion associated reverse transcriptase activity and the amount of intracellular p27 as determined by the fluorometric enzyme immunoassay. An immunocytochemical assay using the same conjugated monoclonal with a different substrate gave visible plaques in infected cell monolayers and was therefore used to titrate FeLV in plaque-forming units. The results obtained by all the procedures followed single hit kinetics for FeLV infection. The fluorometric enzyme immunoassay was adapted to measure FeLV neutralizing antibodies, allowing a sensitive and accurate determination of neutralizing titers.

Detection of antibodies to human immunodeficiency virus by latex agglutination with recombinant antigen.

Recombinant human immunodeficiency virus (HIV) env antigen was attached to polystyrene particles, and these complexes were used to develop the first latex agglutination assay for antibodies to HIV. A total of 95 positive and 116 negative human serum samples were assayed for antibodies to HIV by latex agglutination, and results were compared with those of a commercial enzyme immunoassay. Latex agglutination was also compared with, and found to be completely concordant with, Western blot (immunoblot) analysis with virion antigens.

Enzyme immunoassay using a novel recombinant polypeptide to detect human immunodeficiency virus env antibody.

A unique antigen, CBre3, has been synthesized from a genetically engineered clone to detect human immunodeficiency virus (HIV) env antibodies with high sensitivity and specificity. The antigen contains sequences derived from both envelope proteins of HIV, i.e., gp120 and gp41, and was purified free of Escherichia coli proteins detectable by Coomassie stain or immunoblotting with E. coli antiserum. The purified recombinant polypeptides were used as antigen in an enzyme immunoassay (EIA) to screen serum samples from healthy and HIV-infected individuals. The same samples were also tested by radioimmunoprecipitation (RIP) for gp120 and gp160 HIV antibodies. All samples containing gp120 and gp160 antibodies by RIP had CBre3 EIA values greater than 0.35 (n, 122; range, 0.37 to 2.1+; median, 1.65). All RIP HIV antibody-negative samples had CBre3 EIA values less than 0.25 (n, 140; mean, 0.052; standard deviation, 0.045; range, 0.00 to 0.22). The endpoint titer of a standard positive control serum was 1:10,000 by RIP and by CBre3 EIA. The assay was 100% accurate in three proficiency panels. It easily detected six samples from individuals whose infections were confirmed by culture; these samples were reactive only with p24 by Western blot. The samples also were positive for gp120 and gp160 antibodies by RIP. These data suggest that the CBre3 EIA can detect env antibodies as sensitively and specifically as RIP and with more sensitivity than Western blot.

Efficient purification of mouse monoclonal antibodies from ascites fluid by medium-performance anion exchange chromatography.

Medium-performance anion-exchange chromatography was applied to the purification of murine IgG class monoclonal antibodies from ascites fluid. The separations were performed under mild conditions at pH 8 using relatively low sodium-chloride concentrations. Recoveries for monoclonal antibodies of subclasses IgG1, IgG2a, and IgG2b were about 90%. The IgG preparations were free of other ascites fluid proteins.

Amphiphilic properties of the avian myeloblastosis virus major glycoprotein, gp 80.

The major glycoprotein (gp 80) from avian myeloblastosis virus (AMV) displays significant lipophilic properties, as shown by its strong interactions with acetylated uncharged decylamino agarose in hydrophobic chromatography. In effect, release from binding was achieved only by the added presence of a polarity reducing agent (ethylene glycol) and the strong anionic detergent sodium dodecyl sulfate. The hydrophobic behavior of the glycoprotein, coupled to the high content of hydrophilic carbohydrates, indicates its amphiphilic character. Confirmation of the amphiphilic nature of the AMV gp 80 was obtained by charge shift electrophoresis and crossed hydrophobic interaction immunoelectrophoresis. In both instances, the electrophoretic behavior of the glycoprotein was dependent on the presence of detergents. The AMV gp 80 displays the properties of integral membrane proteins.

Colorimetric determination of agarose-immobilized proteins by formation of copper-protein complexes.

