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PubMed1994

Target cells of cytotoxic T lymphocytes directed to the individual structural proteins of rabies virus.

Target cells of cytotoxic T lymphocytes (CTL) directed to the individual structural proteins (except for the large polymerase (L) protein) of rabies virus were established by expressing only the respective protein in murine neuroblastoma (NA) and murine macrophage (J774-1) cell lines. Mice infected with the ERA strain of rabies virus developed CTL responses to all of these rabies virus proteins. The cytotoxic activity was abrogated by pretreatment of the effector cells with anti-CD8 monoclonal antibody (MAb) and complement but not with anti-CD4 MAb. Cell lysis by CTL was blocked in the presence of anti-major histocompatibility complex (MHC) class 1 antibodies in J774-1 cell lines. Rabies virus-infected cells express these proteins at the surface, which can be recognized and lysed by the respective CTL. Mice immunized with beta-propiolactone-inactivated virus induced a CTL response against glycoprotein but not against internal viral components. This assay system might be useful for further analysis of the possible contribution of these proteins in the cell-mediated immune protection against rabies.

Animals↗

PubMed1986

Effect of various treatments of gamma-globulin (IgG) for achieving intravenous tolerance on the capacity to interact with human monocyte Fc receptors. A comparative study.

Gamma-globulins for intravenous application (IgG-IV), processed by various methods, were tested for their ability to interact with human monocyte Fc receptors by determining the dose required to inhibit monocyte Fc receptor-mediated rosette formation and phagocytosis by half (ID50). Since dimeric and oligomeric IgG were found to be 2-3 times and 5-15 times more potent, respectively, than monomeric IgG, the varying proportions of polymeric IgG in intact IgG-IV were corrected for by calculation. The results of the rosette formation and phagocytosis tests were closely correlated, and insignificant differences between preparations processed by the same procedure were noted, while considerable differences were found between different procedures. The decreasing order of inhibitory activity was DEAE-Sephadex-treated IgG, acid-treated IgG, plasmin-digested IgG, polyethylene glycol(PEG)-precipitated IgG, IgG subjected to reduction/alkylation, IgG that underwent sulfitolysis, IgG treated with beta-propiolactone, and finally pepsin-treated IgG. Thus, while mild procedures preserve the capacity of IgG to interact with monocyte Fc receptors, chemical modification severely interferes with this important effector function.

Humans↗

PubMed1978

Purification and characterization of an endonuclease from Micrococcus luteus that acts on depurinated and carcinogen-modified DNA.

An endonuclease which is active with regard to depurinated, alkylated, arylated, and arylamidated DNA has been purified 500-fold from Micrococcus luteus. In this purification, separation from the pyrimidine-dimer-specific ultraviolet-endonuclease has been achieved. The enzyme has a molecular weight of 30000 on the basis of gel filtration; its activity is not absolutely dependent upon the presence of Mg2+, but 5--30 mM Mg2+ produces a five-fold stimulation. Potassium chloride concentrations of less than 100 mM are optimal, while concentrations exceeding 100 mM inhibit. The enzyme has no effect on native DNA, but introduces single-strand breaks into DNA containing apurinic/apyrimidinic sites produced by heating at an acidic pH. DNA treated with such carcinogens as N-alkyl-N-nitrosoureas, alkyl methanesulfonates, alkyl sulfates, nitrogen mustard, beta-propiolactone, 7-bromomethyl-benz[a]anthracene, N-acetoxy-2-acetylaminofluorene, and 7,12-dimethyl-benz[a]anthracene-5,6-oxide also becomes susceptible to enzymic action. The activity of the enzyme has been detected by making use of the difference in mobility between supercoiled closed-circular DNA of Pseudomonas phage PM2 and its nicked form in agarose gel elctrophoresis. Even depurinated or carcinogen-modified supercoiled PM2 DNA migrated faster than the respective relaxed nicked forms. A comparison of the number of enzyme-catalyzed single-strand breaks with the number of alkali-labile (i.e. apurinic) sites in carcinogen-modified PM2 DNA showed that the enzyme preparation introduced approximately twice as many breaks into the substrates as the number of apurinic sites present. We conclude that the enzyme preparation either recognizes both apurinic sites and DNA bases carrying carcinogenic residues or contains DNA glycosidase activity in addition to the endonuclease activity. Exposure of ultraviolet-irradiated PM2 DNA to the endonuclease preparation showed that pyrimidine dimers were not substrates. The yield of enzyme-catalyzed single-strand breaks found in ultraviolet-irradiated DNA was five times the number of alkali-labile sites present suggesting that minor photoproducts, possibly 5,6-saturated pyrimidine residues, were recognized in addition to apurinic sites.

