Immune response in skunks to a vaccinia virus recombinant expressing the rabies virus glycoprotein.

Striped skunks (Mephitis mephitis) were vaccinated with a vaccinia virus recombinant expressing the rabies virus glycoprotein. Virus neutralizing antibodies to rabies virus were present at 14 days postvaccination by the following routes: scarification (6/6), intramuscular (4/4) and intestinal (5/8). Six out of seven skunks that ate vaccine filled baits had virus neutralizing antibodies at 28 days. When challenged intramuscularly with street virus, the survival rates were 5/7 for the bait-fed group, 4/8 for the intestinal group, 3/4 for the intramuscular group, 5/6 for the animals that were scarified, and 0/8 for controls. This is the first report of a high rate of immunization of skunks with a rabies vaccine administered orally.

Studies of ERA/BHK-21 rabies vaccine in skunks and mice.

ERA rabies vaccine virus grown in BHK-21 13S cells (ERA/BHK-21) and street rabies virus were titrated in mice by intracerebral, intranasal and intramuscular inoculation. Mice were also given undiluted ERA/BHK-21 in baits. Skunks were given undiluted ERA/BHK-21 in baits and by intramuscular, intranasal and intestinal inoculation. Virus neutralizing antibody titers against rabies virus were measured over a three month observation period. The surviving skunks were challenged by intramuscular inoculation with rabies street virus from a skunk salivary gland suspension. When titrated in mice, ERA/BHK-21 had titers of 10(7.0), 10(5.2) and 10(3.9) median lethal doses per mL by the intracerebral, intranasal and intramuscular routes, respectively. All skunks (8/8) inoculated intranasally developed paralytic rabies by 12 days after exposure to ERA/BHK-21 virus. None of the skunks that developed vaccine-induced rabies had infectious virus in the submandibular salivary glands. Vaccine-induced rabies also occurred in 1/8 skunks in the intramuscularly inoculated group and in 1/8 in the intestinally inoculated group. The survival rates of challenged skunks in the various groups were as follows: intramuscular, 7/7; intestinal, 2/7; bait, 0/8; and control, 0/8. These results indicate that ERA/BHK-21 virus has a significant residual pathogenicity in mice and in skunks by some routes of inoculation. Skunks given vaccine intramuscularly were protected against challenge, while those skunks given the vaccine in baits were not.

Mutants of rabies viruses in skunks: immune response and pathogenicity.

In studies to develop an oral rabies vaccine for wildlife, the immune response to and pathogenicity of two types of mutants of rabies viruses were examined. Forty-five small plaque mutants were selected from cultures of ERA rabies virus treated with 8-azaguanine or 5-fluorouracil and tested for pathogenicity in mice. Two of these mutants AZA 1 and AZA 2 (low pathogenicity in mice) were given to skunks by oral (bait), intestinal (endoscope) and intramuscular routes. Additionally, challenge virus standard (CVS) rabies virus and mutants of this and ERA rabies virus (CVS 3766 and 3713, and ERA 3629) that were resistant to neutralization by specific antiglycoprotein monoclonal antibodies (and apathogenic in mice) were tested by various routes in skunks. Skunks given AZA 1 and AZA 2 were challenged at three months postinoculation with street rabies virus. After oral administration, there were very low rates of seroconversion with AZA 1 and AZA 2 and on challenge only 2/7 given AZA 1 and 1/8 given AZA 2 survived. None of the skunks given the other mutants orally seroconverted. AZA 2 produced a high rate of seroconversion (8/8) by the intestinal route and all challenged skunks in this group survived (7/7). All skunks vaccinated intramuscularly with AZA 1 (4/4) or AZA 2 (4/4) developed high levels of rabies neutralizing antibodies and survived challenge. The mutant CVS 3766, while apathogenic when given intracerebrally to adult mice, was consistently pathogenic by this route (and intranasally) in skunks. These results demonstrate that skunks are highly resistant to oral immunization by live rabies virus vaccines and that pathogenicity (by intracerebral route) of the mutant CVS 3766 is markedly different in mice and skunks.

Vaccinia recombinant virus expressing the rabies virus glycoprotein: safety and efficacy trials in Canadian wildlife.