A simple, reliable, and sensitive colorimetric procedure for the determination of proteins bound to agarose is described. The procedure utilizes the capacity of diethyldithiocarbamate to react with cupric ions resulting in a complex of dark yellow color. The extent of reduction in the color, due to chelation of Cu2+ by the immobilized proteins, indicates the amount of protein. The optimum conditions for the determination of immobilized proteins are investigated. The formation of Cu2+-protein complex proceeds stepwise until enough excess of Cu2+ is present to form the final complex. The reliability of the procedure requires that all the protein species, samples and standards, are in the final Cu2+-protein complex form. A comparative study on the determination of different proteins bound to agarose using this method as well as other methods, such as protein balance, modified Lowry reaction, and direct ultraviolet spectrophotometry, is described. The method is substantially more reliable, accurate, and simple than previously described methods.

Stimulation of sugar uptake and glycolysis in chicken embryo fibroblasts by the major glycoprotein from avian myeloblastosis virus.

Addition of purified major glycoprotein from avian myeloblastosis virus to growing or quiescent chicken embryo fibroblasts rapidly stimulates the rate of hexose transport and increases the lactic acid production. These stimulatory effects are dependent on the time of exposure and the dose of viral glycoprotein. In contrast, the glycoprotein only marginally affects hexose transport in chicken cells transformed by Rous sarcoma virus. Some effects of the glycoprotein on serum-starved quiescent cells were similar to those observed upon re-addition of serum; however, the viral glycoprotein did not stimulate DNA synthesis. Quiescent cells stimulated by saturating levels of serum showed little further stimulation of hexose uptake upon exposure to viral glycoprotein for 3 hr. This behavior suggests that the glycoprotein may be acting on a system that is also a target for serum action.

Anomalous behavior of the major avian myeloblastosis virus glycoprotein in the presence of sodium dodecyl sulfate.

The sodium dodecyl sulfate (SDS) complex of the major glycoprotein of avian myeloblastosis virus exhibited an anomalously low free electrophoretic mobility compared with those of non-glycosylated protein standards. The apparent molecular weight of the glycoprotein calculated from the relation between log molecular weight and electrophoretic mobility depended on the acrylamide concentration and reached a lower limit of 80,000. The molecular weight was also estimated from the retardation coefficients of protein standards and the viral glycoprotein. This method yielded a molecular weight of 64,000 for the avian myeloblastosis virus glycoprotein. When gel chromatography in SDS was used to determine the apparent molecular weight of the glycoprotein from its hydrodynamic properties alone, the estimated value was 50,000. The generally assigned value of 80,000 daltons for the avian myeloblastosis virus major glycoprotein, as determined by SDS electrophoresis, may be an overestimate due to its relatively low free electrophoretic mobility and peculiar conformation in SDS.

Cell-free synthesis of the precursor polypeptide for avian myeloblastosis virus DNA polymerase.

High molecular weight RNA (35S) isolated from avian myeloblastosis virus directs the cell-free synthesis of two prominent polypeptides of 180,000 and 76,000 molecular weight. The latter polypeptide has previously been identified as the precursor to the group-specific antigens of the virus ("gag" proteins) [Vogt, V. M., Eisenman, R. & Diggelmann, H. (1975) J. Mol. Biol. 96, 471-493]. Two-dimensional tryptic peptide analyses of the [35S]methionine-labeled peptides demonstrate that the 180,000-dalton product is a polyprotein that can account for all the peptides of the avian myeloblastosis virus DNA polymerase (DNA nucleotidyltransferase, EC 2.7.7.7) and those of the gag viral proteins. This is direct confirmation of the genomic order of the viral structural genes, placing the polymerase gene adjacent to the 5'-proximal gag gene of the virus. Furthermore, our findings suggest that the primary polymerase gene product is the beta subunit of the enzyme. These results are discussed in relation to the proposed structural gene map for the avian retraviruses and suggest a model for the in vivo processing of the viral polymerase.

Inactivation of avian myeloblastosis virus DNA polymerase by specific binding of pyridoxal 5'-phosphate to deoxynucleoside triphosphate binding site.