Carcinogens↗

PubMed1985

Synthesis of poly(ethylene glycol)-bound NADP by selective modification at the 6-amino group of NADP.

The N-1 position of the adenine ring of NADP was selectively alkylated by the reaction of 2',3'-cyclic NADP with 3-propiolactone to yield 2',3'-cyclic 1-(2-carboxyethyl)-NADP (I). Derivative I was converted to a mixture of the isomers of N6-(2-carboxyethyl)-NADP with their phosphate groups at the 2' or 3' position (IIa and IIb) by chemical reduction, alkaline rearrangement and chemical reoxidation. Carbodiimide coupling of the mixture of IIa and IIb to alpha, omega-diaminopoly(ethylene glycol) gave the 2', 3'-cyclic derivative of poly(ethylene glycol)-bound NADP (III), which was enzymically hydrolyzed to yield poly(ethylene glycol)-bound NADP (PEG-NADP). PEG-NADP has good cofactor activity (16-100% of that of NADP) for NADP-specific and NAD(P)-specific dehydrogenases except isocitrate and glucose dehydrogenases. For NAD-specific enzymes, PEG-NADP has higher cofactor activity than NADP: for horse liver alcohol dehydrogenase, the cofactor activity of PEG-NADP is 40 times that of NADP and 14% of that of NAD. Kinetic studies show that for most of enzymes tested, Km values for PEG-NADP are larger than those for NADP and V values for PEG-NADP are similar to those for NADP. PEG-NADP proved to be applicable in a continuous enzyme reactor, in which reactions of glutamate dehydrogenase and glucose-6-phosphate dehydrogenase were coupled by the recycling of PEG-NADP.

Alkylation↗

PubMed1998

Evaluation of Anaplasma marginale from tick cell culture as an immunogen for cattle.

Anaplasma marginale has been propagated and continuously passaged in an Ixodes scapularis cell line. Anaplasma development was characterized and cultures with a high density of rickettsiae were harvested at a predictable rate. Culture-derived A. marginale (CAM) remained infective for cattle and was used effectively as antigen in diagnostic tests with the sensitivity to identify bovine carriers of A. marginale. This study presents results of an initial trial using the CAM as an immunogen for cattle. CAM was mechanically disrupted, frozen at -70 degrees C, and inactivated with beta-propiolactone. Two intact yearling cattle were immunized with CAM and Freund's adjuvant, receiving 4 subcutaneous injections at 3-4 week intervals. Two control yearling cattle received adjuvant and PBS. Serum samples were evaluated by competitive ELISA (C-ELISA) using CAM as antigen and the standard complement fixation test (CFT). All cattle were subsequently challenged with A. marginale-infected blood from a carrier cow. An additional intact calf was inoculated with live CAM from the same passage and screened by C-ELISA and CFT. Sera collected from immunized cattle were negative or suspicious by CFT throughout the immunization study. The same sera were strongly positive by C-ELISA two weeks after the first injection and throughout the study. All cattle became infected following challenge-exposure with blood, but immunized cattle exhibited longer prepatent periods as well as lower parasitemias and percent reduction of packed cell volumes as compared with the controls. The calf receiving live CAM became infected and underwent a mild clinical reaction with positive C-ELISA and CFT results and did not become clinically ill following blood challenge. This preliminary study suggests that the CAM antigen is highly immunogenic in cattle. Furthermore, the CFT did not identify immunized animals whereas the C-ELISA (using CAM) was highly sensitive for detection of both immunized and infected animals.

Anaplasma↗

PubMed1990

Development of a vaccine preventing parvovirus-induced reproductive failure in pigs.

An inactivated porcine parvovirus (PPV) vaccine for the prevention of PPV-induced reproductive failure in pigs was developed, using virus grown in cell culture, inactivated with beta-propiolactone and adjuvanted with aluminum hydroxide. The vaccine was tested for safety by subcutaneous injection into pregnant gilts. There were no signs of abnormal reactions nor evidence of PPV infection in the gilts or their foetuses when they were sacrificed 6 weeks after vaccination. To demonstrate that the vaccine was immunogenic, pigs were immunised either once or twice with 4 weeks between doses. Resulting antibody titres (haemagglutination inhibition - HAI) ranged from less than 8 to 64 (geometric mean of 30) after one dose of vaccine, and from 128 to 512 (geometric mean 256) after two doses. To demonstrate that the vaccine was protective, antibody-negative gilts were vaccinated twice, with 4 weeks between doses, joined after the second dose, and were then infected with virulent PPV 40 to 50 days after joining. In litters from 10 vaccinated gilts, none of 93 foetuses showed evidence of PPV infection. In contrast, in litters from two unvaccinated gilts, all 13 foetuses showed evidence of PPV infection and 10 of these were mummified. The average number of live piglets per litter was 9.2 from vaccinated gilts and 1.5 from unvaccinated gilts. The vaccine was therefore considered to be effective in preventing PPV reproductive failure in susceptible gilts.