Twenty-six meadow voles (Microtus pennsylvanicus), ten woodchucks (Marmota monax), thirteen grey squirrels (Sciurus carolinensis), thirteen ring-billed gulls (Larus delawarensis), six red-tailed hawks (Buteo jamaicensis) and eight great horned owls (Bubo virginianus) received vaccinia virus recombinant expressing the rabies virus glycoprotein (V-RG) by direct instillation into the oral cavity. Each of ten coyotes (Canis latrans) received the virus in two vaccine-laden baits. Several voles and most of the gulls died from diseases unrelated to vaccination during the observation period, but all other animals remained healthy and survived. These deaths from causes other than vaccination and the absence of any lesions suggestive of vaccinia infection indicate that it is unlikely that any animal suffered or died as a result of V-RG administration. In addition several animals showed an unexpected high level of rabies neutralizing antibodies.

Examination of whole cell mounts by transmission electron microscopy using cultures grown on carbon coated coverslips.

Cultures of porcine granulosa cells have been grown on serum coated carbon films deposited on glass coverslips. Intact and detergent-treated cultures were critical point dried, chromium shadowed and removed from the coverslips by dilute hydrofluoric acid. Whole cell mounts were examined by conventional transmission electron microscopy. The advantage of this system over other methods includes the ability to: (1) examine cells with a highly rounded morphology, (2) use high cell densities, (3) study cell projections extending onto the substratum, (4) maintain cultures for long periods, (5) examine large samples easily, (6) readily use the same cultures for transmitted light and fluorescent microscopy, scanning and transmission electron microscopy (SEM and TEM).

Decreased micrococcal nuclease sensitivity of nuclei from nerve growth factor-treated PC12 cells.

Nuclei from nerve growth factor-treated PC12 cells are more resistant to digestion with micrococcal nuclease than are nuclei from control cells. The production of oligosomal fragments is decreased, as is the generation of Mg2+-soluble products. One interpretation of the data is that differentiation of these cells due to treatment with nerve growth factor involves a decrease in the total number of DNA sequences transcribed.

Lectin probes of glycoconjugates in human salivary gland neoplasms: 2.

Lectins are proteins or glycoproteins which exhibit a high affinity for specific sugar molecules. Terminal sugars on cell surface or cytoplasmic oligosaccharides bound to proteins and lipids can be probed with lectins. This study records the lectin-binding characteristics of 20 salivary gland neoplasms and compares them with observations in normal human serous and mucous salivary glands. The results of this study support the current histogenetic concepts for the development of some of the salivary gland neoplasms.

Lectin probes of glycoconjugates in human salivary glands: 1.

Research into the oligosaccharide content of glycoproteins in saliva indicates that serous saliva primarily contains N-glycosidically linked carbohydrate chains with high concentrations of mannose, whereas mucous saliva contains a predominance of O-glycosidically linked carbohydrate chains with high concentrations of terminal fucose and N-acetylneuraminic acid molecules. These differences between serous and mucous saliva can be visualized morphologically in salivary gland tissues by differential lectin binding in acinar cells and duct contents. This study utilizes a fluorescein-labelled lectin-binding method to demonstrate these differences and to study the characteristics of ductal and myoepithelial cells in salivary gland tissues. The results generally confirm the predictable differential binding.

Selective de novo synthesis of tyrosine hydroxylase in organ cultures of rat superior cervical ganglia after in vivo administration of nerve growth factor.

Tyrosine hydroxylase is synthesized de novo in rat superior cervical ganglia in organ culture. The differential rate of synthesis is not increased significantly by the addition of nerve growth factor to the culture. Prior administration of nerve growth factor in vivo, however, leads to an augmented synthesis of tyrosine hydroxylase in ganglia subsequently cultured in vitro. The differential rate of tyrosine hydroxylase synthesis was increased by a factor of between 3 and 4. Increases in the differential rate of synthesis were detected within 6 h; the rate reached a maximum 24 to 36 h after a single injection of nerve growth factor. Administration of actinomycin D or of nerve growth factor antibody in vivo prevented the nerve growth factor-induced increase in the differential rate of tyrosine hydroxylase synthesis in vitro. However, the increase in the synthetic rate of tyrosine hydroxylase was not prevented by the addition of actinomycin D to the culture.

Effects of 12-0-Tetradecanoylphorbol-13-acetate (TPA) on rat pheochromocytoma (PC12) cells: interactions with epidermal growth factor and nerve growth factor.