Avian myeloblastosis virus (AMV) DNA polymerase is inactivated by preincubation with pyridoxal 5'-phosphate. This inactivation is relatively specific since various pyridoxal-5'-P analogs cause no inactivation. This effect is reversible but can be made irreversible by reduction with sodium borohydride; the reduced pyridoxal-5'-P adduct exhibits a new absorbance maximum at 325 nm and a fluorescence emission at 392 nm when excited at 325 nm. The evidence presented suggests the formation of a Schiff base between pyridoxal-5'-P and a nucleophilic residue of AMV DNA polymerase. The presence of a deoxynucleoside 5'-triphosphate (dTTP) protected the enzyme from inactivation. Reduction of the pyridoxal-5'-P enzyme complex in the presence or absence of a deoxynucleoside 5'-triphosphate showed that the alpha subunit possesses five reactive amino groups, one of which is essential for catalytic activity; the beta subunit has three reactive amino groups which are not involved in the deoxynucleoside binding site.

Interactions of concanavalin A with chick embryo fibroblasts transformed by Rous sarcoma virus. Study with an RSV mutant thermosensitive for transformation.

The interactions between concanavalin A and chick embryo fibroblasts, normal and infected with Rous sarcoma virus (RSV-BH) or its thermosensitive mutant RSV-BH-Ta, have been studied. Normal chick embryo cells and RSV-BH transformed cells showed at 4 and 25 degrees C a similar number of concanavalin A receptors per cell. Analysis of the binding data by the Scatchard relation showed that apparent changes in binding as a function of temperature are due to the thermodynamic properties of the process and not to endocytosis. The lectin receptors on the cell surface of normal and RSV-BH infected cells showed homogeneity in their binding properties. Chick cells infected with RSV-BH-Ta showed a lectin binding behavior that was dependent on the temperature at which the cells were grown. At the permissive temperature for transformation (37 degrees C), the binding process was similar to that observed for normal and RSV-BH infected cells. At the nonpermissive temperature (41 degrees C), the cells showed at least two sets of concanavalin A receptors. The new set of receptors on the cell surface had a lower lectin affinity than those observed in the same cells at 37 degrees C. Chick cells infected with RSV-BH showed an enhanced agglutinability by concanavalin A, as compared with normal cells. Cells infected with RSV-BH-Ta showed a reversal of the correlation between increased concanavalin A agglutinability and the transformed state. At the permissive temperature for transformation, the cells were not agglutinable, whereas at the nonpermissive temperature they presented agglutinability indexes as high as those observed with RSV-BH infected cells. This enhanced agglutinability observed with cells maintained at the nonpermissive temperature for transformation may be related to the new set of low affinity receptors present at 41 degrees C.

Characteristics of cell membranes from somatic cell hybrids between rat hepatoma and mouse L-cells.

Cell membranes from mouse L-cells (L-B82), rat hepatoma (HTC-H1), and three clones of their somatic cell hybrids (07, V4a, and V5) showing different degrees of density-dependent inhibition of growth were analyzed by polyacrylamide gel electrophoresis. The membrane polypeptides of the hybrid clones were all similar and all showed higher proportions of polypeptides with molecular weights of 56,000 and 45,000 than their parents of their normal counterparts. The major glycoprotein form cell hybrids appeared to be identical with that of rat liver or rat hepatoma cells and different from that of L-cells. One hybrid showed density-dependent inhibition growth; the other two, like both parents, did not. All produced tumors in nude mice, although tumor production by the hybrids was delayed. A large external protein (M.W. 240,000) iodinated by lactoperoxidase-catalyzed reaction was virtually missing in the parents but was present at high levels in all their hybrid clones. Thus, there was a lack of correlation between the presence of this protein, growth control in vitro, and tumorigenicity. Furthermore, no correlation was seen between agglutination of these cells by concanavalin A and tumorigenicity. The factors controlling these membrane properties thus are independent of density-dependent inhibition of growth and of those controlling the expression of cancer.