Animals↗

PubMed1961

Interaction of chromatid breaks produced by x-rays and radiomimetic compounds.

The results of combination treatments of the roots of Vicia faba with certain radiomimetic compounds (8-ethoxycaffeine, maleic hydrazide, beta-propiolactone, potassium cyanide) and x-ray as well as combination treatments of certain radiomimetic compounds with one another were observed to determine whether interaction will occur between chromosomal breaks induced by different agents. Interaction was observed between breaks induced by x-rays and all of the breaks induced by chemicals but not between breaks induced by any two chemicals. The results are discussed in terms of possible breakage bond differences and the effects of temporal and spatial differences in breaks induced by different agents.

Caffeine↗

PubMed1967

Inactivation of adenovirus and simian virus 40 tumorigenicity in hamsters by vaccine processing methods.

The effectiveness of the adenovirus vaccine inactivation process in destroying the tumorigenic potential for hamsters of adenoviruses, simian virus 40 (SV-40), and adenovirus-SV-40 hybrids was studied. Baby hamsters injected with untreated virus and with samples subjected to the complete inactivation process and to portions of the process were observed for tumor development for periods in excess of 300 days. Over 20,000 hamsters were injected. From 1 to 7 hr of exposure to formaldehyde at a concentration of 0.031 m at 37 C was sufficient to destroy the tumorigenicity observed in the nontreated preparations. Since the inactivation process included 48 hr of exposure at 37 C to 0.031 m formaldehyde plus treatment with ultraviolet (UV) and with beta-propiolactone (BPL), it was concluded that the process has a large margin of safety. Adenovirus isolates free from tumorigenic potential are difficult, if not impossible, to obtain. Therefore, a proven inactivation process appears to provide the best assurance for obtaining adenovirus vaccines free from such potential. Data presented suggest that the tumorigenic property of the viruses studied might be independent of the infectivity of the preparation. The tumorigenic property was found to be highly susceptible to formaldehyde, but less sensitive to BPL or UV treatment. In contrast, treatment with UV or BPL decreased viral infectivity more readily than tumorigenicity. The three-stage inactivation process (formaldehyde, UV, and BPL) inactivated both tumorigenicity and infectivity.

Adenoviridae↗

PubMed1969

Preparation of La Crosse virus hemagglutinating antigen in BHK-21 suspension cell cultures.

Hemagglutinating and complement-fixing antigens of La Crosse virus (California arbovirus group) were produced in serum-free suspension cultures of BHK-21/13S cells. The appearance and production of these antigens were correlated with the titer of infectious virus. No significant differences in antigen titers were produced by varying virus dose 10-fold. Hemagglutinin appeared 6 to 8 hr after inoculation and reached peak titer in 14 to 22 hr. Both beta-propiolactone and Tween 80-ether treatment inactivated infectious virus in the antigens. Unlyophilized antigen was stable at -60, 5 and 24 C for at least 117 days but not for 1 year. Lyophilized antigen was stable for at least a year, however, at -20 and 5 C. Cell culture-produced antigen was more sensitive than brain-produced antigen in detecting hemagglutination inhibition antibody in human sera.

Animals↗

PubMed1970

Purified rabies vaccine (suckling rat brain origin).

A 10% suckling rat brain rabies vaccine free from encephalitogenic activity was prepared and inactivated with 1:8,000 beta-propiolactone (BPL), or ultraviolet light, or a combination of ultraviolet light and BPL, or 1% phenol. Potency was excellent in all samples, with the exception of the phenolized product which was marginal. A purified suckling rat brain (SRB) vaccine prepared by zonal centrifugation and inactivated with 1:8,000 BPL contained about 0.01 the amount of protein nitrogen of the unpurified 10% SRB vaccine. This purified product passed the National Institutes of Health potency test for rabies vaccine after administration of a quantity equivalent to a standard 10% brain suspension.