The phorbol ester tumor promotor 12-0-tetradecanoylphorbol-13-acetate (TPA) specifically inhibited the binding of radioiodinated epidermal growth factor (125I-EGF) to rat pheochromocytoma (PC12) cells in a noncompetitive fashion with an apparent Ki of 11-26 nM. Both TPA and EGF elicited similar biological responses in PC12 cells including enhanced incorporation of 3H-choline and 32 P-orthophosphate into macromolecules, induction of ornithine decarboxylase, and stimulation of the phosphorylation of a 30,000 MW nonhistone, chromosome-associated protein. These effects were also elicited by nerve growth fact (NGF) which, in contrast to the former agents, is a differentiating stimulus for PC12 cells. The effects of TPA were additive or more than additive to the effects of NGF and EGF. When PC12 cells were induced to differentiate by treatment with NGF for 72 hours, the binding of 125I-EGF and responses to EGF were reduced by approximately 70%. The response of PC12 cells to the tumor promoter TPA was unaffected by treatment with NGF. Thus, the qualitatively similar effects of TPA and EGF seemed to be mediated through separate receptor systems with only the EGF receptor system reduced by NGF treatment.

Nerve growth factor-induced decrease in the cell-free phosphorylation of a soluble protein in PC12 cells.

Incubation of cell-free extracts from PC12 cells with [32P]ATP leads to the phosphorylation of a 100,000-dalton protein. In extracts from cells treated with nerve growth factor, the labeling of the 100,000-dalton protein is substantially and selectively reduced. Direct quantitation indicates that the reduction is a minimum of 30-50% in the various experiments. The decrease is evident after as little as 15 min of nerve growth factor treatment, and disappears within 2 h after the removal of nerve growth factor. The decrease is dose dependent; a complete response is seen after treatment with 10 ng of nerve growth factor/ml. Some decrease in phosphorylation is also seen after treatment of the cells with epidermal growth factor, 12-O-tetradecanoylphorbol-13-acetate, or 5'-N-ethylcarboxamideadenosine, a potent adenosine receptor agonist, but not after treatment with insulin. The phosphorylation of the 100,000-dalton protein, in extracts from either control or nerve growth factor-treated cells, leads almost exclusively to the formation of phosphothreonine. The addition of equal amounts of extract from untreated cells and extract from nerve growth factor-treated cells produces a level of phosphorylation exactly intermediate between those of the two extracts used separately, indicating the absence of a soluble kinase inhibitor. The data suggest that nerve growth factor treatment produces either a covalent inhibition or a physical removal of the kinase for the 100,000-dalton protein.

Increased phosphorylation of specific nuclear proteins in superior cervical ganglia and PC12 cells in response to nerve growth factor.

Treatment of rat superior cervical ganglia in culture with nerve growth factor (NGF) increases the amount of radioactive phosphate incorporated into a nuclear protein band. This band migrates coincidentally with H1 histone on 10% sodium dodecyl sulfate/polyacrylamide gels. The increase in phosphate incorporation is at least 70% and occurs only in tissues known to be responsive to NGF. It is not produced by treatment with related peptides, but is observed after the addition of dibutyryl cyclic AMP. An increase in phosphorylation can be detected after 1 h, and can be seen with as little as 10 ng/ml of NGF in the medium. Neither actinomycin D nor cycloheximide inhibits the effect. When the nuclei are extracted with 0.2 M H2SO4 and the extract analyzed on acid-urea/polyacrylamide gels, two NGF-responsive proteins can be detected. One protein again migrates with the H1 histone marker; the other migrates more slowly than H1. These two NGF-responsive proteins have molecular weights of approximately 30,000 and are chromatin-bound. They are not soluble in 5% perchloric acid, but can be extracted from the nuclei with 0.35 M NaCl. No increase in the phosphorylation of these proteins was seen in ganglia from 6-hydroxydopamine-treated rats. The phosphorylation of the proteins in both control and NGF-treated ganglia occurs almost exclusively on serine residues. The amino acid compositions of the two nuclear proteins show that they are different from the H1 histone and different from each other. Both nerve growth factor (NGF) and epidermal growth factor (EGF) increase the incorporation of radioactive phosphate into a specific nuclear protein in cultures of PC12, a clone of rat pheochromocytoma. Purified NGF antibody blocks the effect of NGF, but not that of EGF; EGF antiserum neutralizes the effect of EGF, but not that of NGF. Insulin, glucagon, and dexamethasone are without effect. The increase in phosphorylation due to NGF can be detected within 1 h. Dibutyryl cyclic AMP increases the phosphorylation of this protein, but dibutyryl cyclic GMP does not. Neither the uptake nor the overall incorporation of [32P]orthophosphate is altered by NGF, EGF, or dibutyryl cAMP under the present experimental conditions. The nuclear protein exhibiting increased radioactivity is similar in solubility, size, and amino acid composition to one of the NGF-responsive nuclear proteins from sympathetic ganglia.