Animals↗

PubMed1970

Preparation of noninfectious arbovirus antigens.

Noninfectious arbovirus antigens were prepared from borate-saline suspensions of infected suckling mouse brain buffered with tris(hydroxmethyl)aminomethane and treated with beta-propiolactone (BPL). The activity and stability of these antigens were enhanced by altering the buffering system, by passing the virus seed through a series of four or more continuous passages in the brains of suckling mice, or by a combination of these procedures. The titers of group A and B arbovirus antigens were comparable to titers of antigens extracted by the conventional sucroseacetone-BPL (SA-BPL) method. Antigens prepared from some ungrouped and Bunyamwera arboviruses by either the borate-saline-BPL or SA-BPL method produced inconsistent results and will require the development of more unique procedures to obtain suitable hemagglutinating antigens.

Animals↗

PubMed1971

Zonal-centrifuged purified duck embryo cell culture rabies vaccine for human vaccination.

Rabies virus produced in duck embryo cell culture was concentrated from volumes of 14 to 30 liters to 400 to 800 ml by zonal centrifugation. Virus titers of peak fractions were from 100- to 1,000-fold greater than those of the starting material. Vaccines were prepared by combining fractions with peak virus titers and diluting back to 10 times concentration. The resulting beta-propiolactone-inactivated vaccines, when prepared as lyophilized vaccines with AlPO(4) adjuvant diluents, were low in protein nitrogen (0.01 mg/ml), and three of four lots passed the National Institutes of Health potency test when tested as equivalent to a standard 10% suspension of duck embryo or mouse brain tissue vaccine. These vaccines also induced good sero-conversion in adult rabbits after a single 1-ml dose of vaccine. Guinea pigs sensitized with zonal-centrifuged purified duck embryo vaccine (with AlPO(4) adjuvant) did not exhibit anaphylactic shock reactions when challenged with homologous vaccine. Also, no anaphylactic shock reactions were observed when guinea pigs were sensitized with either a 10% experimental duck embryo vaccine or cell culture vaccine and then challenged with the zonal-purified vaccine. However, guinea pigs sensitized with cell culture or zonal-purified vaccine and then challenged with the 10% experimental vaccine did show slight transitory congestion. The 10% experimental whole duck embryo vaccine was responsible for all observed anaphylactic shock reactions whether homologous or heterologous.

Adjuvants, Immunologic↗

PubMed1978

Delayed-type hypersensitivity to rabies virus in mice: assay of active or passive sensitization by the footpad test.

With a purified beta-propiolactone-inactivated rabies virus, a significant increase in footpad swelling was elicited in normal or in BCG-pretreated mice after immunization with varying doses of rabies vaccine. These footpad reactions were shown to peak at 24 h and to be associated with an infiltration of newly formed blood monocytes demonstrated by histology and [125I]deoxyuridine labeling. A relationship between the lymphoproliferation and the degree of sensitization is described, and the susceptibility to cyclophosphamide treatment is also examined. Adoptive transfer of specific reactivity to normal recipient mice with immune lymphoid cells, but not with immune serum, was demonstrated, and the results represent another argument for a cell-mediated immunological mechanism.

Animals↗

PubMed1979

Kinetics of different specific immunological parameters after rabies vaccination in mice.

A significant protection to an intracerebral challenge of 70 mean lethal doses of a standard live rabies virus strain was obtained in BCG-pretreated mice or in normal mice which had been immunized with a single subcutaneous injection of a beta-propiolactone-inactivated rabies vaccine. Concomitantly, levels of delayed-type hypersensitivity (measured in vivo by the footpad test) and serum-neutralizing activity were evaluated at various times after immunization. All immune criteria were significantly augmented in the BCG-pretreated, rabies-immune mice as compared to normal, rabies-immune mice. However, peak levels of protection, delayed-type hypersensitivity, and serum-neutralizing activity did not occur at the same times. For instance, in the BCG-pretreated, rabies-immune mice, delayed-type hypersensitivity peaked on day 7, protection peaked on day 21, and serum-neutralizing activity peaked on day 60. In BCG-pretreated mice, which did not receive the rabies vaccine, positive delayed-type hypersensitivity, some protection, and serum neutralizing activity were observed 4 to 5 weeks after BCG pretreatment. The possible relationships between specific and nonspecific immunity provoked by rabies virus antigens, tissue culture cell-associated antigens (derived from the bovine fetal kidney cells in which the rabies virus was grown, and BCG are discussed.

Animals↗

PubMed1962

Induction of lysogenic Micrococcus lysodeikticus.