Cell surface labelling with gold colloid particulates: the use of avidin and staphylococcal protein A-coated gold in conjunction with biotin and fc-bearing ligands.

Procedures for preparing gold colloid particles stabilized with either avidin or protein A are described. Methods of using these general utility tracers for localizing biotinylated and fc bearing immunoglobulins are outlined and, as examples of the way in which these methods can be applied, procedures for identifying epidermal growth factor receptors and surface fibronectin on ovarian granulosa cells are described.

Induction of ornithine decarboxylase by renin-free nerve growth factor.

Renin-free nerve growth factor causes the induction of ornithine decarboxylase (L-ornithine carboxy-lyase, EC 4.1.1.17) in superior cervical ganglia from neonatal rats but not in the brain of mature rats. Less pure preparations of nerve growth factor induce the enzyme in both brain and ganglia. The induction of ornithine decarboxylase in the central nervous system appears to be due to renin, not to nerve growth factor itself.

Clinical, metabolic, and antibody responses of adult volunteers to an investigational vaccine composed of pertussis toxin inactivated by hydrogen peroxide.

A toxoid vaccine, composed of purified pertussis toxin inactivated with H2O2 (NICHD-Ptxd), was developed on the basis of evidence that serum neutralizing antibodies (antitoxin) would confer immunity to pertussis. In vivo and in vitro assays of NICHD-Ptxd showed only trace or nondetectable levels of pyrogenic, adenosine diphosphate-ribosyltransferase, binding and pharmacologic activities. Nevertheless, about 40% of the antigenicity of pertussis toxin was retained. Adult volunteers were injected, two times 6 weeks apart, with either 10 (n = 21), 50 (n = 25), or 75 (n = 30) micrograms/dose of one lot, Ptx-06, adsorbed onto AI(OH)3. Neither fever nor changes in the levels of leukocytes, lymphocytes, fasting blood glucose, or insulin were observed in the volunteers. The optimal immunizing dose, 50 micrograms, induced levels of antitoxin (geometric mean (GM) 302 U) comparable to those found in eight adults convalescent from pertussis (GM 269 U) and greater than those found in 18-month-old children after their fourth dose of diphtheria and tetanus toxoids and pertussis vaccine (GM 20.0 U, p less than 0.001). These data indicate that NICHD-Ptxd is safe and immunogenic in adults, and they justify its evaluation in infants and children.

Immunization of foxes against rabies with a vaccinia recombinant virus expressing the rabies glycoprotein.

Foxes were vaccinated orally (by bait), gastrically (by stomach tube) and by scarification with a vaccinia recombinant virus expressing the rabies glycoprotein. Neutralizing antibodies against rabies virus were detected at two weeks postvaccination in 8/8 foxes in the bait-fed group, in 3/6 foxes inoculated by stomach tube and in 2/2 of the scarified foxes. After challenge at three months postvaccination with street rabies virus, all foxes that had developed antibodies were protected. The high rate of seroconversion, high levels of antibodies, and resistance to challenge suggest that this recombinant virus might be a suitable vaccine for oral immunization of foxes against rabies.

Treatment with succinic anhydride improves the immunogenicity of Shigella flexneri type 2a O-specific polysaccharide-protein conjugates in mice.

Seroepidemiological data and a clinical trial with a Shigella sonnei O-specific polysaccharide (O-SP)-Pseudomonas aeruginosa recombinant exoprotein A (rEPA) conjugate provide evidence that a critical level of immunoglobulin G (IgG) lipopolysaccharide (LPS) antibodies in serum confers protection against shigellosis. We evaluated the immunogenicity of conjugates whose carrier proteins and O-SPs were treated with succinic anhydride (SA), which reacts with amino groups at neutral pH to form amide-linked carboxyls (succinylation). Conjugates were synthesized with either of two genetically inactivated medically useful toxins, the diphtheria protein CRM9 or rEPA, bound to the O-SP of Shigella flexneri type 2a. Conjugates composed of the succinylated protein, succinylated O-SP, or both succinylated components were administered to mice by a clinically relevant scheme, and their levels of serum IgG anti-LPS and anti-proteins were assayed 7 days after the second and third injections. CRM9 served as a more immunogenic carrier than rEPA. Conjugates composed of succinylated components were more immunogenic than the conjugates composed of the native components. SA treatment of both the carrier protein and the O-SP did not confer an advantage over the succinylated protein alone. Conjugates prepared with native proteins, in general, elicited slightly higher levels of IgG protein antibodies than conjugates composed of the SA-treated proteins.