Field, A. K. (Cornell University, Ithaca, N.Y.) and H. B. Naylor. Induction of lysogenic Micrococcus lysodeikticus. J. Bacteriol. 84:1129-1133. 1962.-Between 50 and 60% of log-phase cells of Micrococcus lysodeikticus strain ML 53-40, lysogenic for N5 bacteriophage, were induced by an optimal dose of ultraviolet light. Induction of dilute cell suspensions caused a subsequent 200- to 800-fold increase in infective-center concentration. Cells in the stationary growth phase and cells adapted to a chemically defined medium were also induced by ultraviolet irradiation. Dimethyl sulfate induced the lysogenic culture to about the same extent as did ultraviolet light, whereas beta-propiolactone was less effective.

Bacteriophages↗

PubMed1982

Inactivation of Lassa, Marburg, and Ebola viruses by gamma irradiation.

Because of the cumbersome conditions experienced in a maximum containment laboratory, methods for inactivating highly pathogenic viruses were investigated. The infectivity of Lassa, Marburg, and Ebola viruses was inactivated without altering the immunological activity after radiation with Co60 gamma rays. At 4 degrees C, Lassa virus was the most difficult to inactivate with a rate of 5.3 X 10(-6) log 50% tissue culture infective dose per rad of CO60 radiation, as compared with 6.8 X 10(-6) log 50% tissue culture infective dose per rad for Ebola virus and 8.4 X 10(-6) log 50% tissue culture infective dose per rad for Marburg virus. Experimental inactivation curves, as well as curves giving the total radiation needed to inactivate a given concentration of any of the three viruses, are presented. We found this method of inactivation to be superior to UV light or beta-propiolactone inactivation and now routinely use it for preparation of material for protein-chemistry studies or for preparation of immunological reagents.

Arenaviridae↗

PubMed1983

Detection of Rift Valley fever virus antigen by enzyme-linked immunosorbent assay.

A double-antibody (sandwich) enzyme-linked immunosorbent assay (ELISA) was adapted to detect Rift Valley fever virus antigen. Antibodies were purified from hyperimmune mouse and rabbit sera by affinity chromatography, using CNBr-activated Sepharose 4B coupled to a beta-propiolactone-inactivated sucrose-acetone-extracted suckling mouse liver antigen. In the assay, antigen was captured by mouse antibody adsorbed to polystyrene plates and then detected by reacting sequentially with rabbit anti-Rift Valley fever virus antibody and swine anti-rabbit immunoglobulin G conjugated to alkaline phosphatase. ELISA proved to be useful in measuring viral antigen in different animal systems. However, great variation was found in the amount of antigen per PFU encountered in different circumstances. The ELISA system was optimized using supernatant fluids from infected Vero cell cultures and had a sensitivity of 10(5) PFU/ml. Hamsters develop progressive viremia, much as seen in susceptible domestic animals, such as lambs; ELISA could reliably detect 10(6) PFU/ml of viremic hamster serum. Rhesus monkeys with Rift Valley fever infection were positive by ELISA even when viremias were only 5 X 10(3) PFU/ml. ELISA also proved to be useful in measuring viral antigen in infected mosquitoes.

Animals↗

PubMed1972

Cell fusion by canine distemper virus-infected cells.

AV3 cells (continuous human amnion) infected with the Onderstepoort strain of canine distemper virus produced cell fusion within 2 to 5 hr when added to AV3 cell monolayers. An apparent requirement for intact, infected cells was demonstrated by showing that (i) frozen-and-thawed infected cells failed to induce fusion, (ii) infected cells frozen in the presence of glycerol retained their ability to induce fusion, (iii) infected cells subjected to swelling in hypotonic buffer and homogenization lost their ability to fuse cells, and (iv) semipurified and concentrated virus preparations with infectivity titers as high as 10(7.5) mean tissue culture doses per ml failed to induce fusion within 5 hr. Preparations of intact, infected cells had a mean log(10) ratio of infectivity to fusion activity of 3.6. Treatment with beta-propiolactone rendered the active preparations free from detectable infectivity while they retained their ability to cause cell fusion. Cycloheximide did not block the formation of syncytia in assay cells. This type of cell fusion was neutralized by canine distemper virus immune antisera, and measles virus immune sera showed a slight degree of cross-neutralization. Other cell lines, HEp-2, MA 139 (embryonic ferret lung), MA 104 (embryonic rhesus monkey kidney), and Vero (African green monkey kidney) were also susceptible.

Amnion↗